Research ArticleThe Hydrogen Sulfide Donor NaHS Delays Programmed CellDeath in Barley Aleurone Layers by Acting as an Antioxidant

Ying-Xin Zhang1 Kang-Di Hu1 Kai Lv2 Yan-Hong Li1 Lan-Ying Hu1 Xi-Qi Zhang2

Long Ruan2 Yong-Sheng Liu1 and Hua Zhang1

1School of Biotechnology and Food Engineering Hefei University of Technology Hefei 230009 China2Anhui Academy of Agricultural Sciences Hefei 230031 China

Correspondence should be addressed to Hua Zhang hzhanglabgmailcom

Received 18 November 2014 Revised 4 January 2015 Accepted 4 January 2015

Academic Editor Guangdong Yang

Copyright copy 2015 Ying-Xin Zhang et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

H2S is a signalingmolecule in plants and animals Here we investigated the effects ofH

2S on programmed cell death (PCD) in barley

(Hordeum vulgare L) aleurone layers The H2S donor NaHS significantly delayed PCD in aleurone layers isolated from imbibed

embryoless barley grain NaHS at 025mM effectively reduced the accumulation of superoxide anion (sdotO2

minus) hydrogen peroxide(H2O2) and malondialdehyde (MDA) promoted the activity of superoxide dismutase (SOD) guaiacol peroxidase (POD) catalase

(CAT) and ascorbate peroxidase (APX) and decreased those of lipoxygenase (LOX) in isolated aleurone layers Quantitative-PCRshowed thatNaHS treatment of aleurone tissue led to enhanced transcript levels of the antioxidant genesHvSOD1 HvAPXHvCAT1andHvCAT2 and repressed transcript levels ofHvLOX (lipoxygenase gene) and of two cysteine protease genesHvEPA andHvCP3-31 NaHS treatment in gibberellic acid- (GA-) treated aleurone layers also delayed the PCDprocess reduced the content of sdotO

2

minus andincreased POD activity while decreasing LOX activity Furthermore 120572-amylase secretion in barley aleurone layers was enhancedby NaHS treatment regardless of the presence or absence of GAThese data imply that H

2S acted as an antioxidant in delaying PCD

and enhances 120572-amylase secretion regardless of the presence of GA in barley aleurone layers

1 Introduction

Programmed cell death (PCD) a response of plants tobiotic and abiotic stresses can also occur during the normalcourse of development [1] Cereal aleurone layers undergogibberellic acid- (GA-) stimulated PCD process followinggermination and therefore provide a convenient model forstudying PCD [2] PCD in barley aleurone layers occurs onlyafter cells become highly vacuolated and is accompanied withloss of plasma membrane integrity and increased cysteineprotease activity [3] However the hallmarks of apoptosisin animal cells including internucleosomal DNA laddersand formation of apoptotic bodies are not observed inaleurone cells [4] Reactive oxygen species (ROS) such assuperoxide anion (sdotO

2

minus) hydrogen peroxide (H2O2) and

hydroxyl radicals are key players in the PCD process inboth plants and animals [1 4 5] For instance intracellu-lar H

2O2overproduction or exogenous H

2O2application

results in a rapid death in GA-treated aleurone protoplasts[6] Antioxidant enzymes such as catalase (CAT) guaiacolperoxidase (POD) ascorbate peroxidase (APX) and super-oxide dismutase (SOD) are responsible for ROS scavengingthereby keeping homeostatic levels of ROS PCD in plantsis also accompanied by increased protease activity [2 7]For instance cysteine protease activation is instrumental inPCD of soybean cells while ectopic expression of cystatina cysteine protease inhibitor gene inhibits cysteine proteaseactivity and blocks PCD [7] What is more the increasein cysteine protease and aspartic protease activities is alsoobserved in GA-treated barley aleurone layers [2]

Hydrogen sulfide (H2S) similar to nitric oxide (NO)

and carbon monoxide (CO) is an important endogenousgaseous signaling molecule in animal cells [8] Accumulatingevidence shows that H

2S is involved in various processes

in plants such as response to pathogen attack seed germi-nation root organogenesis abiotic stress tolerance guard

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 714756 11 pageshttpdxdoiorg1011552015714756

2 Oxidative Medicine and Cellular Longevity

cell movement and postharvest senescence of fruits andvegetables [9ndash17] In particular during abiotic stresses andpostharvest storage H

2S acts as an antioxidant to counteract

excessive ROS to promote seed germination and alleviatepostharvest senescence [10 15 16] More recently H

2S is

found to delay PCD in GA-treated wheat aleurone layersby modulation of glutathione (GSH) and heme oxygenase 1expression [18] However whether H

2S has a role in regulat-

ing PCD in barley aleurone layers treated with GA or not andwhether ROS andROS-scavenging enzymes participate in therole of H

2S are still unknown In the present research we find

that the H2S donor NaHS effectively delays PCD in barley

aleurone layers regardless of the presence of GA through theenhancement in antioxidant enzyme genes expression andantioxidant enzyme activity and decrease in protease geneexpression

2 Materials and Methods

21 Materials and Treatments Grains of barley (Hordeumvulgare L) were kindly supplied by Jiangsu Academy ofAgricultural Sciences Jiangsu Province China Grains weresurface-sterilized as described by Chrispeels and Varner [19]In brief embryo end of the grain was removed and fifteenhalf-grains were imbibed in distilled water at 25∘C for 3 d onPetri dishes and further used for NaHS or gibberellic acid(GA) plus CaCl

2treatment H

2S donor NaHS and GA were

purchased from Sigma

22 Cell Viability Analysis in Barley Aleurone Layers Barleyhalf-grains pretreated with water for 3 d were incubated indifferent concentrations of NaHS (0 0005 0025 005 025or 05mM) or 025mM NaHS + 5 120583MGA (in 10mM CaCl

2)

at 25∘C for 5 d prior to isolation of aleurone layers from half-grains To determine the number of dead cells three aleuronelayers per treatment were stained with 04 trypan blue [20]for 10min and observed with Nikon Eclipse 80 i fluorescencemicroscope (Nikon Japan) The percentage of dead cellswas determined by the calculation of blue or purple cellscompared to the total number of cells in randomly selectedfields from three different aleurone layers per treatment

23 Detection of Reactive Oxygen Species Using FluorescentProbe A reactive oxygen species kit 2101584071015840-dichlorodifluores-cein diacetate (DCHF-DA) (Cayman Chemical America)which is a fluorogenic probe in living cells was used todetect ROS content [21] Three aleurone layers per treatmentwere rinsed with water three times and incubated with 5120583MDCHF-DA for 20min at 37∘C in the dark according tomanufacturerrsquos instructions The fluorescence of dichloroflu-orescein DCF (excitation at 488 nm emission at 525 nm) wasobserved using a Nikon Eclipse 80 i fluorescence microscope(Nikon Japan) Nonstained aleurone layers were used asnegative control The experiment was repeated three timesand similar results were obtained

24 Determination of the Contents of Superoxide AnionHydrogen Peroxide and Malondialdehyde sdotO

2

minus H2O2 and

MDA contents were measured according to the methodin [22] Embryoless half-grains were pretreated with sterilewater for 3 d and incubated in sterile H

2O 025mM NaHS

and 025mM NaHS + 5 120583M GA Three independent experi-ments with three replicates of 15 half-grains (045 plusmn 0001 g)were sampled for each treatment every 24 h until the fifthday

25 Assays of the Activity of Antioxidant Enzymes and Lipoxy-genase Activity of SOD (EC 11511) CAT (EC 11116)APX (EC 111111) and POD (EC 11117) was determinedaccording to Garcıa-Limones et al [23] and that of LOX(EC 1131112) followed the description by Surrey [24]Frozen grain samples (045 plusmn 0001 g) were homogenizedwith 1mL of 200mM ice-cold phosphate buffer (pH 78)containing 10mM ethylenediaminetetraacetic acid (EDTA)The homogenate was centrifuged at 12000 g at 4∘C for20min and the supernatant was used for activity measure-ment

For LOX three independent replicates of 15 half-grains(045 plusmn 0001 g) in three independent experiments per treat-ment were homogenized with 1mL of 200mM phosphatebuffer (pH 60) The homogenate was centrifuged at 15000 gat 4∘C for 10min and the supernatant was used for theenzyme assay The assay mixture in a total volume of 3mLcontained 200mMborate buffer (pH60) 025 linoleic acid025 tween-20 and 50120583L of enzyme extract The reactionwas carried out at 25∘C for 5min and the activity of LOXwasdetermined in the presence of linoleic acid bymonitoring thechanges in absorbance at 234 nm

26 Quantitative PCR Analysis Total RNAwas isolated fromfive aleurone layers using the plant RNeasy kit (ForgeneChina) according to the manufacturerrsquos instructions TotalRNA (500 ng) from different treatments was used for first-strand cDNA synthesis in a 20120583L reaction volume containing4 120583L 5 times PrimeScript RT Master Mix (TaKaRa) QuantitativePCR was performed using a StepOnePlus Real-Time PCRSystem (Applied Biosystems Foster City CA USA) withSYBR Premix Ex Taq (TaKaRa Bio Inc China) accord-ing to the manufacturerrsquos instructions cDNA was ampli-fied by PCR using the following primers HvActin (acces-sion number LOC548170) forward (51015840-TCTCACGGA-CTCCCTTT-31015840) and HvActin reverse (51015840-CACTGAGCA-CGATGTTTC-31015840)HvCAT1 (accession number HVU20777)forward (51015840-AAGACCGTTTCCTCCAGC-31015840) and reverse(51015840ATTCAAGGCTACCGCACA-31015840) HvCAT2 (accessionnumber HVU20778) forward (51015840-CGCCTTCAAGCCCAA-CCCA-31015840) and reverse (51015840-TTCTCCCTCTTTCCAACCAC-31015840) HvSOD1 (accession number JQ364454) forward (51015840-CGATAGCCAGATTCCTTTG-31015840) and reverse (51015840-TCC-ACCAGCATTTCCAGTA-31015840) HvAPX (accession numberAJ006358) forward (51015840-CTACTACTGCTGCTACTATGC-G-31015840) and reverse (51015840-CACTGACAGCGTTCAAGGTAT-31015840) HvLOX (accession number AJ966349) forward (51015840-CCGCTCTGACCCATTTCG-31015840) and reverse (51015840-TGC-TCCTTGACCTCCACCTT-31015840) HvICY (accession numberAJ536590) forward (51015840-TCGTCGTGCCGTTTACTC-31015840) and

Oxidative Medicine and Cellular Longevity 3

reverse (51015840-TTGGCCTTCTTGTTGTGC-31015840) HvEPA (acces-sion number HVU94591) forward (51015840-CCCGTGTCGGTG-GCAATA-31015840) and reverse (51015840-GCATCCTGATGTAAC-CCTTCTC-31015840) HvCP3-31 (accession number AB377533)forward (51015840-ACAACCTCCGCTACATCG-31015840) and reverse(51015840-CCCTTCTTCCTCCAGTCG-31015840) Relative gene expres-sion was presented as values relative to control HvActintranscript level after normalization to the control HvActintranscript levels

27 Assays of Secreted 120572-Amylase Activity Embryoless barleyhalf-grains were incubated in distilled water for 3 d and thentreated with various concentrations of NaHS in presence orabsence of 20 120583MGA and 10mM CaCl

2

Agar-starch medium (containing 4 agar and 01starch) was used to detect 120572-amylase activity secreted byaleurone layers in NaHS treatment without GA and CaCl

2

for 24 h Aleurone layers which were prepared as describedabovewere placed on agar-starchmedium for 16 h after whichthe agar-starch was stained with 06 I

2and 6 KI solution

to show digested starch zones The experiment was repeatedthree times and similar results were obtained

Twenty embryoless half-grains were imbibed in distilledwater at 25∘C for 3 d on Petri dishes and incubated inErlenmeyer flasks which contained different concentrationsofNaHS in 20120583MGAand 10mMCaCl

2 Incubationmedium

was sampled after 24 h and heated at 70∘C for 15min to elim-inate 120573-amylase activity Amylase secreted to the mediumwas visualized in 10 native PAGE gels by the starch-iodinemethod according to [25] To visualize 120572-amylase activity thegel was incubated at 25∘C for 30min in 50mM PBS (pH 70)containing 1 boiled soluble starch After being washed threetimeswith distilledwater the gel was stainedwith 06 I

2and

6KI solutionThe experiment was repeated three times andsimilar results were obtained

The DNS method for the determination of secreted 120572-amylase activity in mediumwas performed in 001M sodiumacetate buffer pH 54 The reaction mixture containing 1soluble starch was incubated at 25∘C for 5min withoutsubstrate Then the reaction was initiated by adding thesubstrate and was continued for an additional 10min at 37∘CThe reaction was terminated and hydrolysis was determinedwith 35-dinitrosalicylic acid reagent as modified by Noeltingand Bernfeld [26]

28 Statistical Analysis Statistical significance in all experi-ments was tested by one-way analysis of variance (ANOVA)and the results are expressed as the mean values plusmn standarddeviation (SD) of three independent experiments with threereplicates for each Fisherrsquos least significant differences (LSD)were calculated following a significant (119875 lt 001 or 119875 lt 005)119905-test

3 Results

31 Programmed Cell Death in Barley Aleurone Layers IsDelayed by the H

2S Donor NaHS To test the effect of H

2S

on the PCD process water-pretreated barley half-grains were

incubated in different concentrations of NaHS for 5 daysAleurone layers are isolated from half-grains and stainedwith trypan blue to visualize dead cells NaHS treatmentsranging from 0005 to 05mM significantly decrease celldeath compared with water controls (Figures 1(a) and 1(b))Only 9 of cells die in aleurone layers treated with 025mMNaHS while approximately 67 of cells of aleurone layersincubated in water undergo PCD As shown in Figure 1NaHS at 025mM is most effective in delaying PCD in barleyaleurone layers and this concentration is used in subsequentexperiments

A time course of cell death in aleurone layers treatedwith 025mM NaHS is shown in Figures 1(c) and 1(d) After7 days incubation in water about 90 aleurone cells aredead in contrast to only 45 cell death in NaHS-treatedlayers Together these findings show that barley aleurone cellsundergo PCDnaturally in the absence of GA and that theH

2S

donor NaHS effectively delays the PCD process

32 NaHS Treatment Reduces the Accumulation of ReactiveOxygen Species in Non-GA-Treated Barley Aleurone LayersBecause ROS are tightly associated with the promotion ofPCD in barley aleurone cells [6] we examine the contentsof sdotO2

minus H2O2 and MDA in non-GA-treated barley aleurone

layers in the presence and absence of NaHS As shown inFigure 2(a) sdotO

2

minus content in control barley aleurone layersaccumulates rapidly during the 5 days of incubation How-ever sdotO

2

minus content in NaHS-treated layers accumulates slowlyuntil day 3 and keeps stable on day 5

The assay of H2O2

shows that layers incubated inNaHS produce less H

2O2than those incubated in water

(Figure 2(b)) H2O2content increases rapidly in control

aleurone layers during the whole incubating time whereasa slower increase in H

2O2content was observed in NaHS

treatment on the first two days followed by a plateauMDA is determined as an index of lipid peroxidation As

shown in Figure 2(c)MDAcontent increases rapidly inwatercontrols until day 4 followed by a decrease In contrast NaHStreatment significantly lowers the level ofMDA (Figure 2(c))

We use the ROS-sensitive fluorescent probe DCHF-DAto visualize the production of ROS in barley aleurone layers(Figure 2(d)) Fluorescence from layers which are incubatedin 005 and 025mM NaHS is much less intense thanwater controls More weak fluorescence is detected in tissueincubated in 025mM NaHS

33 Effects ofNaHS onAntioxidant Enzymes and Lipoxygenasein Non-GA-Treated Barley Aleurone Layers We examine theactivity of the ROS metabolizing enzymes SOD POD CATAPX and LOX in barley aleurone layers that are incubatedin 025mM NaHS and water (Figure 3) The activity of SODincreases to maximum on day 3 and then declines in NaHS-treated layers In contrast SOD activity in water controlsfluctuates slightly up to day 3 followed by a significantdecrease (Figure 3(a))

Figure 3(b) shows changes in POD activity in NaHS-treated and water control layers NaHS significantly increasesPOD activity on day 1 and remains high until day 4

4 Oxidative Medicine and Cellular Longevity

0 0005 0025

005 025 05

(mM)

(a)

0

20

40

60

80

0 0005 0025 005 025 05

Dea

d ce

lls (

)

NaHS concentration (mM)

(b)

0d 3d 5d 7d

Con

trol

H2S

(c)

0

20

40

60

80

100

0 3 5 7

Dea

d ce

lls (

)

Treatment time (d)

ControlH2S

(d)

Figure 1 Effect of H2S donor NaHS on cell viability in barley aleurone layers ((a) (b)) Aleurone layers are incubated in different

concentrations of NaHS (0 0005 0025 005 025 and 05mM) for 5 d at 25∘C After staining with trypan blue the images are obtainedby light microscopy with blue or purple indicating dead cells ((c) (d)) Time course of PCD in barley aleurone layers treated with NaHS(H2S) or water (Control) Aleurone layers are incubated in 025mM NaHS or water for 0 1 3 5 and 7 d at 25∘C and are stained with trypan

blue Digital images of barley aleurone layers ((a) (c)) and percentages of dead cells ((b) (d)) are shown Bar 50120583m Data are means plusmn SD ofthree different aleurone layers per treatment

In comparison POD activity in water controls increasesgradually and peaks on day 3 followed by a sharp declineNaHS treatment maintains significantly higher levels of PODactivity compared with water control during the wholetreatment time APX activity increases during the first 3 daysof incubation and peaks on day 3 followed by a decreasein both NaHS-treated and water controls However APXactivity in NaHS treatment is always significantly higher thanthat of control (Figure 3(c))

Changes inCAT activity are shown in Figure 3(d) In bothNaHS andwater controls CAT activity increases gradually upto day 3 and then decreases sharply However CAT activity

from NaHS-treated layers is always significantly higher thanthose in control layers

Figure 3(e) shows the changes in LOX activity in barleyaleurone layers LOX activity in water control increasesdramatically and peaks on day 3 followed by a decreaseIn contrast NaHS treatment significantly decreases LOXactivity being about 50 of that of water control on day 3

34 Transcript Analysis of HvSOD1 HvCAT1 HvCAT2HvLOX Cysteine Protease (HvCP3-31 and HvEPA) and Cys-tatin (HvICY) in Non-GA-Treated Barley Aleurone LayersWe examine the expression of HvSOD1 HvCAT1 HvCAT2

Oxidative Medicine and Cellular Longevity 5

20

30

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50

60

70

80

0 1 2 3 4 5Treatment time (d)

ControlH2S

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

Con

tent

of

(120583gmiddot

gminus1

FW)

middotO2minus

(a)

100

140

180

220

260

0 1 2 3 4 5Treatment time (d)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Con

tent

ofH

2O2

(120583m

olmiddotgminus1

FW)

ControlH2S

(b)

0

2

4

6

8

10

12

0 1 2 3 4 5Treatment time (d)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

ControlH2S

Con

tent

of M

DA

(nm

olmiddotgminus1

FW)

(c)

0 005 025

(mM)

DCH

F-D

AN

o D

CHF-

DA

(d)

Figure 2 Effects of NaHS on the contents of sdotO2

minus (a) H2O2(b) and MDA (c) in barley aleurone layers Aleurone layers treated with 0

005 and 025mMNaHS for 1 d are incubated DCHF-DA and are observed by fluorescence microscopy (d) Bar 100 120583m Data are expressedas means plusmn SD of three independent experiments with three replicates of 15 grains per treatment The symbols lowast and lowastlowast mean significantdifference at 119875 lt 005 and 119875 lt 001 between NaHS (H

2S) and water (control) treatment respectively

HvLOX the cysteine proteases HvCP3-31 and HvEPA andcystatin (HvICY) in NaHS-treated barley aleurone layersand water controls (Figure 4) Compared with water con-trols NaHS induces higher expression of HvSOD1 HvAPXHvCAT1 and HvCAT2 on days 1 and 5 HvLOX expressionincreases in water control layers on days 1 and 5 comparedwith day 0 while NaHS treatment sustains lower transcriptof HvLOX than water control especially on day 5 PCDin barley aleurone layers is accompanied with increasedcysteine protease activity [3] Accordingly we determine theexpression of the cysteine proteinases HvEPA and HvCP3-31and the cystatinHvICY inNaHS treatment andwater controlThe expression of HvEPA and HvCP3-31 increases in water

controls on days 1 and 5 whereas their expression is muchlower in NaHS-treated tissue The expression of HvICY wasenhanced in NaHS-treated layers whereas less transcript ofHvICY is observed in water controls

35 NaHS Delays PCD in GA-Treated Barley Aleurone LayersPCD in barley aleurone layers is tightly regulated by GAand abscisic acid (ABA) We therefore assess whether H

2S

can ameliorate PCD in GA-treated barley aleurone layers Asshown in Figure 5(a) the accumulation of dead cells increasesrapidly from 24 to 96 h in GA-treated barley aleurone layerswhereas 025mM NaHS treatment significantly delays therate of PCD After incubation for 96 h about 90 cells in

6 Oxidative Medicine and Cellular Longevity

10

20

30

40

50

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f SO

D (U

middotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

50

90

130

170

210

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f PO

D (U

middotgminus1

FW)

lowastlowast

lowastlowast lowastlowast lowastlowast

lowastlowast

(b)

80

130

180

230

280

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f APX

(Umiddotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)

35

55

75

95

115

135

0 2 4Treatment time (d)

ControlH2S

Activ

ity o

f CAT

(Umiddotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(d)

70

110

150

190

230

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f LO

X (U

middotgminus1

FW)

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(e)

Figure 3 Effects of NaHS on the activity of SOD (a) POD (b) APX (c) CAT (d) and LOX (e) in barley aleurone layers Data are expressedas means plusmn SD of three independent experiments with three replicates of 15 grains per treatment The symbols lowast and lowastlowast mean significantdifference at 119875 lt 005 and 119875 lt 001 between control and T respectively

GA-treated layers are dead while half of cells are still alivein NaHS-treated layers (Figure 5(b)) In water control muchless cells undergo PCD compared with the counterpart of GAand GA plus NaHS

Determination of sdotO2

minus content shows that NaHS treat-ment maintains lower levels of sdotO

2

minus in GA-treated barleyaleurone layers (Figure 5(c)) After a rapid increase duringthe first 2 days of incubation the content of sdotO

2

minus in GA-treated layers decreases till day 4 In contrast sdotO

2

minus content inNaHS plus GA treatment increases more slowly until day 3A comparable but lower sdotO

2

minus content was observed in watercontrol compared with NaHS plus GA treatment

Figure 5(d) shows the effect of H2S on POD activity in

GA-treated barley aleurone layers In both NaHS treatmentand GA control POD activity in GA-treated barley aleurone

layers increases gradually up to day 3 and day 2 respectivelyand decreases thereafter However the activity of POD inNaHS-treated aleurone layers is always significantly higherthan those in water controls and GA treatment alone

Figure 5(e) shows that NaHS treatment maintains lowerlevels of LOX activity compared with water control duringthe first 2 days of GA treatment LOX activity in GAtreatment increases dramatically on day 1 followed by agradual decrease while in NaHS-treated tissue the activityincreases more slowly till day 3 followed by a decline Afterday 3 LOX activity in NaHS plus GA is higher than that inGA treatment

36 H2S Donor Promotes 120572-Amylase Secretion in Barley

Aleurone Layers Regardless of GA Secretion of 120572-amylase is

Oxidative Medicine and Cellular Longevity 7

0

2

4

6

8

10

12

14

C0 C1 T1 C5 T5

ab

d

b

c

020406080

100120140160180

C0 C1 T1 C5 T5

a a

c

b

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05

10152025303540

C0 C1 T1 C5 T5

a a

bc

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0102030405060708090

C0 C1 T1 C5 T5

T5

a

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00005

0010015

0020025

0030035

0040045

C0 C1 T1 C5 T5

ab

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aa

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Relat

ive e

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ress

ion

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tive e

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nRe

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e exp

ress

ion

Rela

tive e

xpre

ssio

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e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

HvSOD1 HvAPX

HvCAT1 HvCAT2

HvLOX

HvEPA HvCP3-31

HvICY

Figure 4 Effects of H2S on the expression of HvSOD1 HvCAT1 HvCAT2 HvLOX cystatin (HvICY) and the cysteine proteases HvCP3-31

andHvEPA in barley aleurone layers Aleurone layers are incubated in NaHS (T) or water (C) and total RNA is obtained at 0 1 and 5 dMeansand SD values are calculated from three independent experiments Within each identified gene bars with different letters are significantlydifferent in comparison with the corresponding control at 119875 lt 001 according to Fisherrsquos least significant differences (LSD)

a characteristic response of aleurone cells toGAWe thereforetest whether the ameliorating effect of H

2S on PCD affects

the release of 120572-amylase As shown in Figure 6(a) NaHS pro-motes 120572-amylase release in water-treated aleurone layers Inthe presence ofGANaHS treatment also enhances120572-amylaserelease frombarley aleurone layers (Figure 6(b)) with 05mMNaHS exhibiting optimal effect Figure 6(c) shows the time

changes in 120572-amylase secretion in water control GA-treatedaleurone layers and GA plus 025mM NaHS treatment Theaccumulation of 120572-amylase in incubation medium in GA-treated layers increases and peaks on day 3 followed by aplateau but addition of 025mM NaHS brings about a morerapid increase till day 4 followed by a decrease on day 5(Figure 6(c)) The activity of 120572-amylase released following

8 Oxidative Medicine and Cellular Longevity

Con

trol

GA

GA+

H2S

24h 48h 72h 96h

(a)

0

20

40

60

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120

24 72 96

Dea

d ce

lls (

)

Treatment time (d)

GA

ControlGA + H2S

(b)

0

50

100

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250

0 1 2 3 4Treatment time (d)

GA

ControlGA + H2S

Con

tent

of

(120583gmiddot

gminus1

FW)

middotO2minus

(c)

40

60

80

100

120

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160

180

0 1 2 3 4Treatment time (d)

Activ

ity o

f PO

D (U

middotgminus1

FW)

GA

ControlGA + H2S

(d)

GA

0

50

100

150

200

250

300

0 1 2 3 4Treatment time (d)

ControlGA + H2S

Activ

ity o

f LO

X (U

middotgminus1

FW)

(e)

Figure 5 NaHS delays PCD in GA-treated barley aleurone layers Aleurone layers are incubated in water GA or GA + NaHS (GA + H2S)

and after being stained with trypan blue images are obtained by lightmicroscopy (a) and the percentage of dead cells is shown in (b) Contentof sdotO2

minus (c) activity of POD (d) and LOX (e) are measured on 0 1 2 3 and 4 d Bar 100 120583m Data in (c) and (d) are expressed as means plusmn SDof three independent experiments with three replicates of 15 grains per treatment

Oxidative Medicine and Cellular Longevity 9

0 0005 005 025 05

(mM)

(a)

0 0005 005 025 05

(mM)

(b)

0

50

100

150

200

0 1 2 3 4 5Treatment time (d)

GA

ControlGA + H2S

Activ

ity o

f120572-a

myl

ase (

Umiddotgminus1

FW)

(c)

Figure 6 NaHS increases the activity of 120572-amylase in incubation medium of barley aleurone layers in the absence (a) and presence ((b)(c)) of GA Barley aleurone layers isolated from treated embryoless half grains are incubated in 0 0005 005 025 or 05mMNaHS for 24 h((a) (b)) After incubation aleurone layers are placed on agar-starch medium (containing 4 agar and 01 starch) for 16 h Agar plates arestained with I

2-KI solution to detect the activity of 120572-amylase (a) (b) shows native PAGE analysis of 120572-amylase activity in incubationmedium

surrounding the aleurone layers (c) indicates secreted 120572-amylase activity in incubation medium surrounding barley aleurone layers treatedwith water (control) GA or GA + NaHS (GA + H

2S) at different times of incubation Data in (c) are expressed as means plusmn SD of three

independent experiments with three replicates of 20 embryoless half grains per treatment

GA + NaHS treatment is significantly higher than that oflayers incubated only in GA during the whole treatmenttime As expected much lower 120572-amylase activity was onlyobserved inwater control after 3 days of incubation Togetherthis result indicates that H

2S delays PCD in barley aleurone

layers and meanwhile promotes 120572-amylase release regardlessof the presence of GA

4 Discussion

H2S participates in multiple processes in plants [27] In

this paper we show that H2S delays PCD in barley aleu-

rone layers regardless of the presence or absence of GAIn the absence of GA PCD in barley aleurone layers isevident on day 3 of incubation while H

2S delays PCD at

an optimal concentration of 025mM (Figure 1) Consistentwith the reports that GA accelerates PCD process in barley

aleurone layers GA treatment triggers cell death in about90 aleurone cells at 72 h (Figure 5(a)) GA-induced celldeath is slowed by the addition of NaHS and this H

2S

donor also prolongs the phase of 120572-amylase productionin GA-treated layers The promoting effect of NaHS on120572-amylase synthesis and its prolongation of cell survivalindicate that 25mMNaHS does not affect aleurone cell func-tion

ROS such as sdotO2

minus andH2O2 are inducers of PCD in plant

and animal cells [1] It is reported that the peroxidation ofmembrane lipids and damage to the plasma membrane canoccur when the rate of ROS production overcomes the cellsrsquoability for scavenging ROS [27] Overproduction of ROS andoxidative damage are universal events in PCD in plant cells[28] In this paper we show that the content of sdotO

2

minus increasesin parallel with cell death in GA-treated layers (Figure 5(c))In non-GA-treated layers the burst of sdotO

2

minus and H2O2and

10 Oxidative Medicine and Cellular Longevity

the accumulation of MDA are also accompanied by PCD(Figure 2)These results suggest that ROS play a key role bothin GA-treated [29] and in non-GA-treated aleurone layers(Figures 2(a) 2(b) and 5(c))

Aleurone cells contain a suite of ROS-metabolizingenzymes GA-induced PCD in layers is accompanied by adecline in activity of ROS metabolizing enzymes which leadsto increased susceptibility of aleurone cells to ROS [29] Anovel aspect of our work is that NaHS treatment effectivelyreduces the accumulation of ROS in barley aleurone layersregardless the presence of GA (Figures 2 and 5) therebydelaying PCD process in these cells We propose that theH2S donor reduces ROS accumulation in layers by increasing

the activity of ROS-scavenging enzymes The data in presentstudy show that H

2S treatment maintains significantly higher

POD activity in GA-treated layers (Figure 5(d)) and higherSOD POD CAT and APX activity in non-GA-treatedlayers (Figure 3) The increased activity of ROS-scavengingenzymes in NaHS-treated likely promotes the cellrsquos abilityto metabolize ROS In addition LOX activities which areresponsible for lipid peroxidation are downregulated inNaHS-treated aleurone layers at early stage of treatment(Figures 3(e) and 5(e)) Meanwhile quantitative-PCR anal-ysis shows that expression of HvSOD1 HvAPX HvCAT1and HvCAT2 genes in non-GA-treated layers is maintainedat higher levels in NaHS treatment compared with watercontrols (Figure 4) Consistent with lower LOX activity inNaHS-treated aleurone layers the accumulation of LOXtranscripts is also reduced (Figure 4) In summary H

2S slows

down ROS-induced PCD in barley aleurone layers probablyby enhancing the activity and expression of ROS-scavengingenzymes and reducing the peroxidation of membrane lipids

Consistently Xie et al [18] found that H2S delayed GA-

triggered PCD in wheat aleurone layers by increasing GSHcontent and heme oxygenase-1 gene expression Here weprovide evidence thatH

2S can alleviate both natural PCD and

GA-triggered PCD through the modulation of antioxidantenzyme activities and their expression Compared with theslow natural PCDprocess the present study also confirms thepivotal role of GA in triggering PCD (Figure 5)

The role of ROS in GA and ABA signaling in barleyaleurone cells is recently clarified [30] in which they foundthat the production of H

2O2 a type of ROS was induced by

GA in aleurone cells but suppressed by ABA Furthermoreexogenous H

2O2appeared to promote the induction of 120572-

amylases by GA by promoting the expression of GAMyb and120572-amylase genes whereas antioxidants suppressed the induc-tion of 120572-amylase Unexpectedly we found that H

2S reduces

ROS accumulation and delays PCD process in barley aleu-rone layers in the presence or absence of GA and meanwhilepromotes the secretion of 120572-amylase suggesting that theantioxidant H

2S works through an unknown way to regulate

120572-amylase secretion and antioxidants do not always suppressthe induction of 120572-amylase Besides the activation of 120572-amylase by H

2S in the absence of GA implies that 120572-amylase

can be secreted independent of GA signaling pathwayTherefore the present findings advance our knowledge onthe relations between PCD process and 120572-amylase secretion

and the independence of 120572-amylase secretion and GA path-way

The activation of cysteine proteases was instrumental inthe PCD of soybean cells while cystatin an endogenouscysteine protease inhibitor gene inhibited cysteine proteaseactivity and blocked PCD in these cells [7] In this paperwe show that H

2S downregulates the transcriptions of two

barley cysteine proteinases HvEPA and HvCP3-31 in non-GA-treated barley aleurone layers thereby delaying cellcomponent degradation and PCD process

5 Conclusion

In summary we report the role of H2S in delaying PCD in

barley aleurone layers regardless of the presence or absence ofGA without repressing 120572-amylase induction suggesting thatthe function ofH

2Smay be universal in regulating plant PCD

PCD in plant cells is regulated by many internal and externalfactors such as the hormones (GA andABA) Ca2+ ROS andNO [6] It will be interesting to knowwhether H

2S is involved

in other signals and how 120572-amylase is induced by H2S in the

presence or absence of GA in cereal aleurone cells

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ying-Xin Zhang and Kang-Di Hu contributed equally to thiswork

Acknowledgments

Theauthors acknowledge Russell Jones University of Califor-nia at Berkeley for editing the paperThisworkwas supportedby the Natural Science Foundation of China (nos 3127180331301820 31300133 and 31470013) the Scientific ResearchFoundation for Returned Overseas Chinese Scholars (SRFfor ROCS MOE) the Natural Science Foundations of AnhuiProvince (11040606M85) and the Anhui Provincial Educa-tion Department (nos 2012AJZR0028 ZD200910)

References

[1] T Jabs ldquoReactive oxygen intermediates as mediators of pro-grammed cell death in plants and animalsrdquo Biochemical Phar-macology vol 57 no 3 pp 231ndash245 1999

[2] A Fath P Bethke V Beligni and R Jones ldquoActive oxygenand cell death in cereal aleurone cellsrdquo Journal of ExperimentalBotany vol 53 no 372 pp 1273ndash1282 2002

[3] A Fath P Bethke J Lonsdale R Meza-Romero and R JonesldquoProgrammed cell death in cereal aleuronerdquo Plant MolecularBiology vol 44 no 3 pp 255ndash266 2000

[4] P C Bethke J E Lonsdale A Fath and R L Jones ldquoHormon-ally regulated programmed cell death in barley aleurone cellsrdquoThe Plant Cell vol 11 no 16 pp 1033ndash1045 1999

Oxidative Medicine and Cellular Longevity 11

[5] P EHockberger T A Skimina V E Centonze et al ldquoActivationof flavin-containing oxidases underlies light-induced produc-tion of H

2O2in mammalian cellsrdquo Proceedings of the National

Academy of Sciences of the United States of America vol 96 no11 pp 6255ndash6260 1999

[6] P C Bethke and R L Jones ldquoCell death of barley aleuroneprotoplasts is mediated by reactive oxygen speciesrdquo The PlantJournal vol 25 no 1 pp 19ndash29 2001

[7] M Solomon B Belenghi M Delledonne E Menachem andA Levine ldquoThe involvement of cysteine proteases and proteaseinhibitor genes in the regulation of programmed cell death inplantsrdquoThe Plant Cell vol 11 no 3 pp 431ndash443 1999

[8] R Wang ldquoTworsquos company threersquos a crowd can H2S be the third

endogenous gaseous transmitterrdquo The FASEB Journal vol 16no 13 pp 1792ndash1798 2002

[9] E Bloem A Riemenschneider J Volker et al ldquoSulphur supplyand infection with Pyrenopeziza brassicae influence L-cysteinedesulphydrase activity in Brassica napus Lrdquo Journal of Experi-mental Botany vol 55 no 406 pp 2305ndash2312 2004

[10] H Zhang L-Y Hu K-D Hu Y-D He S-H Wang and J-P Luo ldquoHydrogen sulfide promotes wheat seed germinationand alleviates oxidative damage against copper stressrdquo Journalof Integrative Plant Biology vol 50 no 12 pp 1518ndash1529 2008

[11] H Zhang J Tang X-P Liu et al ldquoHydrogen sulfide promotesroot organogenesis in Ipomoea batatas Salix matsudana andGlycine maxrdquo Journal of Integrative Plant Biology vol 51 no 12pp 1086ndash1094 2009

[12] Z P Jin J J Shen Z J Qiao G D Yang R Wang andY X Pei ldquoHydrogen sulfide improves drought resistance inArabidopsis thalianardquo Biochemical and Biophysical ResearchCommunications vol 414 no 3 pp 481ndash486 2011

[13] Z P Jin SWXue YN Luo et al ldquoHydrogen sulfide interactingwith abscisic acid in stomatal regulation responses to droughtstress inArabidopsisrdquoPlant Physiology and Biochemistry vol 62pp 41ndash46 2013

[14] C Garcıa-Mata and L Lamattina ldquoHydrogen sulphide anovel gasotransmitter involved in guard cell signallingrdquo NewPhytologist vol 188 no 4 pp 977ndash984 2010

[15] L-Y Hu S-L Hu J Wu et al ldquoHydrogen sulfide prolongspostharvest shelf life of strawberry and plays an antioxidativerole in fruitsrdquo Journal of Agricultural and Food Chemistry vol60 no 35 pp 8684ndash8693 2012

[16] S-P Gao K-D Hu L-Y Hu et al ldquoHydrogen sulfide delayspostharvest senescence and plays an antioxidative role in fresh-cut kiwifruitrdquo HortScience vol 49 no 10 pp 1385ndash1392 2013

[17] S-P Li K-D Hu L-Y Hu et al ldquoHydrogen sulfide alleviatespostharvest senescence of broccoli by modulating antioxidantdefense and senescence-related gene expressionrdquo Journal ofAgricultural and Food Chemistry vol 62 no 5 pp 1119ndash11292014

[18] Y Xie C Zhang D Lai et al ldquoHydrogen sulfide delays GA-triggered programmed cell death inwheat aleurone layers by themodulation of glutathione homeostasis and heme oxygenase-1expressionrdquo Journal of Plant Physiology vol 171 no 2 pp 53ndash622014

[19] M J Chrispeels and J E Varner ldquoGibberellic acid enhancedsynthesis and release of 120572-amylase and ribonuclease by isolatedbarley and aleurone layersrdquo Plant Physiology vol 42 no 3 pp398ndash406 1967

[20] J R Tennant ldquoEvaluation of the trypan blue technique fordetermination of cell viabilityrdquo Transplantation vol 2 pp 685ndash694 1964

[21] M Tsuchiya M Suematsu and H Suzuki ldquoIn vivo visualiza-tion of oxygen radical-dependent photoemissionrdquo Methods inEnzymology vol 233 pp 128ndash140 1994

[22] R F del Maestro J Bjork and K-E Arfors ldquoIncrease inmicrovascular permeability induced by enzymatically gener-ated free radicals I In vivo studyrdquoMicrovascular Research vol22 no 3 pp 239ndash254 1981

[23] C Garcıa-Limones A Hervas J A Navas-Cortes R MJimenez-Dıaz and M Tena ldquoInduction of an antioxidantenzyme system and other oxidative stress markers associatedwith compatible and incompatible interactions between chick-pea (Cicer arietinum L) and Fusarium oxysporum f sp cicerisrdquoPhysiological and Molecular Plant Pathology vol 61 no 6 pp325ndash337 2002

[24] K Surrey ldquoSpectrophotometric method for determination oflipoxidase activityrdquo Plant Physiology vol 39 no 1 pp 65ndash701964

[25] G G Collins C F Jenner and L G Paleg ldquoThe metabolismof soluble nucleotides in wheat aleurone layers treated withgibberellic acidrdquo Plant P hysiology vol 49 no 3 pp 404ndash4101972

[26] G Noelting and P Bernfeld ldquoSur les enzymes amylolytiqucsIII La 120573-amylase dosage drsquoactivite et controle de lrsquoabsence drsquo120572-amylaserdquo Helvetica Chimica Acta vol 31 no 1 pp 286ndash2901948

[27] M Lisjak T Teklic I D Wilson M Whiteman and J THancock ldquoHydrogen sulfide environmental factor or signallingmoleculerdquo Plant Cell and Environment vol 36 no 9 pp 1607ndash1616 2013

[28] M C de Pinto A Paradiso P Leonetti and L de Gara ldquoHydro-gen peroxide nitric oxide and cytosolic ascorbate peroxidaseat the crossroad between defence and cell deathrdquo The PlantJournal vol 48 no 5 pp 784ndash795 2006

[29] A Fath P C Bethke and R L Jones ldquoEnzymes that scavengereactive oxygen species are down-regulated prior to gibberellicacid-induced programmed cell death in barley aleuronerdquo PlantPhysiology vol 126 no 1 pp 156ndash166 2001

[30] Y Ishibashi T Tawaratsumida K Kondo et al ldquoReactive oxy-gen species are involved in gibberellinabscisic acid signaling inbarley aleurone cellsrdquo Plant Physiology vol 158 no 4 pp 1705ndash1714 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Oxidative Medicine and Cellular Longevity

cell movement and postharvest senescence of fruits andvegetables [9ndash17] In particular during abiotic stresses andpostharvest storage H

2S acts as an antioxidant to counteract

excessive ROS to promote seed germination and alleviatepostharvest senescence [10 15 16] More recently H

2S is

found to delay PCD in GA-treated wheat aleurone layersby modulation of glutathione (GSH) and heme oxygenase 1expression [18] However whether H

2S has a role in regulat-

ing PCD in barley aleurone layers treated with GA or not andwhether ROS andROS-scavenging enzymes participate in therole of H

2S are still unknown In the present research we find

that the H2S donor NaHS effectively delays PCD in barley

aleurone layers regardless of the presence of GA through theenhancement in antioxidant enzyme genes expression andantioxidant enzyme activity and decrease in protease geneexpression

2 Materials and Methods

21 Materials and Treatments Grains of barley (Hordeumvulgare L) were kindly supplied by Jiangsu Academy ofAgricultural Sciences Jiangsu Province China Grains weresurface-sterilized as described by Chrispeels and Varner [19]In brief embryo end of the grain was removed and fifteenhalf-grains were imbibed in distilled water at 25∘C for 3 d onPetri dishes and further used for NaHS or gibberellic acid(GA) plus CaCl

2treatment H

2S donor NaHS and GA were

purchased from Sigma

22 Cell Viability Analysis in Barley Aleurone Layers Barleyhalf-grains pretreated with water for 3 d were incubated indifferent concentrations of NaHS (0 0005 0025 005 025or 05mM) or 025mM NaHS + 5 120583MGA (in 10mM CaCl

2)

at 25∘C for 5 d prior to isolation of aleurone layers from half-grains To determine the number of dead cells three aleuronelayers per treatment were stained with 04 trypan blue [20]for 10min and observed with Nikon Eclipse 80 i fluorescencemicroscope (Nikon Japan) The percentage of dead cellswas determined by the calculation of blue or purple cellscompared to the total number of cells in randomly selectedfields from three different aleurone layers per treatment

23 Detection of Reactive Oxygen Species Using FluorescentProbe A reactive oxygen species kit 2101584071015840-dichlorodifluores-cein diacetate (DCHF-DA) (Cayman Chemical America)which is a fluorogenic probe in living cells was used todetect ROS content [21] Three aleurone layers per treatmentwere rinsed with water three times and incubated with 5120583MDCHF-DA for 20min at 37∘C in the dark according tomanufacturerrsquos instructions The fluorescence of dichloroflu-orescein DCF (excitation at 488 nm emission at 525 nm) wasobserved using a Nikon Eclipse 80 i fluorescence microscope(Nikon Japan) Nonstained aleurone layers were used asnegative control The experiment was repeated three timesand similar results were obtained

24 Determination of the Contents of Superoxide AnionHydrogen Peroxide and Malondialdehyde sdotO

2

minus H2O2 and

MDA contents were measured according to the methodin [22] Embryoless half-grains were pretreated with sterilewater for 3 d and incubated in sterile H

2O 025mM NaHS

and 025mM NaHS + 5 120583M GA Three independent experi-ments with three replicates of 15 half-grains (045 plusmn 0001 g)were sampled for each treatment every 24 h until the fifthday

25 Assays of the Activity of Antioxidant Enzymes and Lipoxy-genase Activity of SOD (EC 11511) CAT (EC 11116)APX (EC 111111) and POD (EC 11117) was determinedaccording to Garcıa-Limones et al [23] and that of LOX(EC 1131112) followed the description by Surrey [24]Frozen grain samples (045 plusmn 0001 g) were homogenizedwith 1mL of 200mM ice-cold phosphate buffer (pH 78)containing 10mM ethylenediaminetetraacetic acid (EDTA)The homogenate was centrifuged at 12000 g at 4∘C for20min and the supernatant was used for activity measure-ment

For LOX three independent replicates of 15 half-grains(045 plusmn 0001 g) in three independent experiments per treat-ment were homogenized with 1mL of 200mM phosphatebuffer (pH 60) The homogenate was centrifuged at 15000 gat 4∘C for 10min and the supernatant was used for theenzyme assay The assay mixture in a total volume of 3mLcontained 200mMborate buffer (pH60) 025 linoleic acid025 tween-20 and 50120583L of enzyme extract The reactionwas carried out at 25∘C for 5min and the activity of LOXwasdetermined in the presence of linoleic acid bymonitoring thechanges in absorbance at 234 nm

26 Quantitative PCR Analysis Total RNAwas isolated fromfive aleurone layers using the plant RNeasy kit (ForgeneChina) according to the manufacturerrsquos instructions TotalRNA (500 ng) from different treatments was used for first-strand cDNA synthesis in a 20120583L reaction volume containing4 120583L 5 times PrimeScript RT Master Mix (TaKaRa) QuantitativePCR was performed using a StepOnePlus Real-Time PCRSystem (Applied Biosystems Foster City CA USA) withSYBR Premix Ex Taq (TaKaRa Bio Inc China) accord-ing to the manufacturerrsquos instructions cDNA was ampli-fied by PCR using the following primers HvActin (acces-sion number LOC548170) forward (51015840-TCTCACGGA-CTCCCTTT-31015840) and HvActin reverse (51015840-CACTGAGCA-CGATGTTTC-31015840)HvCAT1 (accession number HVU20777)forward (51015840-AAGACCGTTTCCTCCAGC-31015840) and reverse(51015840ATTCAAGGCTACCGCACA-31015840) HvCAT2 (accessionnumber HVU20778) forward (51015840-CGCCTTCAAGCCCAA-CCCA-31015840) and reverse (51015840-TTCTCCCTCTTTCCAACCAC-31015840) HvSOD1 (accession number JQ364454) forward (51015840-CGATAGCCAGATTCCTTTG-31015840) and reverse (51015840-TCC-ACCAGCATTTCCAGTA-31015840) HvAPX (accession numberAJ006358) forward (51015840-CTACTACTGCTGCTACTATGC-G-31015840) and reverse (51015840-CACTGACAGCGTTCAAGGTAT-31015840) HvLOX (accession number AJ966349) forward (51015840-CCGCTCTGACCCATTTCG-31015840) and reverse (51015840-TGC-TCCTTGACCTCCACCTT-31015840) HvICY (accession numberAJ536590) forward (51015840-TCGTCGTGCCGTTTACTC-31015840) and

Oxidative Medicine and Cellular Longevity 3

reverse (51015840-TTGGCCTTCTTGTTGTGC-31015840) HvEPA (acces-sion number HVU94591) forward (51015840-CCCGTGTCGGTG-GCAATA-31015840) and reverse (51015840-GCATCCTGATGTAAC-CCTTCTC-31015840) HvCP3-31 (accession number AB377533)forward (51015840-ACAACCTCCGCTACATCG-31015840) and reverse(51015840-CCCTTCTTCCTCCAGTCG-31015840) Relative gene expres-sion was presented as values relative to control HvActintranscript level after normalization to the control HvActintranscript levels

27 Assays of Secreted 120572-Amylase Activity Embryoless barleyhalf-grains were incubated in distilled water for 3 d and thentreated with various concentrations of NaHS in presence orabsence of 20 120583MGA and 10mM CaCl

2

Agar-starch medium (containing 4 agar and 01starch) was used to detect 120572-amylase activity secreted byaleurone layers in NaHS treatment without GA and CaCl

2

for 24 h Aleurone layers which were prepared as describedabovewere placed on agar-starchmedium for 16 h after whichthe agar-starch was stained with 06 I

2and 6 KI solution

to show digested starch zones The experiment was repeatedthree times and similar results were obtained

Twenty embryoless half-grains were imbibed in distilledwater at 25∘C for 3 d on Petri dishes and incubated inErlenmeyer flasks which contained different concentrationsofNaHS in 20120583MGAand 10mMCaCl

2 Incubationmedium

was sampled after 24 h and heated at 70∘C for 15min to elim-inate 120573-amylase activity Amylase secreted to the mediumwas visualized in 10 native PAGE gels by the starch-iodinemethod according to [25] To visualize 120572-amylase activity thegel was incubated at 25∘C for 30min in 50mM PBS (pH 70)containing 1 boiled soluble starch After being washed threetimeswith distilledwater the gel was stainedwith 06 I

2and

6KI solutionThe experiment was repeated three times andsimilar results were obtained

The DNS method for the determination of secreted 120572-amylase activity in mediumwas performed in 001M sodiumacetate buffer pH 54 The reaction mixture containing 1soluble starch was incubated at 25∘C for 5min withoutsubstrate Then the reaction was initiated by adding thesubstrate and was continued for an additional 10min at 37∘CThe reaction was terminated and hydrolysis was determinedwith 35-dinitrosalicylic acid reagent as modified by Noeltingand Bernfeld [26]

28 Statistical Analysis Statistical significance in all experi-ments was tested by one-way analysis of variance (ANOVA)and the results are expressed as the mean values plusmn standarddeviation (SD) of three independent experiments with threereplicates for each Fisherrsquos least significant differences (LSD)were calculated following a significant (119875 lt 001 or 119875 lt 005)119905-test

3 Results

31 Programmed Cell Death in Barley Aleurone Layers IsDelayed by the H

2S Donor NaHS To test the effect of H

2S

on the PCD process water-pretreated barley half-grains were

incubated in different concentrations of NaHS for 5 daysAleurone layers are isolated from half-grains and stainedwith trypan blue to visualize dead cells NaHS treatmentsranging from 0005 to 05mM significantly decrease celldeath compared with water controls (Figures 1(a) and 1(b))Only 9 of cells die in aleurone layers treated with 025mMNaHS while approximately 67 of cells of aleurone layersincubated in water undergo PCD As shown in Figure 1NaHS at 025mM is most effective in delaying PCD in barleyaleurone layers and this concentration is used in subsequentexperiments

A time course of cell death in aleurone layers treatedwith 025mM NaHS is shown in Figures 1(c) and 1(d) After7 days incubation in water about 90 aleurone cells aredead in contrast to only 45 cell death in NaHS-treatedlayers Together these findings show that barley aleurone cellsundergo PCDnaturally in the absence of GA and that theH

2S

donor NaHS effectively delays the PCD process

32 NaHS Treatment Reduces the Accumulation of ReactiveOxygen Species in Non-GA-Treated Barley Aleurone LayersBecause ROS are tightly associated with the promotion ofPCD in barley aleurone cells [6] we examine the contentsof sdotO2

minus H2O2 and MDA in non-GA-treated barley aleurone

layers in the presence and absence of NaHS As shown inFigure 2(a) sdotO

2

minus content in control barley aleurone layersaccumulates rapidly during the 5 days of incubation How-ever sdotO

2

minus content in NaHS-treated layers accumulates slowlyuntil day 3 and keeps stable on day 5

The assay of H2O2

shows that layers incubated inNaHS produce less H

2O2than those incubated in water

(Figure 2(b)) H2O2content increases rapidly in control

aleurone layers during the whole incubating time whereasa slower increase in H

2O2content was observed in NaHS

treatment on the first two days followed by a plateauMDA is determined as an index of lipid peroxidation As

shown in Figure 2(c)MDAcontent increases rapidly inwatercontrols until day 4 followed by a decrease In contrast NaHStreatment significantly lowers the level ofMDA (Figure 2(c))

We use the ROS-sensitive fluorescent probe DCHF-DAto visualize the production of ROS in barley aleurone layers(Figure 2(d)) Fluorescence from layers which are incubatedin 005 and 025mM NaHS is much less intense thanwater controls More weak fluorescence is detected in tissueincubated in 025mM NaHS

33 Effects ofNaHS onAntioxidant Enzymes and Lipoxygenasein Non-GA-Treated Barley Aleurone Layers We examine theactivity of the ROS metabolizing enzymes SOD POD CATAPX and LOX in barley aleurone layers that are incubatedin 025mM NaHS and water (Figure 3) The activity of SODincreases to maximum on day 3 and then declines in NaHS-treated layers In contrast SOD activity in water controlsfluctuates slightly up to day 3 followed by a significantdecrease (Figure 3(a))

Figure 3(b) shows changes in POD activity in NaHS-treated and water control layers NaHS significantly increasesPOD activity on day 1 and remains high until day 4

4 Oxidative Medicine and Cellular Longevity

0 0005 0025

005 025 05

(mM)

(a)

0

20

40

60

80

0 0005 0025 005 025 05

Dea

d ce

lls (

)

NaHS concentration (mM)

(b)

0d 3d 5d 7d

Con

trol

H2S

(c)

0

20

40

60

80

100

0 3 5 7

Dea

d ce

lls (

)

Treatment time (d)

ControlH2S

(d)

Figure 1 Effect of H2S donor NaHS on cell viability in barley aleurone layers ((a) (b)) Aleurone layers are incubated in different

concentrations of NaHS (0 0005 0025 005 025 and 05mM) for 5 d at 25∘C After staining with trypan blue the images are obtainedby light microscopy with blue or purple indicating dead cells ((c) (d)) Time course of PCD in barley aleurone layers treated with NaHS(H2S) or water (Control) Aleurone layers are incubated in 025mM NaHS or water for 0 1 3 5 and 7 d at 25∘C and are stained with trypan

blue Digital images of barley aleurone layers ((a) (c)) and percentages of dead cells ((b) (d)) are shown Bar 50120583m Data are means plusmn SD ofthree different aleurone layers per treatment

In comparison POD activity in water controls increasesgradually and peaks on day 3 followed by a sharp declineNaHS treatment maintains significantly higher levels of PODactivity compared with water control during the wholetreatment time APX activity increases during the first 3 daysof incubation and peaks on day 3 followed by a decreasein both NaHS-treated and water controls However APXactivity in NaHS treatment is always significantly higher thanthat of control (Figure 3(c))

Changes inCAT activity are shown in Figure 3(d) In bothNaHS andwater controls CAT activity increases gradually upto day 3 and then decreases sharply However CAT activity

from NaHS-treated layers is always significantly higher thanthose in control layers

Figure 3(e) shows the changes in LOX activity in barleyaleurone layers LOX activity in water control increasesdramatically and peaks on day 3 followed by a decreaseIn contrast NaHS treatment significantly decreases LOXactivity being about 50 of that of water control on day 3

34 Transcript Analysis of HvSOD1 HvCAT1 HvCAT2HvLOX Cysteine Protease (HvCP3-31 and HvEPA) and Cys-tatin (HvICY) in Non-GA-Treated Barley Aleurone LayersWe examine the expression of HvSOD1 HvCAT1 HvCAT2

Oxidative Medicine and Cellular Longevity 5

20

30

40

50

60

70

80

0 1 2 3 4 5Treatment time (d)

ControlH2S

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

Con

tent

of

(120583gmiddot

gminus1

FW)

middotO2minus

(a)

100

140

180

220

260

0 1 2 3 4 5Treatment time (d)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Con

tent

ofH

2O2

(120583m

olmiddotgminus1

FW)

ControlH2S

(b)

0

2

4

6

8

10

12

0 1 2 3 4 5Treatment time (d)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

ControlH2S

Con

tent

of M

DA

(nm

olmiddotgminus1

FW)

(c)

0 005 025

(mM)

DCH

F-D

AN

o D

CHF-

DA

(d)

Figure 2 Effects of NaHS on the contents of sdotO2

minus (a) H2O2(b) and MDA (c) in barley aleurone layers Aleurone layers treated with 0

005 and 025mMNaHS for 1 d are incubated DCHF-DA and are observed by fluorescence microscopy (d) Bar 100 120583m Data are expressedas means plusmn SD of three independent experiments with three replicates of 15 grains per treatment The symbols lowast and lowastlowast mean significantdifference at 119875 lt 005 and 119875 lt 001 between NaHS (H

2S) and water (control) treatment respectively

HvLOX the cysteine proteases HvCP3-31 and HvEPA andcystatin (HvICY) in NaHS-treated barley aleurone layersand water controls (Figure 4) Compared with water con-trols NaHS induces higher expression of HvSOD1 HvAPXHvCAT1 and HvCAT2 on days 1 and 5 HvLOX expressionincreases in water control layers on days 1 and 5 comparedwith day 0 while NaHS treatment sustains lower transcriptof HvLOX than water control especially on day 5 PCDin barley aleurone layers is accompanied with increasedcysteine protease activity [3] Accordingly we determine theexpression of the cysteine proteinases HvEPA and HvCP3-31and the cystatinHvICY inNaHS treatment andwater controlThe expression of HvEPA and HvCP3-31 increases in water

controls on days 1 and 5 whereas their expression is muchlower in NaHS-treated tissue The expression of HvICY wasenhanced in NaHS-treated layers whereas less transcript ofHvICY is observed in water controls

35 NaHS Delays PCD in GA-Treated Barley Aleurone LayersPCD in barley aleurone layers is tightly regulated by GAand abscisic acid (ABA) We therefore assess whether H

2S

can ameliorate PCD in GA-treated barley aleurone layers Asshown in Figure 5(a) the accumulation of dead cells increasesrapidly from 24 to 96 h in GA-treated barley aleurone layerswhereas 025mM NaHS treatment significantly delays therate of PCD After incubation for 96 h about 90 cells in

6 Oxidative Medicine and Cellular Longevity

10

20

30

40

50

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f SO

D (U

middotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

50

90

130

170

210

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f PO

D (U

middotgminus1

FW)

lowastlowast

lowastlowast lowastlowast lowastlowast

lowastlowast

(b)

80

130

180

230

280

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f APX

(Umiddotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)

35

55

75

95

115

135

0 2 4Treatment time (d)

ControlH2S

Activ

ity o

f CAT

(Umiddotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(d)

70

110

150

190

230

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f LO

X (U

middotgminus1

FW)

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(e)

Figure 3 Effects of NaHS on the activity of SOD (a) POD (b) APX (c) CAT (d) and LOX (e) in barley aleurone layers Data are expressedas means plusmn SD of three independent experiments with three replicates of 15 grains per treatment The symbols lowast and lowastlowast mean significantdifference at 119875 lt 005 and 119875 lt 001 between control and T respectively

GA-treated layers are dead while half of cells are still alivein NaHS-treated layers (Figure 5(b)) In water control muchless cells undergo PCD compared with the counterpart of GAand GA plus NaHS

Determination of sdotO2

minus content shows that NaHS treat-ment maintains lower levels of sdotO

2

minus in GA-treated barleyaleurone layers (Figure 5(c)) After a rapid increase duringthe first 2 days of incubation the content of sdotO

2

minus in GA-treated layers decreases till day 4 In contrast sdotO

2

minus content inNaHS plus GA treatment increases more slowly until day 3A comparable but lower sdotO

2

minus content was observed in watercontrol compared with NaHS plus GA treatment

Figure 5(d) shows the effect of H2S on POD activity in

GA-treated barley aleurone layers In both NaHS treatmentand GA control POD activity in GA-treated barley aleurone

layers increases gradually up to day 3 and day 2 respectivelyand decreases thereafter However the activity of POD inNaHS-treated aleurone layers is always significantly higherthan those in water controls and GA treatment alone

Figure 5(e) shows that NaHS treatment maintains lowerlevels of LOX activity compared with water control duringthe first 2 days of GA treatment LOX activity in GAtreatment increases dramatically on day 1 followed by agradual decrease while in NaHS-treated tissue the activityincreases more slowly till day 3 followed by a decline Afterday 3 LOX activity in NaHS plus GA is higher than that inGA treatment

36 H2S Donor Promotes 120572-Amylase Secretion in Barley

Aleurone Layers Regardless of GA Secretion of 120572-amylase is

Oxidative Medicine and Cellular Longevity 7

0

2

4

6

8

10

12

14

C0 C1 T1 C5 T5

ab

d

b

c

020406080

100120140160180

C0 C1 T1 C5 T5

a a

c

b

d

05

10152025303540

C0 C1 T1 C5 T5

a a

bc

d

0102030405060708090

C0 C1 T1 C5 T5

T5

a

b

c

d

e

00005

0010015

0020025

0030035

0040045

C0 C1 T1 C5 T5

ab

b

cd

0

50

100

150

200

250

C0 C1 T1 C5

aa

b

cc

0

200

400

600

C0 C1 T1 C5 T5a ab

c

d

0

4

8

12

C0 C1 T1 C5 T5

aa

b

cd

Relat

ive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

HvSOD1 HvAPX

HvCAT1 HvCAT2

HvLOX

HvEPA HvCP3-31

HvICY

Figure 4 Effects of H2S on the expression of HvSOD1 HvCAT1 HvCAT2 HvLOX cystatin (HvICY) and the cysteine proteases HvCP3-31

andHvEPA in barley aleurone layers Aleurone layers are incubated in NaHS (T) or water (C) and total RNA is obtained at 0 1 and 5 dMeansand SD values are calculated from three independent experiments Within each identified gene bars with different letters are significantlydifferent in comparison with the corresponding control at 119875 lt 001 according to Fisherrsquos least significant differences (LSD)

a characteristic response of aleurone cells toGAWe thereforetest whether the ameliorating effect of H

2S on PCD affects

the release of 120572-amylase As shown in Figure 6(a) NaHS pro-motes 120572-amylase release in water-treated aleurone layers Inthe presence ofGANaHS treatment also enhances120572-amylaserelease frombarley aleurone layers (Figure 6(b)) with 05mMNaHS exhibiting optimal effect Figure 6(c) shows the time

changes in 120572-amylase secretion in water control GA-treatedaleurone layers and GA plus 025mM NaHS treatment Theaccumulation of 120572-amylase in incubation medium in GA-treated layers increases and peaks on day 3 followed by aplateau but addition of 025mM NaHS brings about a morerapid increase till day 4 followed by a decrease on day 5(Figure 6(c)) The activity of 120572-amylase released following

8 Oxidative Medicine and Cellular Longevity

Con

trol

GA

GA+

H2S

24h 48h 72h 96h

(a)

0

20

40

60

80

100

120

24 72 96

Dea

d ce

lls (

)

Treatment time (d)

GA

ControlGA + H2S

(b)

0

50

100

150

200

250

0 1 2 3 4Treatment time (d)

GA

ControlGA + H2S

Con

tent

of

(120583gmiddot

gminus1

FW)

middotO2minus

(c)

40

60

80

100

120

140

160

180

0 1 2 3 4Treatment time (d)

Activ

ity o

f PO

D (U

middotgminus1

FW)

GA

ControlGA + H2S

(d)

GA

0

50

100

150

200

250

300

0 1 2 3 4Treatment time (d)

ControlGA + H2S

Activ

ity o

f LO

X (U

middotgminus1

FW)

(e)

Figure 5 NaHS delays PCD in GA-treated barley aleurone layers Aleurone layers are incubated in water GA or GA + NaHS (GA + H2S)

and after being stained with trypan blue images are obtained by lightmicroscopy (a) and the percentage of dead cells is shown in (b) Contentof sdotO2

minus (c) activity of POD (d) and LOX (e) are measured on 0 1 2 3 and 4 d Bar 100 120583m Data in (c) and (d) are expressed as means plusmn SDof three independent experiments with three replicates of 15 grains per treatment

Oxidative Medicine and Cellular Longevity 9

0 0005 005 025 05

(mM)

(a)

0 0005 005 025 05

(mM)

(b)

0

50

100

150

200

0 1 2 3 4 5Treatment time (d)

GA

ControlGA + H2S

Activ

ity o

f120572-a

myl

ase (

Umiddotgminus1

FW)

(c)

Figure 6 NaHS increases the activity of 120572-amylase in incubation medium of barley aleurone layers in the absence (a) and presence ((b)(c)) of GA Barley aleurone layers isolated from treated embryoless half grains are incubated in 0 0005 005 025 or 05mMNaHS for 24 h((a) (b)) After incubation aleurone layers are placed on agar-starch medium (containing 4 agar and 01 starch) for 16 h Agar plates arestained with I

2-KI solution to detect the activity of 120572-amylase (a) (b) shows native PAGE analysis of 120572-amylase activity in incubationmedium

surrounding the aleurone layers (c) indicates secreted 120572-amylase activity in incubation medium surrounding barley aleurone layers treatedwith water (control) GA or GA + NaHS (GA + H

2S) at different times of incubation Data in (c) are expressed as means plusmn SD of three

independent experiments with three replicates of 20 embryoless half grains per treatment

GA + NaHS treatment is significantly higher than that oflayers incubated only in GA during the whole treatmenttime As expected much lower 120572-amylase activity was onlyobserved inwater control after 3 days of incubation Togetherthis result indicates that H

2S delays PCD in barley aleurone

layers and meanwhile promotes 120572-amylase release regardlessof the presence of GA

4 Discussion

H2S participates in multiple processes in plants [27] In

this paper we show that H2S delays PCD in barley aleu-

rone layers regardless of the presence or absence of GAIn the absence of GA PCD in barley aleurone layers isevident on day 3 of incubation while H

2S delays PCD at

an optimal concentration of 025mM (Figure 1) Consistentwith the reports that GA accelerates PCD process in barley

aleurone layers GA treatment triggers cell death in about90 aleurone cells at 72 h (Figure 5(a)) GA-induced celldeath is slowed by the addition of NaHS and this H

2S

donor also prolongs the phase of 120572-amylase productionin GA-treated layers The promoting effect of NaHS on120572-amylase synthesis and its prolongation of cell survivalindicate that 25mMNaHS does not affect aleurone cell func-tion

ROS such as sdotO2

minus andH2O2 are inducers of PCD in plant

and animal cells [1] It is reported that the peroxidation ofmembrane lipids and damage to the plasma membrane canoccur when the rate of ROS production overcomes the cellsrsquoability for scavenging ROS [27] Overproduction of ROS andoxidative damage are universal events in PCD in plant cells[28] In this paper we show that the content of sdotO

2

minus increasesin parallel with cell death in GA-treated layers (Figure 5(c))In non-GA-treated layers the burst of sdotO

2

minus and H2O2and

10 Oxidative Medicine and Cellular Longevity

the accumulation of MDA are also accompanied by PCD(Figure 2)These results suggest that ROS play a key role bothin GA-treated [29] and in non-GA-treated aleurone layers(Figures 2(a) 2(b) and 5(c))

Aleurone cells contain a suite of ROS-metabolizingenzymes GA-induced PCD in layers is accompanied by adecline in activity of ROS metabolizing enzymes which leadsto increased susceptibility of aleurone cells to ROS [29] Anovel aspect of our work is that NaHS treatment effectivelyreduces the accumulation of ROS in barley aleurone layersregardless the presence of GA (Figures 2 and 5) therebydelaying PCD process in these cells We propose that theH2S donor reduces ROS accumulation in layers by increasing

the activity of ROS-scavenging enzymes The data in presentstudy show that H

2S treatment maintains significantly higher

POD activity in GA-treated layers (Figure 5(d)) and higherSOD POD CAT and APX activity in non-GA-treatedlayers (Figure 3) The increased activity of ROS-scavengingenzymes in NaHS-treated likely promotes the cellrsquos abilityto metabolize ROS In addition LOX activities which areresponsible for lipid peroxidation are downregulated inNaHS-treated aleurone layers at early stage of treatment(Figures 3(e) and 5(e)) Meanwhile quantitative-PCR anal-ysis shows that expression of HvSOD1 HvAPX HvCAT1and HvCAT2 genes in non-GA-treated layers is maintainedat higher levels in NaHS treatment compared with watercontrols (Figure 4) Consistent with lower LOX activity inNaHS-treated aleurone layers the accumulation of LOXtranscripts is also reduced (Figure 4) In summary H

2S slows

down ROS-induced PCD in barley aleurone layers probablyby enhancing the activity and expression of ROS-scavengingenzymes and reducing the peroxidation of membrane lipids

Consistently Xie et al [18] found that H2S delayed GA-

triggered PCD in wheat aleurone layers by increasing GSHcontent and heme oxygenase-1 gene expression Here weprovide evidence thatH

2S can alleviate both natural PCD and

GA-triggered PCD through the modulation of antioxidantenzyme activities and their expression Compared with theslow natural PCDprocess the present study also confirms thepivotal role of GA in triggering PCD (Figure 5)

The role of ROS in GA and ABA signaling in barleyaleurone cells is recently clarified [30] in which they foundthat the production of H

2O2 a type of ROS was induced by

GA in aleurone cells but suppressed by ABA Furthermoreexogenous H

2O2appeared to promote the induction of 120572-

amylases by GA by promoting the expression of GAMyb and120572-amylase genes whereas antioxidants suppressed the induc-tion of 120572-amylase Unexpectedly we found that H

2S reduces

ROS accumulation and delays PCD process in barley aleu-rone layers in the presence or absence of GA and meanwhilepromotes the secretion of 120572-amylase suggesting that theantioxidant H

2S works through an unknown way to regulate

120572-amylase secretion and antioxidants do not always suppressthe induction of 120572-amylase Besides the activation of 120572-amylase by H

2S in the absence of GA implies that 120572-amylase

can be secreted independent of GA signaling pathwayTherefore the present findings advance our knowledge onthe relations between PCD process and 120572-amylase secretion

and the independence of 120572-amylase secretion and GA path-way

The activation of cysteine proteases was instrumental inthe PCD of soybean cells while cystatin an endogenouscysteine protease inhibitor gene inhibited cysteine proteaseactivity and blocked PCD in these cells [7] In this paperwe show that H

2S downregulates the transcriptions of two

barley cysteine proteinases HvEPA and HvCP3-31 in non-GA-treated barley aleurone layers thereby delaying cellcomponent degradation and PCD process

5 Conclusion

In summary we report the role of H2S in delaying PCD in

barley aleurone layers regardless of the presence or absence ofGA without repressing 120572-amylase induction suggesting thatthe function ofH

2Smay be universal in regulating plant PCD

PCD in plant cells is regulated by many internal and externalfactors such as the hormones (GA andABA) Ca2+ ROS andNO [6] It will be interesting to knowwhether H

2S is involved

in other signals and how 120572-amylase is induced by H2S in the

presence or absence of GA in cereal aleurone cells

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ying-Xin Zhang and Kang-Di Hu contributed equally to thiswork

Acknowledgments

Theauthors acknowledge Russell Jones University of Califor-nia at Berkeley for editing the paperThisworkwas supportedby the Natural Science Foundation of China (nos 3127180331301820 31300133 and 31470013) the Scientific ResearchFoundation for Returned Overseas Chinese Scholars (SRFfor ROCS MOE) the Natural Science Foundations of AnhuiProvince (11040606M85) and the Anhui Provincial Educa-tion Department (nos 2012AJZR0028 ZD200910)

References

[1] T Jabs ldquoReactive oxygen intermediates as mediators of pro-grammed cell death in plants and animalsrdquo Biochemical Phar-macology vol 57 no 3 pp 231ndash245 1999

[2] A Fath P Bethke V Beligni and R Jones ldquoActive oxygenand cell death in cereal aleurone cellsrdquo Journal of ExperimentalBotany vol 53 no 372 pp 1273ndash1282 2002

[3] A Fath P Bethke J Lonsdale R Meza-Romero and R JonesldquoProgrammed cell death in cereal aleuronerdquo Plant MolecularBiology vol 44 no 3 pp 255ndash266 2000

[4] P C Bethke J E Lonsdale A Fath and R L Jones ldquoHormon-ally regulated programmed cell death in barley aleurone cellsrdquoThe Plant Cell vol 11 no 16 pp 1033ndash1045 1999

Oxidative Medicine and Cellular Longevity 11

[5] P EHockberger T A Skimina V E Centonze et al ldquoActivationof flavin-containing oxidases underlies light-induced produc-tion of H

2O2in mammalian cellsrdquo Proceedings of the National

Academy of Sciences of the United States of America vol 96 no11 pp 6255ndash6260 1999

[6] P C Bethke and R L Jones ldquoCell death of barley aleuroneprotoplasts is mediated by reactive oxygen speciesrdquo The PlantJournal vol 25 no 1 pp 19ndash29 2001

[7] M Solomon B Belenghi M Delledonne E Menachem andA Levine ldquoThe involvement of cysteine proteases and proteaseinhibitor genes in the regulation of programmed cell death inplantsrdquoThe Plant Cell vol 11 no 3 pp 431ndash443 1999

[8] R Wang ldquoTworsquos company threersquos a crowd can H2S be the third

endogenous gaseous transmitterrdquo The FASEB Journal vol 16no 13 pp 1792ndash1798 2002

[9] E Bloem A Riemenschneider J Volker et al ldquoSulphur supplyand infection with Pyrenopeziza brassicae influence L-cysteinedesulphydrase activity in Brassica napus Lrdquo Journal of Experi-mental Botany vol 55 no 406 pp 2305ndash2312 2004

[10] H Zhang L-Y Hu K-D Hu Y-D He S-H Wang and J-P Luo ldquoHydrogen sulfide promotes wheat seed germinationand alleviates oxidative damage against copper stressrdquo Journalof Integrative Plant Biology vol 50 no 12 pp 1518ndash1529 2008

[11] H Zhang J Tang X-P Liu et al ldquoHydrogen sulfide promotesroot organogenesis in Ipomoea batatas Salix matsudana andGlycine maxrdquo Journal of Integrative Plant Biology vol 51 no 12pp 1086ndash1094 2009

[12] Z P Jin J J Shen Z J Qiao G D Yang R Wang andY X Pei ldquoHydrogen sulfide improves drought resistance inArabidopsis thalianardquo Biochemical and Biophysical ResearchCommunications vol 414 no 3 pp 481ndash486 2011

[13] Z P Jin SWXue YN Luo et al ldquoHydrogen sulfide interactingwith abscisic acid in stomatal regulation responses to droughtstress inArabidopsisrdquoPlant Physiology and Biochemistry vol 62pp 41ndash46 2013

[14] C Garcıa-Mata and L Lamattina ldquoHydrogen sulphide anovel gasotransmitter involved in guard cell signallingrdquo NewPhytologist vol 188 no 4 pp 977ndash984 2010

[15] L-Y Hu S-L Hu J Wu et al ldquoHydrogen sulfide prolongspostharvest shelf life of strawberry and plays an antioxidativerole in fruitsrdquo Journal of Agricultural and Food Chemistry vol60 no 35 pp 8684ndash8693 2012

[16] S-P Gao K-D Hu L-Y Hu et al ldquoHydrogen sulfide delayspostharvest senescence and plays an antioxidative role in fresh-cut kiwifruitrdquo HortScience vol 49 no 10 pp 1385ndash1392 2013

[17] S-P Li K-D Hu L-Y Hu et al ldquoHydrogen sulfide alleviatespostharvest senescence of broccoli by modulating antioxidantdefense and senescence-related gene expressionrdquo Journal ofAgricultural and Food Chemistry vol 62 no 5 pp 1119ndash11292014

[18] Y Xie C Zhang D Lai et al ldquoHydrogen sulfide delays GA-triggered programmed cell death inwheat aleurone layers by themodulation of glutathione homeostasis and heme oxygenase-1expressionrdquo Journal of Plant Physiology vol 171 no 2 pp 53ndash622014

[19] M J Chrispeels and J E Varner ldquoGibberellic acid enhancedsynthesis and release of 120572-amylase and ribonuclease by isolatedbarley and aleurone layersrdquo Plant Physiology vol 42 no 3 pp398ndash406 1967

[20] J R Tennant ldquoEvaluation of the trypan blue technique fordetermination of cell viabilityrdquo Transplantation vol 2 pp 685ndash694 1964

[21] M Tsuchiya M Suematsu and H Suzuki ldquoIn vivo visualiza-tion of oxygen radical-dependent photoemissionrdquo Methods inEnzymology vol 233 pp 128ndash140 1994

[22] R F del Maestro J Bjork and K-E Arfors ldquoIncrease inmicrovascular permeability induced by enzymatically gener-ated free radicals I In vivo studyrdquoMicrovascular Research vol22 no 3 pp 239ndash254 1981

[23] C Garcıa-Limones A Hervas J A Navas-Cortes R MJimenez-Dıaz and M Tena ldquoInduction of an antioxidantenzyme system and other oxidative stress markers associatedwith compatible and incompatible interactions between chick-pea (Cicer arietinum L) and Fusarium oxysporum f sp cicerisrdquoPhysiological and Molecular Plant Pathology vol 61 no 6 pp325ndash337 2002

[24] K Surrey ldquoSpectrophotometric method for determination oflipoxidase activityrdquo Plant Physiology vol 39 no 1 pp 65ndash701964

[25] G G Collins C F Jenner and L G Paleg ldquoThe metabolismof soluble nucleotides in wheat aleurone layers treated withgibberellic acidrdquo Plant P hysiology vol 49 no 3 pp 404ndash4101972

[26] G Noelting and P Bernfeld ldquoSur les enzymes amylolytiqucsIII La 120573-amylase dosage drsquoactivite et controle de lrsquoabsence drsquo120572-amylaserdquo Helvetica Chimica Acta vol 31 no 1 pp 286ndash2901948

[27] M Lisjak T Teklic I D Wilson M Whiteman and J THancock ldquoHydrogen sulfide environmental factor or signallingmoleculerdquo Plant Cell and Environment vol 36 no 9 pp 1607ndash1616 2013

[28] M C de Pinto A Paradiso P Leonetti and L de Gara ldquoHydro-gen peroxide nitric oxide and cytosolic ascorbate peroxidaseat the crossroad between defence and cell deathrdquo The PlantJournal vol 48 no 5 pp 784ndash795 2006

[29] A Fath P C Bethke and R L Jones ldquoEnzymes that scavengereactive oxygen species are down-regulated prior to gibberellicacid-induced programmed cell death in barley aleuronerdquo PlantPhysiology vol 126 no 1 pp 156ndash166 2001

[30] Y Ishibashi T Tawaratsumida K Kondo et al ldquoReactive oxy-gen species are involved in gibberellinabscisic acid signaling inbarley aleurone cellsrdquo Plant Physiology vol 158 no 4 pp 1705ndash1714 2012

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Oxidative Medicine and Cellular Longevity

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 3

reverse (51015840-TTGGCCTTCTTGTTGTGC-31015840) HvEPA (acces-sion number HVU94591) forward (51015840-CCCGTGTCGGTG-GCAATA-31015840) and reverse (51015840-GCATCCTGATGTAAC-CCTTCTC-31015840) HvCP3-31 (accession number AB377533)forward (51015840-ACAACCTCCGCTACATCG-31015840) and reverse(51015840-CCCTTCTTCCTCCAGTCG-31015840) Relative gene expres-sion was presented as values relative to control HvActintranscript level after normalization to the control HvActintranscript levels

27 Assays of Secreted 120572-Amylase Activity Embryoless barleyhalf-grains were incubated in distilled water for 3 d and thentreated with various concentrations of NaHS in presence orabsence of 20 120583MGA and 10mM CaCl

2

Agar-starch medium (containing 4 agar and 01starch) was used to detect 120572-amylase activity secreted byaleurone layers in NaHS treatment without GA and CaCl

2

for 24 h Aleurone layers which were prepared as describedabovewere placed on agar-starchmedium for 16 h after whichthe agar-starch was stained with 06 I

2and 6 KI solution

to show digested starch zones The experiment was repeatedthree times and similar results were obtained

Twenty embryoless half-grains were imbibed in distilledwater at 25∘C for 3 d on Petri dishes and incubated inErlenmeyer flasks which contained different concentrationsofNaHS in 20120583MGAand 10mMCaCl

2 Incubationmedium

was sampled after 24 h and heated at 70∘C for 15min to elim-inate 120573-amylase activity Amylase secreted to the mediumwas visualized in 10 native PAGE gels by the starch-iodinemethod according to [25] To visualize 120572-amylase activity thegel was incubated at 25∘C for 30min in 50mM PBS (pH 70)containing 1 boiled soluble starch After being washed threetimeswith distilledwater the gel was stainedwith 06 I

2and

6KI solutionThe experiment was repeated three times andsimilar results were obtained

The DNS method for the determination of secreted 120572-amylase activity in mediumwas performed in 001M sodiumacetate buffer pH 54 The reaction mixture containing 1soluble starch was incubated at 25∘C for 5min withoutsubstrate Then the reaction was initiated by adding thesubstrate and was continued for an additional 10min at 37∘CThe reaction was terminated and hydrolysis was determinedwith 35-dinitrosalicylic acid reagent as modified by Noeltingand Bernfeld [26]

28 Statistical Analysis Statistical significance in all experi-ments was tested by one-way analysis of variance (ANOVA)and the results are expressed as the mean values plusmn standarddeviation (SD) of three independent experiments with threereplicates for each Fisherrsquos least significant differences (LSD)were calculated following a significant (119875 lt 001 or 119875 lt 005)119905-test

3 Results

31 Programmed Cell Death in Barley Aleurone Layers IsDelayed by the H

2S Donor NaHS To test the effect of H

2S

on the PCD process water-pretreated barley half-grains were

incubated in different concentrations of NaHS for 5 daysAleurone layers are isolated from half-grains and stainedwith trypan blue to visualize dead cells NaHS treatmentsranging from 0005 to 05mM significantly decrease celldeath compared with water controls (Figures 1(a) and 1(b))Only 9 of cells die in aleurone layers treated with 025mMNaHS while approximately 67 of cells of aleurone layersincubated in water undergo PCD As shown in Figure 1NaHS at 025mM is most effective in delaying PCD in barleyaleurone layers and this concentration is used in subsequentexperiments

A time course of cell death in aleurone layers treatedwith 025mM NaHS is shown in Figures 1(c) and 1(d) After7 days incubation in water about 90 aleurone cells aredead in contrast to only 45 cell death in NaHS-treatedlayers Together these findings show that barley aleurone cellsundergo PCDnaturally in the absence of GA and that theH

2S

donor NaHS effectively delays the PCD process

32 NaHS Treatment Reduces the Accumulation of ReactiveOxygen Species in Non-GA-Treated Barley Aleurone LayersBecause ROS are tightly associated with the promotion ofPCD in barley aleurone cells [6] we examine the contentsof sdotO2

minus H2O2 and MDA in non-GA-treated barley aleurone

layers in the presence and absence of NaHS As shown inFigure 2(a) sdotO

2

minus content in control barley aleurone layersaccumulates rapidly during the 5 days of incubation How-ever sdotO

2

minus content in NaHS-treated layers accumulates slowlyuntil day 3 and keeps stable on day 5

The assay of H2O2

shows that layers incubated inNaHS produce less H

2O2than those incubated in water

(Figure 2(b)) H2O2content increases rapidly in control

aleurone layers during the whole incubating time whereasa slower increase in H

2O2content was observed in NaHS

treatment on the first two days followed by a plateauMDA is determined as an index of lipid peroxidation As

shown in Figure 2(c)MDAcontent increases rapidly inwatercontrols until day 4 followed by a decrease In contrast NaHStreatment significantly lowers the level ofMDA (Figure 2(c))

We use the ROS-sensitive fluorescent probe DCHF-DAto visualize the production of ROS in barley aleurone layers(Figure 2(d)) Fluorescence from layers which are incubatedin 005 and 025mM NaHS is much less intense thanwater controls More weak fluorescence is detected in tissueincubated in 025mM NaHS

33 Effects ofNaHS onAntioxidant Enzymes and Lipoxygenasein Non-GA-Treated Barley Aleurone Layers We examine theactivity of the ROS metabolizing enzymes SOD POD CATAPX and LOX in barley aleurone layers that are incubatedin 025mM NaHS and water (Figure 3) The activity of SODincreases to maximum on day 3 and then declines in NaHS-treated layers In contrast SOD activity in water controlsfluctuates slightly up to day 3 followed by a significantdecrease (Figure 3(a))

Figure 3(b) shows changes in POD activity in NaHS-treated and water control layers NaHS significantly increasesPOD activity on day 1 and remains high until day 4

4 Oxidative Medicine and Cellular Longevity

0 0005 0025

005 025 05

(mM)

(a)

0

20

40

60

80

0 0005 0025 005 025 05

Dea

d ce

lls (

)

NaHS concentration (mM)

(b)

0d 3d 5d 7d

Con

trol

H2S

(c)

0

20

40

60

80

100

0 3 5 7

Dea

d ce

lls (

)

Treatment time (d)

ControlH2S

(d)

Figure 1 Effect of H2S donor NaHS on cell viability in barley aleurone layers ((a) (b)) Aleurone layers are incubated in different

concentrations of NaHS (0 0005 0025 005 025 and 05mM) for 5 d at 25∘C After staining with trypan blue the images are obtainedby light microscopy with blue or purple indicating dead cells ((c) (d)) Time course of PCD in barley aleurone layers treated with NaHS(H2S) or water (Control) Aleurone layers are incubated in 025mM NaHS or water for 0 1 3 5 and 7 d at 25∘C and are stained with trypan

blue Digital images of barley aleurone layers ((a) (c)) and percentages of dead cells ((b) (d)) are shown Bar 50120583m Data are means plusmn SD ofthree different aleurone layers per treatment

In comparison POD activity in water controls increasesgradually and peaks on day 3 followed by a sharp declineNaHS treatment maintains significantly higher levels of PODactivity compared with water control during the wholetreatment time APX activity increases during the first 3 daysof incubation and peaks on day 3 followed by a decreasein both NaHS-treated and water controls However APXactivity in NaHS treatment is always significantly higher thanthat of control (Figure 3(c))

Changes inCAT activity are shown in Figure 3(d) In bothNaHS andwater controls CAT activity increases gradually upto day 3 and then decreases sharply However CAT activity

from NaHS-treated layers is always significantly higher thanthose in control layers

Figure 3(e) shows the changes in LOX activity in barleyaleurone layers LOX activity in water control increasesdramatically and peaks on day 3 followed by a decreaseIn contrast NaHS treatment significantly decreases LOXactivity being about 50 of that of water control on day 3

34 Transcript Analysis of HvSOD1 HvCAT1 HvCAT2HvLOX Cysteine Protease (HvCP3-31 and HvEPA) and Cys-tatin (HvICY) in Non-GA-Treated Barley Aleurone LayersWe examine the expression of HvSOD1 HvCAT1 HvCAT2

Oxidative Medicine and Cellular Longevity 5

20

30

40

50

60

70

80

0 1 2 3 4 5Treatment time (d)

ControlH2S

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

Con

tent

of

(120583gmiddot

gminus1

FW)

middotO2minus

(a)

100

140

180

220

260

0 1 2 3 4 5Treatment time (d)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Con

tent

ofH

2O2

(120583m

olmiddotgminus1

FW)

ControlH2S

(b)

0

2

4

6

8

10

12

0 1 2 3 4 5Treatment time (d)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

ControlH2S

Con

tent

of M

DA

(nm

olmiddotgminus1

FW)

(c)

0 005 025

(mM)

DCH

F-D

AN

o D

CHF-

DA

(d)

Figure 2 Effects of NaHS on the contents of sdotO2

minus (a) H2O2(b) and MDA (c) in barley aleurone layers Aleurone layers treated with 0

005 and 025mMNaHS for 1 d are incubated DCHF-DA and are observed by fluorescence microscopy (d) Bar 100 120583m Data are expressedas means plusmn SD of three independent experiments with three replicates of 15 grains per treatment The symbols lowast and lowastlowast mean significantdifference at 119875 lt 005 and 119875 lt 001 between NaHS (H

2S) and water (control) treatment respectively

HvLOX the cysteine proteases HvCP3-31 and HvEPA andcystatin (HvICY) in NaHS-treated barley aleurone layersand water controls (Figure 4) Compared with water con-trols NaHS induces higher expression of HvSOD1 HvAPXHvCAT1 and HvCAT2 on days 1 and 5 HvLOX expressionincreases in water control layers on days 1 and 5 comparedwith day 0 while NaHS treatment sustains lower transcriptof HvLOX than water control especially on day 5 PCDin barley aleurone layers is accompanied with increasedcysteine protease activity [3] Accordingly we determine theexpression of the cysteine proteinases HvEPA and HvCP3-31and the cystatinHvICY inNaHS treatment andwater controlThe expression of HvEPA and HvCP3-31 increases in water

controls on days 1 and 5 whereas their expression is muchlower in NaHS-treated tissue The expression of HvICY wasenhanced in NaHS-treated layers whereas less transcript ofHvICY is observed in water controls

35 NaHS Delays PCD in GA-Treated Barley Aleurone LayersPCD in barley aleurone layers is tightly regulated by GAand abscisic acid (ABA) We therefore assess whether H

2S

can ameliorate PCD in GA-treated barley aleurone layers Asshown in Figure 5(a) the accumulation of dead cells increasesrapidly from 24 to 96 h in GA-treated barley aleurone layerswhereas 025mM NaHS treatment significantly delays therate of PCD After incubation for 96 h about 90 cells in

6 Oxidative Medicine and Cellular Longevity

10

20

30

40

50

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f SO

D (U

middotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

50

90

130

170

210

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f PO

D (U

middotgminus1

FW)

lowastlowast

lowastlowast lowastlowast lowastlowast

lowastlowast

(b)

80

130

180

230

280

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f APX

(Umiddotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)

35

55

75

95

115

135

0 2 4Treatment time (d)

ControlH2S

Activ

ity o

f CAT

(Umiddotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(d)

70

110

150

190

230

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f LO

X (U

middotgminus1

FW)

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(e)

Figure 3 Effects of NaHS on the activity of SOD (a) POD (b) APX (c) CAT (d) and LOX (e) in barley aleurone layers Data are expressedas means plusmn SD of three independent experiments with three replicates of 15 grains per treatment The symbols lowast and lowastlowast mean significantdifference at 119875 lt 005 and 119875 lt 001 between control and T respectively

GA-treated layers are dead while half of cells are still alivein NaHS-treated layers (Figure 5(b)) In water control muchless cells undergo PCD compared with the counterpart of GAand GA plus NaHS

Determination of sdotO2

minus content shows that NaHS treat-ment maintains lower levels of sdotO

2

minus in GA-treated barleyaleurone layers (Figure 5(c)) After a rapid increase duringthe first 2 days of incubation the content of sdotO

2

minus in GA-treated layers decreases till day 4 In contrast sdotO

2

minus content inNaHS plus GA treatment increases more slowly until day 3A comparable but lower sdotO

2

minus content was observed in watercontrol compared with NaHS plus GA treatment

Figure 5(d) shows the effect of H2S on POD activity in

GA-treated barley aleurone layers In both NaHS treatmentand GA control POD activity in GA-treated barley aleurone

layers increases gradually up to day 3 and day 2 respectivelyand decreases thereafter However the activity of POD inNaHS-treated aleurone layers is always significantly higherthan those in water controls and GA treatment alone

Figure 5(e) shows that NaHS treatment maintains lowerlevels of LOX activity compared with water control duringthe first 2 days of GA treatment LOX activity in GAtreatment increases dramatically on day 1 followed by agradual decrease while in NaHS-treated tissue the activityincreases more slowly till day 3 followed by a decline Afterday 3 LOX activity in NaHS plus GA is higher than that inGA treatment

36 H2S Donor Promotes 120572-Amylase Secretion in Barley

Aleurone Layers Regardless of GA Secretion of 120572-amylase is

Oxidative Medicine and Cellular Longevity 7

0

2

4

6

8

10

12

14

C0 C1 T1 C5 T5

ab

d

b

c

020406080

100120140160180

C0 C1 T1 C5 T5

a a

c

b

d

05

10152025303540

C0 C1 T1 C5 T5

a a

bc

d

0102030405060708090

C0 C1 T1 C5 T5

T5

a

b

c

d

e

00005

0010015

0020025

0030035

0040045

C0 C1 T1 C5 T5

ab

b

cd

0

50

100

150

200

250

C0 C1 T1 C5

aa

b

cc

0

200

400

600

C0 C1 T1 C5 T5a ab

c

d

0

4

8

12

C0 C1 T1 C5 T5

aa

b

cd

Relat

ive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

HvSOD1 HvAPX

HvCAT1 HvCAT2

HvLOX

HvEPA HvCP3-31

HvICY

Figure 4 Effects of H2S on the expression of HvSOD1 HvCAT1 HvCAT2 HvLOX cystatin (HvICY) and the cysteine proteases HvCP3-31

andHvEPA in barley aleurone layers Aleurone layers are incubated in NaHS (T) or water (C) and total RNA is obtained at 0 1 and 5 dMeansand SD values are calculated from three independent experiments Within each identified gene bars with different letters are significantlydifferent in comparison with the corresponding control at 119875 lt 001 according to Fisherrsquos least significant differences (LSD)

a characteristic response of aleurone cells toGAWe thereforetest whether the ameliorating effect of H

2S on PCD affects

the release of 120572-amylase As shown in Figure 6(a) NaHS pro-motes 120572-amylase release in water-treated aleurone layers Inthe presence ofGANaHS treatment also enhances120572-amylaserelease frombarley aleurone layers (Figure 6(b)) with 05mMNaHS exhibiting optimal effect Figure 6(c) shows the time

changes in 120572-amylase secretion in water control GA-treatedaleurone layers and GA plus 025mM NaHS treatment Theaccumulation of 120572-amylase in incubation medium in GA-treated layers increases and peaks on day 3 followed by aplateau but addition of 025mM NaHS brings about a morerapid increase till day 4 followed by a decrease on day 5(Figure 6(c)) The activity of 120572-amylase released following

8 Oxidative Medicine and Cellular Longevity

Con

trol

GA

GA+

H2S

24h 48h 72h 96h

(a)

0

20

40

60

80

100

120

24 72 96

Dea

d ce

lls (

)

Treatment time (d)

GA

ControlGA + H2S

(b)

0

50

100

150

200

250

0 1 2 3 4Treatment time (d)

GA

ControlGA + H2S

Con

tent

of

(120583gmiddot

gminus1

FW)

middotO2minus

(c)

40

60

80

100

120

140

160

180

0 1 2 3 4Treatment time (d)

Activ

ity o

f PO

D (U

middotgminus1

FW)

GA

ControlGA + H2S

(d)

GA

0

50

100

150

200

250

300

0 1 2 3 4Treatment time (d)

ControlGA + H2S

Activ

ity o

f LO

X (U

middotgminus1

FW)

(e)

Figure 5 NaHS delays PCD in GA-treated barley aleurone layers Aleurone layers are incubated in water GA or GA + NaHS (GA + H2S)

and after being stained with trypan blue images are obtained by lightmicroscopy (a) and the percentage of dead cells is shown in (b) Contentof sdotO2

minus (c) activity of POD (d) and LOX (e) are measured on 0 1 2 3 and 4 d Bar 100 120583m Data in (c) and (d) are expressed as means plusmn SDof three independent experiments with three replicates of 15 grains per treatment

Oxidative Medicine and Cellular Longevity 9

0 0005 005 025 05

(mM)

(a)

0 0005 005 025 05

(mM)

(b)

0

50

100

150

200

0 1 2 3 4 5Treatment time (d)

GA

ControlGA + H2S

Activ

ity o

f120572-a

myl

ase (

Umiddotgminus1

FW)

(c)

Figure 6 NaHS increases the activity of 120572-amylase in incubation medium of barley aleurone layers in the absence (a) and presence ((b)(c)) of GA Barley aleurone layers isolated from treated embryoless half grains are incubated in 0 0005 005 025 or 05mMNaHS for 24 h((a) (b)) After incubation aleurone layers are placed on agar-starch medium (containing 4 agar and 01 starch) for 16 h Agar plates arestained with I

2-KI solution to detect the activity of 120572-amylase (a) (b) shows native PAGE analysis of 120572-amylase activity in incubationmedium

surrounding the aleurone layers (c) indicates secreted 120572-amylase activity in incubation medium surrounding barley aleurone layers treatedwith water (control) GA or GA + NaHS (GA + H

2S) at different times of incubation Data in (c) are expressed as means plusmn SD of three

independent experiments with three replicates of 20 embryoless half grains per treatment

GA + NaHS treatment is significantly higher than that oflayers incubated only in GA during the whole treatmenttime As expected much lower 120572-amylase activity was onlyobserved inwater control after 3 days of incubation Togetherthis result indicates that H

2S delays PCD in barley aleurone

layers and meanwhile promotes 120572-amylase release regardlessof the presence of GA

4 Discussion

H2S participates in multiple processes in plants [27] In

this paper we show that H2S delays PCD in barley aleu-

rone layers regardless of the presence or absence of GAIn the absence of GA PCD in barley aleurone layers isevident on day 3 of incubation while H

2S delays PCD at

an optimal concentration of 025mM (Figure 1) Consistentwith the reports that GA accelerates PCD process in barley

aleurone layers GA treatment triggers cell death in about90 aleurone cells at 72 h (Figure 5(a)) GA-induced celldeath is slowed by the addition of NaHS and this H

2S

donor also prolongs the phase of 120572-amylase productionin GA-treated layers The promoting effect of NaHS on120572-amylase synthesis and its prolongation of cell survivalindicate that 25mMNaHS does not affect aleurone cell func-tion

ROS such as sdotO2

minus andH2O2 are inducers of PCD in plant

and animal cells [1] It is reported that the peroxidation ofmembrane lipids and damage to the plasma membrane canoccur when the rate of ROS production overcomes the cellsrsquoability for scavenging ROS [27] Overproduction of ROS andoxidative damage are universal events in PCD in plant cells[28] In this paper we show that the content of sdotO

2

minus increasesin parallel with cell death in GA-treated layers (Figure 5(c))In non-GA-treated layers the burst of sdotO

2

minus and H2O2and

10 Oxidative Medicine and Cellular Longevity

the accumulation of MDA are also accompanied by PCD(Figure 2)These results suggest that ROS play a key role bothin GA-treated [29] and in non-GA-treated aleurone layers(Figures 2(a) 2(b) and 5(c))

Aleurone cells contain a suite of ROS-metabolizingenzymes GA-induced PCD in layers is accompanied by adecline in activity of ROS metabolizing enzymes which leadsto increased susceptibility of aleurone cells to ROS [29] Anovel aspect of our work is that NaHS treatment effectivelyreduces the accumulation of ROS in barley aleurone layersregardless the presence of GA (Figures 2 and 5) therebydelaying PCD process in these cells We propose that theH2S donor reduces ROS accumulation in layers by increasing

the activity of ROS-scavenging enzymes The data in presentstudy show that H

2S treatment maintains significantly higher

POD activity in GA-treated layers (Figure 5(d)) and higherSOD POD CAT and APX activity in non-GA-treatedlayers (Figure 3) The increased activity of ROS-scavengingenzymes in NaHS-treated likely promotes the cellrsquos abilityto metabolize ROS In addition LOX activities which areresponsible for lipid peroxidation are downregulated inNaHS-treated aleurone layers at early stage of treatment(Figures 3(e) and 5(e)) Meanwhile quantitative-PCR anal-ysis shows that expression of HvSOD1 HvAPX HvCAT1and HvCAT2 genes in non-GA-treated layers is maintainedat higher levels in NaHS treatment compared with watercontrols (Figure 4) Consistent with lower LOX activity inNaHS-treated aleurone layers the accumulation of LOXtranscripts is also reduced (Figure 4) In summary H

2S slows

down ROS-induced PCD in barley aleurone layers probablyby enhancing the activity and expression of ROS-scavengingenzymes and reducing the peroxidation of membrane lipids

Consistently Xie et al [18] found that H2S delayed GA-

triggered PCD in wheat aleurone layers by increasing GSHcontent and heme oxygenase-1 gene expression Here weprovide evidence thatH

2S can alleviate both natural PCD and

GA-triggered PCD through the modulation of antioxidantenzyme activities and their expression Compared with theslow natural PCDprocess the present study also confirms thepivotal role of GA in triggering PCD (Figure 5)

The role of ROS in GA and ABA signaling in barleyaleurone cells is recently clarified [30] in which they foundthat the production of H

2O2 a type of ROS was induced by

GA in aleurone cells but suppressed by ABA Furthermoreexogenous H

2O2appeared to promote the induction of 120572-

amylases by GA by promoting the expression of GAMyb and120572-amylase genes whereas antioxidants suppressed the induc-tion of 120572-amylase Unexpectedly we found that H

2S reduces

ROS accumulation and delays PCD process in barley aleu-rone layers in the presence or absence of GA and meanwhilepromotes the secretion of 120572-amylase suggesting that theantioxidant H

2S works through an unknown way to regulate

120572-amylase secretion and antioxidants do not always suppressthe induction of 120572-amylase Besides the activation of 120572-amylase by H

2S in the absence of GA implies that 120572-amylase

can be secreted independent of GA signaling pathwayTherefore the present findings advance our knowledge onthe relations between PCD process and 120572-amylase secretion

and the independence of 120572-amylase secretion and GA path-way

The activation of cysteine proteases was instrumental inthe PCD of soybean cells while cystatin an endogenouscysteine protease inhibitor gene inhibited cysteine proteaseactivity and blocked PCD in these cells [7] In this paperwe show that H

2S downregulates the transcriptions of two

barley cysteine proteinases HvEPA and HvCP3-31 in non-GA-treated barley aleurone layers thereby delaying cellcomponent degradation and PCD process

5 Conclusion

In summary we report the role of H2S in delaying PCD in

barley aleurone layers regardless of the presence or absence ofGA without repressing 120572-amylase induction suggesting thatthe function ofH

2Smay be universal in regulating plant PCD

PCD in plant cells is regulated by many internal and externalfactors such as the hormones (GA andABA) Ca2+ ROS andNO [6] It will be interesting to knowwhether H

2S is involved

in other signals and how 120572-amylase is induced by H2S in the

presence or absence of GA in cereal aleurone cells

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ying-Xin Zhang and Kang-Di Hu contributed equally to thiswork

Acknowledgments

Theauthors acknowledge Russell Jones University of Califor-nia at Berkeley for editing the paperThisworkwas supportedby the Natural Science Foundation of China (nos 3127180331301820 31300133 and 31470013) the Scientific ResearchFoundation for Returned Overseas Chinese Scholars (SRFfor ROCS MOE) the Natural Science Foundations of AnhuiProvince (11040606M85) and the Anhui Provincial Educa-tion Department (nos 2012AJZR0028 ZD200910)

References

[1] T Jabs ldquoReactive oxygen intermediates as mediators of pro-grammed cell death in plants and animalsrdquo Biochemical Phar-macology vol 57 no 3 pp 231ndash245 1999

[2] A Fath P Bethke V Beligni and R Jones ldquoActive oxygenand cell death in cereal aleurone cellsrdquo Journal of ExperimentalBotany vol 53 no 372 pp 1273ndash1282 2002

[3] A Fath P Bethke J Lonsdale R Meza-Romero and R JonesldquoProgrammed cell death in cereal aleuronerdquo Plant MolecularBiology vol 44 no 3 pp 255ndash266 2000

[4] P C Bethke J E Lonsdale A Fath and R L Jones ldquoHormon-ally regulated programmed cell death in barley aleurone cellsrdquoThe Plant Cell vol 11 no 16 pp 1033ndash1045 1999

Oxidative Medicine and Cellular Longevity 11

[5] P EHockberger T A Skimina V E Centonze et al ldquoActivationof flavin-containing oxidases underlies light-induced produc-tion of H

2O2in mammalian cellsrdquo Proceedings of the National

Academy of Sciences of the United States of America vol 96 no11 pp 6255ndash6260 1999

[6] P C Bethke and R L Jones ldquoCell death of barley aleuroneprotoplasts is mediated by reactive oxygen speciesrdquo The PlantJournal vol 25 no 1 pp 19ndash29 2001

[7] M Solomon B Belenghi M Delledonne E Menachem andA Levine ldquoThe involvement of cysteine proteases and proteaseinhibitor genes in the regulation of programmed cell death inplantsrdquoThe Plant Cell vol 11 no 3 pp 431ndash443 1999

[8] R Wang ldquoTworsquos company threersquos a crowd can H2S be the third

endogenous gaseous transmitterrdquo The FASEB Journal vol 16no 13 pp 1792ndash1798 2002

[9] E Bloem A Riemenschneider J Volker et al ldquoSulphur supplyand infection with Pyrenopeziza brassicae influence L-cysteinedesulphydrase activity in Brassica napus Lrdquo Journal of Experi-mental Botany vol 55 no 406 pp 2305ndash2312 2004

[10] H Zhang L-Y Hu K-D Hu Y-D He S-H Wang and J-P Luo ldquoHydrogen sulfide promotes wheat seed germinationand alleviates oxidative damage against copper stressrdquo Journalof Integrative Plant Biology vol 50 no 12 pp 1518ndash1529 2008

[11] H Zhang J Tang X-P Liu et al ldquoHydrogen sulfide promotesroot organogenesis in Ipomoea batatas Salix matsudana andGlycine maxrdquo Journal of Integrative Plant Biology vol 51 no 12pp 1086ndash1094 2009

[12] Z P Jin J J Shen Z J Qiao G D Yang R Wang andY X Pei ldquoHydrogen sulfide improves drought resistance inArabidopsis thalianardquo Biochemical and Biophysical ResearchCommunications vol 414 no 3 pp 481ndash486 2011

[13] Z P Jin SWXue YN Luo et al ldquoHydrogen sulfide interactingwith abscisic acid in stomatal regulation responses to droughtstress inArabidopsisrdquoPlant Physiology and Biochemistry vol 62pp 41ndash46 2013

[14] C Garcıa-Mata and L Lamattina ldquoHydrogen sulphide anovel gasotransmitter involved in guard cell signallingrdquo NewPhytologist vol 188 no 4 pp 977ndash984 2010

[15] L-Y Hu S-L Hu J Wu et al ldquoHydrogen sulfide prolongspostharvest shelf life of strawberry and plays an antioxidativerole in fruitsrdquo Journal of Agricultural and Food Chemistry vol60 no 35 pp 8684ndash8693 2012

[16] S-P Gao K-D Hu L-Y Hu et al ldquoHydrogen sulfide delayspostharvest senescence and plays an antioxidative role in fresh-cut kiwifruitrdquo HortScience vol 49 no 10 pp 1385ndash1392 2013

[17] S-P Li K-D Hu L-Y Hu et al ldquoHydrogen sulfide alleviatespostharvest senescence of broccoli by modulating antioxidantdefense and senescence-related gene expressionrdquo Journal ofAgricultural and Food Chemistry vol 62 no 5 pp 1119ndash11292014

[18] Y Xie C Zhang D Lai et al ldquoHydrogen sulfide delays GA-triggered programmed cell death inwheat aleurone layers by themodulation of glutathione homeostasis and heme oxygenase-1expressionrdquo Journal of Plant Physiology vol 171 no 2 pp 53ndash622014

[19] M J Chrispeels and J E Varner ldquoGibberellic acid enhancedsynthesis and release of 120572-amylase and ribonuclease by isolatedbarley and aleurone layersrdquo Plant Physiology vol 42 no 3 pp398ndash406 1967

[20] J R Tennant ldquoEvaluation of the trypan blue technique fordetermination of cell viabilityrdquo Transplantation vol 2 pp 685ndash694 1964

[21] M Tsuchiya M Suematsu and H Suzuki ldquoIn vivo visualiza-tion of oxygen radical-dependent photoemissionrdquo Methods inEnzymology vol 233 pp 128ndash140 1994

[22] R F del Maestro J Bjork and K-E Arfors ldquoIncrease inmicrovascular permeability induced by enzymatically gener-ated free radicals I In vivo studyrdquoMicrovascular Research vol22 no 3 pp 239ndash254 1981

[23] C Garcıa-Limones A Hervas J A Navas-Cortes R MJimenez-Dıaz and M Tena ldquoInduction of an antioxidantenzyme system and other oxidative stress markers associatedwith compatible and incompatible interactions between chick-pea (Cicer arietinum L) and Fusarium oxysporum f sp cicerisrdquoPhysiological and Molecular Plant Pathology vol 61 no 6 pp325ndash337 2002

[24] K Surrey ldquoSpectrophotometric method for determination oflipoxidase activityrdquo Plant Physiology vol 39 no 1 pp 65ndash701964

[25] G G Collins C F Jenner and L G Paleg ldquoThe metabolismof soluble nucleotides in wheat aleurone layers treated withgibberellic acidrdquo Plant P hysiology vol 49 no 3 pp 404ndash4101972

[26] G Noelting and P Bernfeld ldquoSur les enzymes amylolytiqucsIII La 120573-amylase dosage drsquoactivite et controle de lrsquoabsence drsquo120572-amylaserdquo Helvetica Chimica Acta vol 31 no 1 pp 286ndash2901948

[27] M Lisjak T Teklic I D Wilson M Whiteman and J THancock ldquoHydrogen sulfide environmental factor or signallingmoleculerdquo Plant Cell and Environment vol 36 no 9 pp 1607ndash1616 2013

[28] M C de Pinto A Paradiso P Leonetti and L de Gara ldquoHydro-gen peroxide nitric oxide and cytosolic ascorbate peroxidaseat the crossroad between defence and cell deathrdquo The PlantJournal vol 48 no 5 pp 784ndash795 2006

[29] A Fath P C Bethke and R L Jones ldquoEnzymes that scavengereactive oxygen species are down-regulated prior to gibberellicacid-induced programmed cell death in barley aleuronerdquo PlantPhysiology vol 126 no 1 pp 156ndash166 2001

[30] Y Ishibashi T Tawaratsumida K Kondo et al ldquoReactive oxy-gen species are involved in gibberellinabscisic acid signaling inbarley aleurone cellsrdquo Plant Physiology vol 158 no 4 pp 1705ndash1714 2012

Submit your manuscripts athttpwwwhindawicom

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

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OncologyJournal of

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Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Oxidative Medicine and Cellular Longevity

0 0005 0025

005 025 05

(mM)

(a)

0

20

40

60

80

0 0005 0025 005 025 05

Dea

d ce

lls (

)

NaHS concentration (mM)

(b)

0d 3d 5d 7d

Con

trol

H2S

(c)

0

20

40

60

80

100

0 3 5 7

Dea

d ce

lls (

)

Treatment time (d)

ControlH2S

(d)

Figure 1 Effect of H2S donor NaHS on cell viability in barley aleurone layers ((a) (b)) Aleurone layers are incubated in different

concentrations of NaHS (0 0005 0025 005 025 and 05mM) for 5 d at 25∘C After staining with trypan blue the images are obtainedby light microscopy with blue or purple indicating dead cells ((c) (d)) Time course of PCD in barley aleurone layers treated with NaHS(H2S) or water (Control) Aleurone layers are incubated in 025mM NaHS or water for 0 1 3 5 and 7 d at 25∘C and are stained with trypan

blue Digital images of barley aleurone layers ((a) (c)) and percentages of dead cells ((b) (d)) are shown Bar 50120583m Data are means plusmn SD ofthree different aleurone layers per treatment

In comparison POD activity in water controls increasesgradually and peaks on day 3 followed by a sharp declineNaHS treatment maintains significantly higher levels of PODactivity compared with water control during the wholetreatment time APX activity increases during the first 3 daysof incubation and peaks on day 3 followed by a decreasein both NaHS-treated and water controls However APXactivity in NaHS treatment is always significantly higher thanthat of control (Figure 3(c))

Changes inCAT activity are shown in Figure 3(d) In bothNaHS andwater controls CAT activity increases gradually upto day 3 and then decreases sharply However CAT activity

from NaHS-treated layers is always significantly higher thanthose in control layers

Figure 3(e) shows the changes in LOX activity in barleyaleurone layers LOX activity in water control increasesdramatically and peaks on day 3 followed by a decreaseIn contrast NaHS treatment significantly decreases LOXactivity being about 50 of that of water control on day 3

34 Transcript Analysis of HvSOD1 HvCAT1 HvCAT2HvLOX Cysteine Protease (HvCP3-31 and HvEPA) and Cys-tatin (HvICY) in Non-GA-Treated Barley Aleurone LayersWe examine the expression of HvSOD1 HvCAT1 HvCAT2

Oxidative Medicine and Cellular Longevity 5

20

30

40

50

60

70

80

0 1 2 3 4 5Treatment time (d)

ControlH2S

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

Con

tent

of

(120583gmiddot

gminus1

FW)

middotO2minus

(a)

100

140

180

220

260

0 1 2 3 4 5Treatment time (d)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Con

tent

ofH

2O2

(120583m

olmiddotgminus1

FW)

ControlH2S

(b)

0

2

4

6

8

10

12

0 1 2 3 4 5Treatment time (d)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

ControlH2S

Con

tent

of M

DA

(nm

olmiddotgminus1

FW)

(c)

0 005 025

(mM)

DCH

F-D

AN

o D

CHF-

DA

(d)

Figure 2 Effects of NaHS on the contents of sdotO2

minus (a) H2O2(b) and MDA (c) in barley aleurone layers Aleurone layers treated with 0

005 and 025mMNaHS for 1 d are incubated DCHF-DA and are observed by fluorescence microscopy (d) Bar 100 120583m Data are expressedas means plusmn SD of three independent experiments with three replicates of 15 grains per treatment The symbols lowast and lowastlowast mean significantdifference at 119875 lt 005 and 119875 lt 001 between NaHS (H

2S) and water (control) treatment respectively

HvLOX the cysteine proteases HvCP3-31 and HvEPA andcystatin (HvICY) in NaHS-treated barley aleurone layersand water controls (Figure 4) Compared with water con-trols NaHS induces higher expression of HvSOD1 HvAPXHvCAT1 and HvCAT2 on days 1 and 5 HvLOX expressionincreases in water control layers on days 1 and 5 comparedwith day 0 while NaHS treatment sustains lower transcriptof HvLOX than water control especially on day 5 PCDin barley aleurone layers is accompanied with increasedcysteine protease activity [3] Accordingly we determine theexpression of the cysteine proteinases HvEPA and HvCP3-31and the cystatinHvICY inNaHS treatment andwater controlThe expression of HvEPA and HvCP3-31 increases in water

controls on days 1 and 5 whereas their expression is muchlower in NaHS-treated tissue The expression of HvICY wasenhanced in NaHS-treated layers whereas less transcript ofHvICY is observed in water controls

35 NaHS Delays PCD in GA-Treated Barley Aleurone LayersPCD in barley aleurone layers is tightly regulated by GAand abscisic acid (ABA) We therefore assess whether H

2S

can ameliorate PCD in GA-treated barley aleurone layers Asshown in Figure 5(a) the accumulation of dead cells increasesrapidly from 24 to 96 h in GA-treated barley aleurone layerswhereas 025mM NaHS treatment significantly delays therate of PCD After incubation for 96 h about 90 cells in

6 Oxidative Medicine and Cellular Longevity

10

20

30

40

50

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f SO

D (U

middotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

50

90

130

170

210

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f PO

D (U

middotgminus1

FW)

lowastlowast

lowastlowast lowastlowast lowastlowast

lowastlowast

(b)

80

130

180

230

280

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f APX

(Umiddotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)

35

55

75

95

115

135

0 2 4Treatment time (d)

ControlH2S

Activ

ity o

f CAT

(Umiddotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(d)

70

110

150

190

230

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f LO

X (U

middotgminus1

FW)

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(e)

Figure 3 Effects of NaHS on the activity of SOD (a) POD (b) APX (c) CAT (d) and LOX (e) in barley aleurone layers Data are expressedas means plusmn SD of three independent experiments with three replicates of 15 grains per treatment The symbols lowast and lowastlowast mean significantdifference at 119875 lt 005 and 119875 lt 001 between control and T respectively

GA-treated layers are dead while half of cells are still alivein NaHS-treated layers (Figure 5(b)) In water control muchless cells undergo PCD compared with the counterpart of GAand GA plus NaHS

Determination of sdotO2

minus content shows that NaHS treat-ment maintains lower levels of sdotO

2

minus in GA-treated barleyaleurone layers (Figure 5(c)) After a rapid increase duringthe first 2 days of incubation the content of sdotO

2

minus in GA-treated layers decreases till day 4 In contrast sdotO

2

minus content inNaHS plus GA treatment increases more slowly until day 3A comparable but lower sdotO

2

minus content was observed in watercontrol compared with NaHS plus GA treatment

Figure 5(d) shows the effect of H2S on POD activity in

GA-treated barley aleurone layers In both NaHS treatmentand GA control POD activity in GA-treated barley aleurone

layers increases gradually up to day 3 and day 2 respectivelyand decreases thereafter However the activity of POD inNaHS-treated aleurone layers is always significantly higherthan those in water controls and GA treatment alone

Figure 5(e) shows that NaHS treatment maintains lowerlevels of LOX activity compared with water control duringthe first 2 days of GA treatment LOX activity in GAtreatment increases dramatically on day 1 followed by agradual decrease while in NaHS-treated tissue the activityincreases more slowly till day 3 followed by a decline Afterday 3 LOX activity in NaHS plus GA is higher than that inGA treatment

36 H2S Donor Promotes 120572-Amylase Secretion in Barley

Aleurone Layers Regardless of GA Secretion of 120572-amylase is

Oxidative Medicine and Cellular Longevity 7

0

2

4

6

8

10

12

14

C0 C1 T1 C5 T5

ab

d

b

c

020406080

100120140160180

C0 C1 T1 C5 T5

a a

c

b

d

05

10152025303540

C0 C1 T1 C5 T5

a a

bc

d

0102030405060708090

C0 C1 T1 C5 T5

T5

a

b

c

d

e

00005

0010015

0020025

0030035

0040045

C0 C1 T1 C5 T5

ab

b

cd

0

50

100

150

200

250

C0 C1 T1 C5

aa

b

cc

0

200

400

600

C0 C1 T1 C5 T5a ab

c

d

0

4

8

12

C0 C1 T1 C5 T5

aa

b

cd

Relat

ive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

HvSOD1 HvAPX

HvCAT1 HvCAT2

HvLOX

HvEPA HvCP3-31

HvICY

Figure 4 Effects of H2S on the expression of HvSOD1 HvCAT1 HvCAT2 HvLOX cystatin (HvICY) and the cysteine proteases HvCP3-31

andHvEPA in barley aleurone layers Aleurone layers are incubated in NaHS (T) or water (C) and total RNA is obtained at 0 1 and 5 dMeansand SD values are calculated from three independent experiments Within each identified gene bars with different letters are significantlydifferent in comparison with the corresponding control at 119875 lt 001 according to Fisherrsquos least significant differences (LSD)

a characteristic response of aleurone cells toGAWe thereforetest whether the ameliorating effect of H

2S on PCD affects

the release of 120572-amylase As shown in Figure 6(a) NaHS pro-motes 120572-amylase release in water-treated aleurone layers Inthe presence ofGANaHS treatment also enhances120572-amylaserelease frombarley aleurone layers (Figure 6(b)) with 05mMNaHS exhibiting optimal effect Figure 6(c) shows the time

changes in 120572-amylase secretion in water control GA-treatedaleurone layers and GA plus 025mM NaHS treatment Theaccumulation of 120572-amylase in incubation medium in GA-treated layers increases and peaks on day 3 followed by aplateau but addition of 025mM NaHS brings about a morerapid increase till day 4 followed by a decrease on day 5(Figure 6(c)) The activity of 120572-amylase released following

8 Oxidative Medicine and Cellular Longevity

Con

trol

GA

GA+

H2S

24h 48h 72h 96h

(a)

0

20

40

60

80

100

120

24 72 96

Dea

d ce

lls (

)

Treatment time (d)

GA

ControlGA + H2S

(b)

0

50

100

150

200

250

0 1 2 3 4Treatment time (d)

GA

ControlGA + H2S

Con

tent

of

(120583gmiddot

gminus1

FW)

middotO2minus

(c)

40

60

80

100

120

140

160

180

0 1 2 3 4Treatment time (d)

Activ

ity o

f PO

D (U

middotgminus1

FW)

GA

ControlGA + H2S

(d)

GA

0

50

100

150

200

250

300

0 1 2 3 4Treatment time (d)

ControlGA + H2S

Activ

ity o

f LO

X (U

middotgminus1

FW)

(e)

Figure 5 NaHS delays PCD in GA-treated barley aleurone layers Aleurone layers are incubated in water GA or GA + NaHS (GA + H2S)

and after being stained with trypan blue images are obtained by lightmicroscopy (a) and the percentage of dead cells is shown in (b) Contentof sdotO2

minus (c) activity of POD (d) and LOX (e) are measured on 0 1 2 3 and 4 d Bar 100 120583m Data in (c) and (d) are expressed as means plusmn SDof three independent experiments with three replicates of 15 grains per treatment

Oxidative Medicine and Cellular Longevity 9

0 0005 005 025 05

(mM)

(a)

0 0005 005 025 05

(mM)

(b)

0

50

100

150

200

0 1 2 3 4 5Treatment time (d)

GA

ControlGA + H2S

Activ

ity o

f120572-a

myl

ase (

Umiddotgminus1

FW)

(c)

Figure 6 NaHS increases the activity of 120572-amylase in incubation medium of barley aleurone layers in the absence (a) and presence ((b)(c)) of GA Barley aleurone layers isolated from treated embryoless half grains are incubated in 0 0005 005 025 or 05mMNaHS for 24 h((a) (b)) After incubation aleurone layers are placed on agar-starch medium (containing 4 agar and 01 starch) for 16 h Agar plates arestained with I

2-KI solution to detect the activity of 120572-amylase (a) (b) shows native PAGE analysis of 120572-amylase activity in incubationmedium

surrounding the aleurone layers (c) indicates secreted 120572-amylase activity in incubation medium surrounding barley aleurone layers treatedwith water (control) GA or GA + NaHS (GA + H

2S) at different times of incubation Data in (c) are expressed as means plusmn SD of three

independent experiments with three replicates of 20 embryoless half grains per treatment

GA + NaHS treatment is significantly higher than that oflayers incubated only in GA during the whole treatmenttime As expected much lower 120572-amylase activity was onlyobserved inwater control after 3 days of incubation Togetherthis result indicates that H

2S delays PCD in barley aleurone

layers and meanwhile promotes 120572-amylase release regardlessof the presence of GA

4 Discussion

H2S participates in multiple processes in plants [27] In

this paper we show that H2S delays PCD in barley aleu-

rone layers regardless of the presence or absence of GAIn the absence of GA PCD in barley aleurone layers isevident on day 3 of incubation while H

2S delays PCD at

an optimal concentration of 025mM (Figure 1) Consistentwith the reports that GA accelerates PCD process in barley

aleurone layers GA treatment triggers cell death in about90 aleurone cells at 72 h (Figure 5(a)) GA-induced celldeath is slowed by the addition of NaHS and this H

2S

donor also prolongs the phase of 120572-amylase productionin GA-treated layers The promoting effect of NaHS on120572-amylase synthesis and its prolongation of cell survivalindicate that 25mMNaHS does not affect aleurone cell func-tion

ROS such as sdotO2

minus andH2O2 are inducers of PCD in plant

and animal cells [1] It is reported that the peroxidation ofmembrane lipids and damage to the plasma membrane canoccur when the rate of ROS production overcomes the cellsrsquoability for scavenging ROS [27] Overproduction of ROS andoxidative damage are universal events in PCD in plant cells[28] In this paper we show that the content of sdotO

2

minus increasesin parallel with cell death in GA-treated layers (Figure 5(c))In non-GA-treated layers the burst of sdotO

2

minus and H2O2and

10 Oxidative Medicine and Cellular Longevity

the accumulation of MDA are also accompanied by PCD(Figure 2)These results suggest that ROS play a key role bothin GA-treated [29] and in non-GA-treated aleurone layers(Figures 2(a) 2(b) and 5(c))

Aleurone cells contain a suite of ROS-metabolizingenzymes GA-induced PCD in layers is accompanied by adecline in activity of ROS metabolizing enzymes which leadsto increased susceptibility of aleurone cells to ROS [29] Anovel aspect of our work is that NaHS treatment effectivelyreduces the accumulation of ROS in barley aleurone layersregardless the presence of GA (Figures 2 and 5) therebydelaying PCD process in these cells We propose that theH2S donor reduces ROS accumulation in layers by increasing

the activity of ROS-scavenging enzymes The data in presentstudy show that H

2S treatment maintains significantly higher

POD activity in GA-treated layers (Figure 5(d)) and higherSOD POD CAT and APX activity in non-GA-treatedlayers (Figure 3) The increased activity of ROS-scavengingenzymes in NaHS-treated likely promotes the cellrsquos abilityto metabolize ROS In addition LOX activities which areresponsible for lipid peroxidation are downregulated inNaHS-treated aleurone layers at early stage of treatment(Figures 3(e) and 5(e)) Meanwhile quantitative-PCR anal-ysis shows that expression of HvSOD1 HvAPX HvCAT1and HvCAT2 genes in non-GA-treated layers is maintainedat higher levels in NaHS treatment compared with watercontrols (Figure 4) Consistent with lower LOX activity inNaHS-treated aleurone layers the accumulation of LOXtranscripts is also reduced (Figure 4) In summary H

2S slows

down ROS-induced PCD in barley aleurone layers probablyby enhancing the activity and expression of ROS-scavengingenzymes and reducing the peroxidation of membrane lipids

Consistently Xie et al [18] found that H2S delayed GA-

triggered PCD in wheat aleurone layers by increasing GSHcontent and heme oxygenase-1 gene expression Here weprovide evidence thatH

2S can alleviate both natural PCD and

GA-triggered PCD through the modulation of antioxidantenzyme activities and their expression Compared with theslow natural PCDprocess the present study also confirms thepivotal role of GA in triggering PCD (Figure 5)

The role of ROS in GA and ABA signaling in barleyaleurone cells is recently clarified [30] in which they foundthat the production of H

2O2 a type of ROS was induced by

GA in aleurone cells but suppressed by ABA Furthermoreexogenous H

2O2appeared to promote the induction of 120572-

amylases by GA by promoting the expression of GAMyb and120572-amylase genes whereas antioxidants suppressed the induc-tion of 120572-amylase Unexpectedly we found that H

2S reduces

ROS accumulation and delays PCD process in barley aleu-rone layers in the presence or absence of GA and meanwhilepromotes the secretion of 120572-amylase suggesting that theantioxidant H

2S works through an unknown way to regulate

120572-amylase secretion and antioxidants do not always suppressthe induction of 120572-amylase Besides the activation of 120572-amylase by H

2S in the absence of GA implies that 120572-amylase

can be secreted independent of GA signaling pathwayTherefore the present findings advance our knowledge onthe relations between PCD process and 120572-amylase secretion

and the independence of 120572-amylase secretion and GA path-way

The activation of cysteine proteases was instrumental inthe PCD of soybean cells while cystatin an endogenouscysteine protease inhibitor gene inhibited cysteine proteaseactivity and blocked PCD in these cells [7] In this paperwe show that H

2S downregulates the transcriptions of two

barley cysteine proteinases HvEPA and HvCP3-31 in non-GA-treated barley aleurone layers thereby delaying cellcomponent degradation and PCD process

5 Conclusion

In summary we report the role of H2S in delaying PCD in

barley aleurone layers regardless of the presence or absence ofGA without repressing 120572-amylase induction suggesting thatthe function ofH

2Smay be universal in regulating plant PCD

PCD in plant cells is regulated by many internal and externalfactors such as the hormones (GA andABA) Ca2+ ROS andNO [6] It will be interesting to knowwhether H

2S is involved

in other signals and how 120572-amylase is induced by H2S in the

presence or absence of GA in cereal aleurone cells

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ying-Xin Zhang and Kang-Di Hu contributed equally to thiswork

Acknowledgments

Theauthors acknowledge Russell Jones University of Califor-nia at Berkeley for editing the paperThisworkwas supportedby the Natural Science Foundation of China (nos 3127180331301820 31300133 and 31470013) the Scientific ResearchFoundation for Returned Overseas Chinese Scholars (SRFfor ROCS MOE) the Natural Science Foundations of AnhuiProvince (11040606M85) and the Anhui Provincial Educa-tion Department (nos 2012AJZR0028 ZD200910)

References

[1] T Jabs ldquoReactive oxygen intermediates as mediators of pro-grammed cell death in plants and animalsrdquo Biochemical Phar-macology vol 57 no 3 pp 231ndash245 1999

[2] A Fath P Bethke V Beligni and R Jones ldquoActive oxygenand cell death in cereal aleurone cellsrdquo Journal of ExperimentalBotany vol 53 no 372 pp 1273ndash1282 2002

[3] A Fath P Bethke J Lonsdale R Meza-Romero and R JonesldquoProgrammed cell death in cereal aleuronerdquo Plant MolecularBiology vol 44 no 3 pp 255ndash266 2000

[4] P C Bethke J E Lonsdale A Fath and R L Jones ldquoHormon-ally regulated programmed cell death in barley aleurone cellsrdquoThe Plant Cell vol 11 no 16 pp 1033ndash1045 1999

Oxidative Medicine and Cellular Longevity 11

[5] P EHockberger T A Skimina V E Centonze et al ldquoActivationof flavin-containing oxidases underlies light-induced produc-tion of H

2O2in mammalian cellsrdquo Proceedings of the National

Academy of Sciences of the United States of America vol 96 no11 pp 6255ndash6260 1999

[6] P C Bethke and R L Jones ldquoCell death of barley aleuroneprotoplasts is mediated by reactive oxygen speciesrdquo The PlantJournal vol 25 no 1 pp 19ndash29 2001

[7] M Solomon B Belenghi M Delledonne E Menachem andA Levine ldquoThe involvement of cysteine proteases and proteaseinhibitor genes in the regulation of programmed cell death inplantsrdquoThe Plant Cell vol 11 no 3 pp 431ndash443 1999

[8] R Wang ldquoTworsquos company threersquos a crowd can H2S be the third

endogenous gaseous transmitterrdquo The FASEB Journal vol 16no 13 pp 1792ndash1798 2002

[9] E Bloem A Riemenschneider J Volker et al ldquoSulphur supplyand infection with Pyrenopeziza brassicae influence L-cysteinedesulphydrase activity in Brassica napus Lrdquo Journal of Experi-mental Botany vol 55 no 406 pp 2305ndash2312 2004

[10] H Zhang L-Y Hu K-D Hu Y-D He S-H Wang and J-P Luo ldquoHydrogen sulfide promotes wheat seed germinationand alleviates oxidative damage against copper stressrdquo Journalof Integrative Plant Biology vol 50 no 12 pp 1518ndash1529 2008

[11] H Zhang J Tang X-P Liu et al ldquoHydrogen sulfide promotesroot organogenesis in Ipomoea batatas Salix matsudana andGlycine maxrdquo Journal of Integrative Plant Biology vol 51 no 12pp 1086ndash1094 2009

[12] Z P Jin J J Shen Z J Qiao G D Yang R Wang andY X Pei ldquoHydrogen sulfide improves drought resistance inArabidopsis thalianardquo Biochemical and Biophysical ResearchCommunications vol 414 no 3 pp 481ndash486 2011

[13] Z P Jin SWXue YN Luo et al ldquoHydrogen sulfide interactingwith abscisic acid in stomatal regulation responses to droughtstress inArabidopsisrdquoPlant Physiology and Biochemistry vol 62pp 41ndash46 2013

[14] C Garcıa-Mata and L Lamattina ldquoHydrogen sulphide anovel gasotransmitter involved in guard cell signallingrdquo NewPhytologist vol 188 no 4 pp 977ndash984 2010

[15] L-Y Hu S-L Hu J Wu et al ldquoHydrogen sulfide prolongspostharvest shelf life of strawberry and plays an antioxidativerole in fruitsrdquo Journal of Agricultural and Food Chemistry vol60 no 35 pp 8684ndash8693 2012

[16] S-P Gao K-D Hu L-Y Hu et al ldquoHydrogen sulfide delayspostharvest senescence and plays an antioxidative role in fresh-cut kiwifruitrdquo HortScience vol 49 no 10 pp 1385ndash1392 2013

[17] S-P Li K-D Hu L-Y Hu et al ldquoHydrogen sulfide alleviatespostharvest senescence of broccoli by modulating antioxidantdefense and senescence-related gene expressionrdquo Journal ofAgricultural and Food Chemistry vol 62 no 5 pp 1119ndash11292014

[18] Y Xie C Zhang D Lai et al ldquoHydrogen sulfide delays GA-triggered programmed cell death inwheat aleurone layers by themodulation of glutathione homeostasis and heme oxygenase-1expressionrdquo Journal of Plant Physiology vol 171 no 2 pp 53ndash622014

[19] M J Chrispeels and J E Varner ldquoGibberellic acid enhancedsynthesis and release of 120572-amylase and ribonuclease by isolatedbarley and aleurone layersrdquo Plant Physiology vol 42 no 3 pp398ndash406 1967

[20] J R Tennant ldquoEvaluation of the trypan blue technique fordetermination of cell viabilityrdquo Transplantation vol 2 pp 685ndash694 1964

[21] M Tsuchiya M Suematsu and H Suzuki ldquoIn vivo visualiza-tion of oxygen radical-dependent photoemissionrdquo Methods inEnzymology vol 233 pp 128ndash140 1994

[22] R F del Maestro J Bjork and K-E Arfors ldquoIncrease inmicrovascular permeability induced by enzymatically gener-ated free radicals I In vivo studyrdquoMicrovascular Research vol22 no 3 pp 239ndash254 1981

[23] C Garcıa-Limones A Hervas J A Navas-Cortes R MJimenez-Dıaz and M Tena ldquoInduction of an antioxidantenzyme system and other oxidative stress markers associatedwith compatible and incompatible interactions between chick-pea (Cicer arietinum L) and Fusarium oxysporum f sp cicerisrdquoPhysiological and Molecular Plant Pathology vol 61 no 6 pp325ndash337 2002

[24] K Surrey ldquoSpectrophotometric method for determination oflipoxidase activityrdquo Plant Physiology vol 39 no 1 pp 65ndash701964

[25] G G Collins C F Jenner and L G Paleg ldquoThe metabolismof soluble nucleotides in wheat aleurone layers treated withgibberellic acidrdquo Plant P hysiology vol 49 no 3 pp 404ndash4101972

[26] G Noelting and P Bernfeld ldquoSur les enzymes amylolytiqucsIII La 120573-amylase dosage drsquoactivite et controle de lrsquoabsence drsquo120572-amylaserdquo Helvetica Chimica Acta vol 31 no 1 pp 286ndash2901948

[27] M Lisjak T Teklic I D Wilson M Whiteman and J THancock ldquoHydrogen sulfide environmental factor or signallingmoleculerdquo Plant Cell and Environment vol 36 no 9 pp 1607ndash1616 2013

[28] M C de Pinto A Paradiso P Leonetti and L de Gara ldquoHydro-gen peroxide nitric oxide and cytosolic ascorbate peroxidaseat the crossroad between defence and cell deathrdquo The PlantJournal vol 48 no 5 pp 784ndash795 2006

[29] A Fath P C Bethke and R L Jones ldquoEnzymes that scavengereactive oxygen species are down-regulated prior to gibberellicacid-induced programmed cell death in barley aleuronerdquo PlantPhysiology vol 126 no 1 pp 156ndash166 2001

[30] Y Ishibashi T Tawaratsumida K Kondo et al ldquoReactive oxy-gen species are involved in gibberellinabscisic acid signaling inbarley aleurone cellsrdquo Plant Physiology vol 158 no 4 pp 1705ndash1714 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 5

20

30

40

50

60

70

80

0 1 2 3 4 5Treatment time (d)

ControlH2S

lowastlowast

lowastlowast

lowastlowastlowastlowast

lowastlowast

Con

tent

of

(120583gmiddot

gminus1

FW)

middotO2minus

(a)

100

140

180

220

260

0 1 2 3 4 5Treatment time (d)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

Con

tent

ofH

2O2

(120583m

olmiddotgminus1

FW)

ControlH2S

(b)

0

2

4

6

8

10

12

0 1 2 3 4 5Treatment time (d)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

ControlH2S

Con

tent

of M

DA

(nm

olmiddotgminus1

FW)

(c)

0 005 025

(mM)

DCH

F-D

AN

o D

CHF-

DA

(d)

Figure 2 Effects of NaHS on the contents of sdotO2

minus (a) H2O2(b) and MDA (c) in barley aleurone layers Aleurone layers treated with 0

005 and 025mMNaHS for 1 d are incubated DCHF-DA and are observed by fluorescence microscopy (d) Bar 100 120583m Data are expressedas means plusmn SD of three independent experiments with three replicates of 15 grains per treatment The symbols lowast and lowastlowast mean significantdifference at 119875 lt 005 and 119875 lt 001 between NaHS (H

2S) and water (control) treatment respectively

HvLOX the cysteine proteases HvCP3-31 and HvEPA andcystatin (HvICY) in NaHS-treated barley aleurone layersand water controls (Figure 4) Compared with water con-trols NaHS induces higher expression of HvSOD1 HvAPXHvCAT1 and HvCAT2 on days 1 and 5 HvLOX expressionincreases in water control layers on days 1 and 5 comparedwith day 0 while NaHS treatment sustains lower transcriptof HvLOX than water control especially on day 5 PCDin barley aleurone layers is accompanied with increasedcysteine protease activity [3] Accordingly we determine theexpression of the cysteine proteinases HvEPA and HvCP3-31and the cystatinHvICY inNaHS treatment andwater controlThe expression of HvEPA and HvCP3-31 increases in water

controls on days 1 and 5 whereas their expression is muchlower in NaHS-treated tissue The expression of HvICY wasenhanced in NaHS-treated layers whereas less transcript ofHvICY is observed in water controls

35 NaHS Delays PCD in GA-Treated Barley Aleurone LayersPCD in barley aleurone layers is tightly regulated by GAand abscisic acid (ABA) We therefore assess whether H

2S

can ameliorate PCD in GA-treated barley aleurone layers Asshown in Figure 5(a) the accumulation of dead cells increasesrapidly from 24 to 96 h in GA-treated barley aleurone layerswhereas 025mM NaHS treatment significantly delays therate of PCD After incubation for 96 h about 90 cells in

6 Oxidative Medicine and Cellular Longevity

10

20

30

40

50

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f SO

D (U

middotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

50

90

130

170

210

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f PO

D (U

middotgminus1

FW)

lowastlowast

lowastlowast lowastlowast lowastlowast

lowastlowast

(b)

80

130

180

230

280

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f APX

(Umiddotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)

35

55

75

95

115

135

0 2 4Treatment time (d)

ControlH2S

Activ

ity o

f CAT

(Umiddotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(d)

70

110

150

190

230

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f LO

X (U

middotgminus1

FW)

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(e)

Figure 3 Effects of NaHS on the activity of SOD (a) POD (b) APX (c) CAT (d) and LOX (e) in barley aleurone layers Data are expressedas means plusmn SD of three independent experiments with three replicates of 15 grains per treatment The symbols lowast and lowastlowast mean significantdifference at 119875 lt 005 and 119875 lt 001 between control and T respectively

GA-treated layers are dead while half of cells are still alivein NaHS-treated layers (Figure 5(b)) In water control muchless cells undergo PCD compared with the counterpart of GAand GA plus NaHS

Determination of sdotO2

minus content shows that NaHS treat-ment maintains lower levels of sdotO

2

minus in GA-treated barleyaleurone layers (Figure 5(c)) After a rapid increase duringthe first 2 days of incubation the content of sdotO

2

minus in GA-treated layers decreases till day 4 In contrast sdotO

2

minus content inNaHS plus GA treatment increases more slowly until day 3A comparable but lower sdotO

2

minus content was observed in watercontrol compared with NaHS plus GA treatment

Figure 5(d) shows the effect of H2S on POD activity in

GA-treated barley aleurone layers In both NaHS treatmentand GA control POD activity in GA-treated barley aleurone

layers increases gradually up to day 3 and day 2 respectivelyand decreases thereafter However the activity of POD inNaHS-treated aleurone layers is always significantly higherthan those in water controls and GA treatment alone

Figure 5(e) shows that NaHS treatment maintains lowerlevels of LOX activity compared with water control duringthe first 2 days of GA treatment LOX activity in GAtreatment increases dramatically on day 1 followed by agradual decrease while in NaHS-treated tissue the activityincreases more slowly till day 3 followed by a decline Afterday 3 LOX activity in NaHS plus GA is higher than that inGA treatment

36 H2S Donor Promotes 120572-Amylase Secretion in Barley

Aleurone Layers Regardless of GA Secretion of 120572-amylase is

Oxidative Medicine and Cellular Longevity 7

0

2

4

6

8

10

12

14

C0 C1 T1 C5 T5

ab

d

b

c

020406080

100120140160180

C0 C1 T1 C5 T5

a a

c

b

d

05

10152025303540

C0 C1 T1 C5 T5

a a

bc

d

0102030405060708090

C0 C1 T1 C5 T5

T5

a

b

c

d

e

00005

0010015

0020025

0030035

0040045

C0 C1 T1 C5 T5

ab

b

cd

0

50

100

150

200

250

C0 C1 T1 C5

aa

b

cc

0

200

400

600

C0 C1 T1 C5 T5a ab

c

d

0

4

8

12

C0 C1 T1 C5 T5

aa

b

cd

Relat

ive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

HvSOD1 HvAPX

HvCAT1 HvCAT2

HvLOX

HvEPA HvCP3-31

HvICY

Figure 4 Effects of H2S on the expression of HvSOD1 HvCAT1 HvCAT2 HvLOX cystatin (HvICY) and the cysteine proteases HvCP3-31

andHvEPA in barley aleurone layers Aleurone layers are incubated in NaHS (T) or water (C) and total RNA is obtained at 0 1 and 5 dMeansand SD values are calculated from three independent experiments Within each identified gene bars with different letters are significantlydifferent in comparison with the corresponding control at 119875 lt 001 according to Fisherrsquos least significant differences (LSD)

a characteristic response of aleurone cells toGAWe thereforetest whether the ameliorating effect of H

2S on PCD affects

the release of 120572-amylase As shown in Figure 6(a) NaHS pro-motes 120572-amylase release in water-treated aleurone layers Inthe presence ofGANaHS treatment also enhances120572-amylaserelease frombarley aleurone layers (Figure 6(b)) with 05mMNaHS exhibiting optimal effect Figure 6(c) shows the time

changes in 120572-amylase secretion in water control GA-treatedaleurone layers and GA plus 025mM NaHS treatment Theaccumulation of 120572-amylase in incubation medium in GA-treated layers increases and peaks on day 3 followed by aplateau but addition of 025mM NaHS brings about a morerapid increase till day 4 followed by a decrease on day 5(Figure 6(c)) The activity of 120572-amylase released following

8 Oxidative Medicine and Cellular Longevity

Con

trol

GA

GA+

H2S

24h 48h 72h 96h

(a)

0

20

40

60

80

100

120

24 72 96

Dea

d ce

lls (

)

Treatment time (d)

GA

ControlGA + H2S

(b)

0

50

100

150

200

250

0 1 2 3 4Treatment time (d)

GA

ControlGA + H2S

Con

tent

of

(120583gmiddot

gminus1

FW)

middotO2minus

(c)

40

60

80

100

120

140

160

180

0 1 2 3 4Treatment time (d)

Activ

ity o

f PO

D (U

middotgminus1

FW)

GA

ControlGA + H2S

(d)

GA

0

50

100

150

200

250

300

0 1 2 3 4Treatment time (d)

ControlGA + H2S

Activ

ity o

f LO

X (U

middotgminus1

FW)

(e)

Figure 5 NaHS delays PCD in GA-treated barley aleurone layers Aleurone layers are incubated in water GA or GA + NaHS (GA + H2S)

and after being stained with trypan blue images are obtained by lightmicroscopy (a) and the percentage of dead cells is shown in (b) Contentof sdotO2

minus (c) activity of POD (d) and LOX (e) are measured on 0 1 2 3 and 4 d Bar 100 120583m Data in (c) and (d) are expressed as means plusmn SDof three independent experiments with three replicates of 15 grains per treatment

Oxidative Medicine and Cellular Longevity 9

0 0005 005 025 05

(mM)

(a)

0 0005 005 025 05

(mM)

(b)

0

50

100

150

200

0 1 2 3 4 5Treatment time (d)

GA

ControlGA + H2S

Activ

ity o

f120572-a

myl

ase (

Umiddotgminus1

FW)

(c)

Figure 6 NaHS increases the activity of 120572-amylase in incubation medium of barley aleurone layers in the absence (a) and presence ((b)(c)) of GA Barley aleurone layers isolated from treated embryoless half grains are incubated in 0 0005 005 025 or 05mMNaHS for 24 h((a) (b)) After incubation aleurone layers are placed on agar-starch medium (containing 4 agar and 01 starch) for 16 h Agar plates arestained with I

2-KI solution to detect the activity of 120572-amylase (a) (b) shows native PAGE analysis of 120572-amylase activity in incubationmedium

surrounding the aleurone layers (c) indicates secreted 120572-amylase activity in incubation medium surrounding barley aleurone layers treatedwith water (control) GA or GA + NaHS (GA + H

2S) at different times of incubation Data in (c) are expressed as means plusmn SD of three

independent experiments with three replicates of 20 embryoless half grains per treatment

GA + NaHS treatment is significantly higher than that oflayers incubated only in GA during the whole treatmenttime As expected much lower 120572-amylase activity was onlyobserved inwater control after 3 days of incubation Togetherthis result indicates that H

2S delays PCD in barley aleurone

layers and meanwhile promotes 120572-amylase release regardlessof the presence of GA

4 Discussion

H2S participates in multiple processes in plants [27] In

this paper we show that H2S delays PCD in barley aleu-

rone layers regardless of the presence or absence of GAIn the absence of GA PCD in barley aleurone layers isevident on day 3 of incubation while H

2S delays PCD at

an optimal concentration of 025mM (Figure 1) Consistentwith the reports that GA accelerates PCD process in barley

aleurone layers GA treatment triggers cell death in about90 aleurone cells at 72 h (Figure 5(a)) GA-induced celldeath is slowed by the addition of NaHS and this H

2S

donor also prolongs the phase of 120572-amylase productionin GA-treated layers The promoting effect of NaHS on120572-amylase synthesis and its prolongation of cell survivalindicate that 25mMNaHS does not affect aleurone cell func-tion

ROS such as sdotO2

minus andH2O2 are inducers of PCD in plant

and animal cells [1] It is reported that the peroxidation ofmembrane lipids and damage to the plasma membrane canoccur when the rate of ROS production overcomes the cellsrsquoability for scavenging ROS [27] Overproduction of ROS andoxidative damage are universal events in PCD in plant cells[28] In this paper we show that the content of sdotO

2

minus increasesin parallel with cell death in GA-treated layers (Figure 5(c))In non-GA-treated layers the burst of sdotO

2

minus and H2O2and

10 Oxidative Medicine and Cellular Longevity

the accumulation of MDA are also accompanied by PCD(Figure 2)These results suggest that ROS play a key role bothin GA-treated [29] and in non-GA-treated aleurone layers(Figures 2(a) 2(b) and 5(c))

Aleurone cells contain a suite of ROS-metabolizingenzymes GA-induced PCD in layers is accompanied by adecline in activity of ROS metabolizing enzymes which leadsto increased susceptibility of aleurone cells to ROS [29] Anovel aspect of our work is that NaHS treatment effectivelyreduces the accumulation of ROS in barley aleurone layersregardless the presence of GA (Figures 2 and 5) therebydelaying PCD process in these cells We propose that theH2S donor reduces ROS accumulation in layers by increasing

the activity of ROS-scavenging enzymes The data in presentstudy show that H

2S treatment maintains significantly higher

POD activity in GA-treated layers (Figure 5(d)) and higherSOD POD CAT and APX activity in non-GA-treatedlayers (Figure 3) The increased activity of ROS-scavengingenzymes in NaHS-treated likely promotes the cellrsquos abilityto metabolize ROS In addition LOX activities which areresponsible for lipid peroxidation are downregulated inNaHS-treated aleurone layers at early stage of treatment(Figures 3(e) and 5(e)) Meanwhile quantitative-PCR anal-ysis shows that expression of HvSOD1 HvAPX HvCAT1and HvCAT2 genes in non-GA-treated layers is maintainedat higher levels in NaHS treatment compared with watercontrols (Figure 4) Consistent with lower LOX activity inNaHS-treated aleurone layers the accumulation of LOXtranscripts is also reduced (Figure 4) In summary H

2S slows

down ROS-induced PCD in barley aleurone layers probablyby enhancing the activity and expression of ROS-scavengingenzymes and reducing the peroxidation of membrane lipids

Consistently Xie et al [18] found that H2S delayed GA-

triggered PCD in wheat aleurone layers by increasing GSHcontent and heme oxygenase-1 gene expression Here weprovide evidence thatH

2S can alleviate both natural PCD and

GA-triggered PCD through the modulation of antioxidantenzyme activities and their expression Compared with theslow natural PCDprocess the present study also confirms thepivotal role of GA in triggering PCD (Figure 5)

The role of ROS in GA and ABA signaling in barleyaleurone cells is recently clarified [30] in which they foundthat the production of H

2O2 a type of ROS was induced by

GA in aleurone cells but suppressed by ABA Furthermoreexogenous H

2O2appeared to promote the induction of 120572-

amylases by GA by promoting the expression of GAMyb and120572-amylase genes whereas antioxidants suppressed the induc-tion of 120572-amylase Unexpectedly we found that H

2S reduces

ROS accumulation and delays PCD process in barley aleu-rone layers in the presence or absence of GA and meanwhilepromotes the secretion of 120572-amylase suggesting that theantioxidant H

2S works through an unknown way to regulate

120572-amylase secretion and antioxidants do not always suppressthe induction of 120572-amylase Besides the activation of 120572-amylase by H

2S in the absence of GA implies that 120572-amylase

can be secreted independent of GA signaling pathwayTherefore the present findings advance our knowledge onthe relations between PCD process and 120572-amylase secretion

and the independence of 120572-amylase secretion and GA path-way

The activation of cysteine proteases was instrumental inthe PCD of soybean cells while cystatin an endogenouscysteine protease inhibitor gene inhibited cysteine proteaseactivity and blocked PCD in these cells [7] In this paperwe show that H

2S downregulates the transcriptions of two

barley cysteine proteinases HvEPA and HvCP3-31 in non-GA-treated barley aleurone layers thereby delaying cellcomponent degradation and PCD process

5 Conclusion

In summary we report the role of H2S in delaying PCD in

barley aleurone layers regardless of the presence or absence ofGA without repressing 120572-amylase induction suggesting thatthe function ofH

2Smay be universal in regulating plant PCD

PCD in plant cells is regulated by many internal and externalfactors such as the hormones (GA andABA) Ca2+ ROS andNO [6] It will be interesting to knowwhether H

2S is involved

in other signals and how 120572-amylase is induced by H2S in the

presence or absence of GA in cereal aleurone cells

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ying-Xin Zhang and Kang-Di Hu contributed equally to thiswork

Acknowledgments

Theauthors acknowledge Russell Jones University of Califor-nia at Berkeley for editing the paperThisworkwas supportedby the Natural Science Foundation of China (nos 3127180331301820 31300133 and 31470013) the Scientific ResearchFoundation for Returned Overseas Chinese Scholars (SRFfor ROCS MOE) the Natural Science Foundations of AnhuiProvince (11040606M85) and the Anhui Provincial Educa-tion Department (nos 2012AJZR0028 ZD200910)

References

[1] T Jabs ldquoReactive oxygen intermediates as mediators of pro-grammed cell death in plants and animalsrdquo Biochemical Phar-macology vol 57 no 3 pp 231ndash245 1999

[2] A Fath P Bethke V Beligni and R Jones ldquoActive oxygenand cell death in cereal aleurone cellsrdquo Journal of ExperimentalBotany vol 53 no 372 pp 1273ndash1282 2002

[3] A Fath P Bethke J Lonsdale R Meza-Romero and R JonesldquoProgrammed cell death in cereal aleuronerdquo Plant MolecularBiology vol 44 no 3 pp 255ndash266 2000

[4] P C Bethke J E Lonsdale A Fath and R L Jones ldquoHormon-ally regulated programmed cell death in barley aleurone cellsrdquoThe Plant Cell vol 11 no 16 pp 1033ndash1045 1999

Oxidative Medicine and Cellular Longevity 11

[5] P EHockberger T A Skimina V E Centonze et al ldquoActivationof flavin-containing oxidases underlies light-induced produc-tion of H

2O2in mammalian cellsrdquo Proceedings of the National

Academy of Sciences of the United States of America vol 96 no11 pp 6255ndash6260 1999

[6] P C Bethke and R L Jones ldquoCell death of barley aleuroneprotoplasts is mediated by reactive oxygen speciesrdquo The PlantJournal vol 25 no 1 pp 19ndash29 2001

[7] M Solomon B Belenghi M Delledonne E Menachem andA Levine ldquoThe involvement of cysteine proteases and proteaseinhibitor genes in the regulation of programmed cell death inplantsrdquoThe Plant Cell vol 11 no 3 pp 431ndash443 1999

[8] R Wang ldquoTworsquos company threersquos a crowd can H2S be the third

endogenous gaseous transmitterrdquo The FASEB Journal vol 16no 13 pp 1792ndash1798 2002

[9] E Bloem A Riemenschneider J Volker et al ldquoSulphur supplyand infection with Pyrenopeziza brassicae influence L-cysteinedesulphydrase activity in Brassica napus Lrdquo Journal of Experi-mental Botany vol 55 no 406 pp 2305ndash2312 2004

[10] H Zhang L-Y Hu K-D Hu Y-D He S-H Wang and J-P Luo ldquoHydrogen sulfide promotes wheat seed germinationand alleviates oxidative damage against copper stressrdquo Journalof Integrative Plant Biology vol 50 no 12 pp 1518ndash1529 2008

[11] H Zhang J Tang X-P Liu et al ldquoHydrogen sulfide promotesroot organogenesis in Ipomoea batatas Salix matsudana andGlycine maxrdquo Journal of Integrative Plant Biology vol 51 no 12pp 1086ndash1094 2009

[12] Z P Jin J J Shen Z J Qiao G D Yang R Wang andY X Pei ldquoHydrogen sulfide improves drought resistance inArabidopsis thalianardquo Biochemical and Biophysical ResearchCommunications vol 414 no 3 pp 481ndash486 2011

[13] Z P Jin SWXue YN Luo et al ldquoHydrogen sulfide interactingwith abscisic acid in stomatal regulation responses to droughtstress inArabidopsisrdquoPlant Physiology and Biochemistry vol 62pp 41ndash46 2013

[14] C Garcıa-Mata and L Lamattina ldquoHydrogen sulphide anovel gasotransmitter involved in guard cell signallingrdquo NewPhytologist vol 188 no 4 pp 977ndash984 2010

[15] L-Y Hu S-L Hu J Wu et al ldquoHydrogen sulfide prolongspostharvest shelf life of strawberry and plays an antioxidativerole in fruitsrdquo Journal of Agricultural and Food Chemistry vol60 no 35 pp 8684ndash8693 2012

[16] S-P Gao K-D Hu L-Y Hu et al ldquoHydrogen sulfide delayspostharvest senescence and plays an antioxidative role in fresh-cut kiwifruitrdquo HortScience vol 49 no 10 pp 1385ndash1392 2013

[17] S-P Li K-D Hu L-Y Hu et al ldquoHydrogen sulfide alleviatespostharvest senescence of broccoli by modulating antioxidantdefense and senescence-related gene expressionrdquo Journal ofAgricultural and Food Chemistry vol 62 no 5 pp 1119ndash11292014

[18] Y Xie C Zhang D Lai et al ldquoHydrogen sulfide delays GA-triggered programmed cell death inwheat aleurone layers by themodulation of glutathione homeostasis and heme oxygenase-1expressionrdquo Journal of Plant Physiology vol 171 no 2 pp 53ndash622014

[19] M J Chrispeels and J E Varner ldquoGibberellic acid enhancedsynthesis and release of 120572-amylase and ribonuclease by isolatedbarley and aleurone layersrdquo Plant Physiology vol 42 no 3 pp398ndash406 1967

[20] J R Tennant ldquoEvaluation of the trypan blue technique fordetermination of cell viabilityrdquo Transplantation vol 2 pp 685ndash694 1964

[21] M Tsuchiya M Suematsu and H Suzuki ldquoIn vivo visualiza-tion of oxygen radical-dependent photoemissionrdquo Methods inEnzymology vol 233 pp 128ndash140 1994

[22] R F del Maestro J Bjork and K-E Arfors ldquoIncrease inmicrovascular permeability induced by enzymatically gener-ated free radicals I In vivo studyrdquoMicrovascular Research vol22 no 3 pp 239ndash254 1981

[23] C Garcıa-Limones A Hervas J A Navas-Cortes R MJimenez-Dıaz and M Tena ldquoInduction of an antioxidantenzyme system and other oxidative stress markers associatedwith compatible and incompatible interactions between chick-pea (Cicer arietinum L) and Fusarium oxysporum f sp cicerisrdquoPhysiological and Molecular Plant Pathology vol 61 no 6 pp325ndash337 2002

[24] K Surrey ldquoSpectrophotometric method for determination oflipoxidase activityrdquo Plant Physiology vol 39 no 1 pp 65ndash701964

[25] G G Collins C F Jenner and L G Paleg ldquoThe metabolismof soluble nucleotides in wheat aleurone layers treated withgibberellic acidrdquo Plant P hysiology vol 49 no 3 pp 404ndash4101972

[26] G Noelting and P Bernfeld ldquoSur les enzymes amylolytiqucsIII La 120573-amylase dosage drsquoactivite et controle de lrsquoabsence drsquo120572-amylaserdquo Helvetica Chimica Acta vol 31 no 1 pp 286ndash2901948

[27] M Lisjak T Teklic I D Wilson M Whiteman and J THancock ldquoHydrogen sulfide environmental factor or signallingmoleculerdquo Plant Cell and Environment vol 36 no 9 pp 1607ndash1616 2013

[28] M C de Pinto A Paradiso P Leonetti and L de Gara ldquoHydro-gen peroxide nitric oxide and cytosolic ascorbate peroxidaseat the crossroad between defence and cell deathrdquo The PlantJournal vol 48 no 5 pp 784ndash795 2006

[29] A Fath P C Bethke and R L Jones ldquoEnzymes that scavengereactive oxygen species are down-regulated prior to gibberellicacid-induced programmed cell death in barley aleuronerdquo PlantPhysiology vol 126 no 1 pp 156ndash166 2001

[30] Y Ishibashi T Tawaratsumida K Kondo et al ldquoReactive oxy-gen species are involved in gibberellinabscisic acid signaling inbarley aleurone cellsrdquo Plant Physiology vol 158 no 4 pp 1705ndash1714 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Oxidative Medicine and Cellular Longevity

10

20

30

40

50

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f SO

D (U

middotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(a)

50

90

130

170

210

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f PO

D (U

middotgminus1

FW)

lowastlowast

lowastlowast lowastlowast lowastlowast

lowastlowast

(b)

80

130

180

230

280

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f APX

(Umiddotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(c)

35

55

75

95

115

135

0 2 4Treatment time (d)

ControlH2S

Activ

ity o

f CAT

(Umiddotgminus1

FW)

lowastlowast

lowastlowast

lowastlowast

lowastlowast

lowastlowast

(d)

70

110

150

190

230

0 1 2 3 4 5Treatment time (d)

ControlH2S

Activ

ity o

f LO

X (U

middotgminus1

FW)

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

(e)

Figure 3 Effects of NaHS on the activity of SOD (a) POD (b) APX (c) CAT (d) and LOX (e) in barley aleurone layers Data are expressedas means plusmn SD of three independent experiments with three replicates of 15 grains per treatment The symbols lowast and lowastlowast mean significantdifference at 119875 lt 005 and 119875 lt 001 between control and T respectively

GA-treated layers are dead while half of cells are still alivein NaHS-treated layers (Figure 5(b)) In water control muchless cells undergo PCD compared with the counterpart of GAand GA plus NaHS

Determination of sdotO2

minus content shows that NaHS treat-ment maintains lower levels of sdotO

2

minus in GA-treated barleyaleurone layers (Figure 5(c)) After a rapid increase duringthe first 2 days of incubation the content of sdotO

2

minus in GA-treated layers decreases till day 4 In contrast sdotO

2

minus content inNaHS plus GA treatment increases more slowly until day 3A comparable but lower sdotO

2

minus content was observed in watercontrol compared with NaHS plus GA treatment

Figure 5(d) shows the effect of H2S on POD activity in

GA-treated barley aleurone layers In both NaHS treatmentand GA control POD activity in GA-treated barley aleurone

layers increases gradually up to day 3 and day 2 respectivelyand decreases thereafter However the activity of POD inNaHS-treated aleurone layers is always significantly higherthan those in water controls and GA treatment alone

Figure 5(e) shows that NaHS treatment maintains lowerlevels of LOX activity compared with water control duringthe first 2 days of GA treatment LOX activity in GAtreatment increases dramatically on day 1 followed by agradual decrease while in NaHS-treated tissue the activityincreases more slowly till day 3 followed by a decline Afterday 3 LOX activity in NaHS plus GA is higher than that inGA treatment

36 H2S Donor Promotes 120572-Amylase Secretion in Barley

Aleurone Layers Regardless of GA Secretion of 120572-amylase is

Oxidative Medicine and Cellular Longevity 7

0

2

4

6

8

10

12

14

C0 C1 T1 C5 T5

ab

d

b

c

020406080

100120140160180

C0 C1 T1 C5 T5

a a

c

b

d

05

10152025303540

C0 C1 T1 C5 T5

a a

bc

d

0102030405060708090

C0 C1 T1 C5 T5

T5

a

b

c

d

e

00005

0010015

0020025

0030035

0040045

C0 C1 T1 C5 T5

ab

b

cd

0

50

100

150

200

250

C0 C1 T1 C5

aa

b

cc

0

200

400

600

C0 C1 T1 C5 T5a ab

c

d

0

4

8

12

C0 C1 T1 C5 T5

aa

b

cd

Relat

ive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

HvSOD1 HvAPX

HvCAT1 HvCAT2

HvLOX

HvEPA HvCP3-31

HvICY

Figure 4 Effects of H2S on the expression of HvSOD1 HvCAT1 HvCAT2 HvLOX cystatin (HvICY) and the cysteine proteases HvCP3-31

andHvEPA in barley aleurone layers Aleurone layers are incubated in NaHS (T) or water (C) and total RNA is obtained at 0 1 and 5 dMeansand SD values are calculated from three independent experiments Within each identified gene bars with different letters are significantlydifferent in comparison with the corresponding control at 119875 lt 001 according to Fisherrsquos least significant differences (LSD)

a characteristic response of aleurone cells toGAWe thereforetest whether the ameliorating effect of H

2S on PCD affects

the release of 120572-amylase As shown in Figure 6(a) NaHS pro-motes 120572-amylase release in water-treated aleurone layers Inthe presence ofGANaHS treatment also enhances120572-amylaserelease frombarley aleurone layers (Figure 6(b)) with 05mMNaHS exhibiting optimal effect Figure 6(c) shows the time

changes in 120572-amylase secretion in water control GA-treatedaleurone layers and GA plus 025mM NaHS treatment Theaccumulation of 120572-amylase in incubation medium in GA-treated layers increases and peaks on day 3 followed by aplateau but addition of 025mM NaHS brings about a morerapid increase till day 4 followed by a decrease on day 5(Figure 6(c)) The activity of 120572-amylase released following

8 Oxidative Medicine and Cellular Longevity

Con

trol

GA

GA+

H2S

24h 48h 72h 96h

(a)

0

20

40

60

80

100

120

24 72 96

Dea

d ce

lls (

)

Treatment time (d)

GA

ControlGA + H2S

(b)

0

50

100

150

200

250

0 1 2 3 4Treatment time (d)

GA

ControlGA + H2S

Con

tent

of

(120583gmiddot

gminus1

FW)

middotO2minus

(c)

40

60

80

100

120

140

160

180

0 1 2 3 4Treatment time (d)

Activ

ity o

f PO

D (U

middotgminus1

FW)

GA

ControlGA + H2S

(d)

GA

0

50

100

150

200

250

300

0 1 2 3 4Treatment time (d)

ControlGA + H2S

Activ

ity o

f LO

X (U

middotgminus1

FW)

(e)

Figure 5 NaHS delays PCD in GA-treated barley aleurone layers Aleurone layers are incubated in water GA or GA + NaHS (GA + H2S)

and after being stained with trypan blue images are obtained by lightmicroscopy (a) and the percentage of dead cells is shown in (b) Contentof sdotO2

minus (c) activity of POD (d) and LOX (e) are measured on 0 1 2 3 and 4 d Bar 100 120583m Data in (c) and (d) are expressed as means plusmn SDof three independent experiments with three replicates of 15 grains per treatment

Oxidative Medicine and Cellular Longevity 9

0 0005 005 025 05

(mM)

(a)

0 0005 005 025 05

(mM)

(b)

0

50

100

150

200

0 1 2 3 4 5Treatment time (d)

GA

ControlGA + H2S

Activ

ity o

f120572-a

myl

ase (

Umiddotgminus1

FW)

(c)

Figure 6 NaHS increases the activity of 120572-amylase in incubation medium of barley aleurone layers in the absence (a) and presence ((b)(c)) of GA Barley aleurone layers isolated from treated embryoless half grains are incubated in 0 0005 005 025 or 05mMNaHS for 24 h((a) (b)) After incubation aleurone layers are placed on agar-starch medium (containing 4 agar and 01 starch) for 16 h Agar plates arestained with I

2-KI solution to detect the activity of 120572-amylase (a) (b) shows native PAGE analysis of 120572-amylase activity in incubationmedium

surrounding the aleurone layers (c) indicates secreted 120572-amylase activity in incubation medium surrounding barley aleurone layers treatedwith water (control) GA or GA + NaHS (GA + H

2S) at different times of incubation Data in (c) are expressed as means plusmn SD of three

independent experiments with three replicates of 20 embryoless half grains per treatment

GA + NaHS treatment is significantly higher than that oflayers incubated only in GA during the whole treatmenttime As expected much lower 120572-amylase activity was onlyobserved inwater control after 3 days of incubation Togetherthis result indicates that H

2S delays PCD in barley aleurone

layers and meanwhile promotes 120572-amylase release regardlessof the presence of GA

4 Discussion

H2S participates in multiple processes in plants [27] In

this paper we show that H2S delays PCD in barley aleu-

rone layers regardless of the presence or absence of GAIn the absence of GA PCD in barley aleurone layers isevident on day 3 of incubation while H

2S delays PCD at

an optimal concentration of 025mM (Figure 1) Consistentwith the reports that GA accelerates PCD process in barley

aleurone layers GA treatment triggers cell death in about90 aleurone cells at 72 h (Figure 5(a)) GA-induced celldeath is slowed by the addition of NaHS and this H

2S

donor also prolongs the phase of 120572-amylase productionin GA-treated layers The promoting effect of NaHS on120572-amylase synthesis and its prolongation of cell survivalindicate that 25mMNaHS does not affect aleurone cell func-tion

ROS such as sdotO2

minus andH2O2 are inducers of PCD in plant

and animal cells [1] It is reported that the peroxidation ofmembrane lipids and damage to the plasma membrane canoccur when the rate of ROS production overcomes the cellsrsquoability for scavenging ROS [27] Overproduction of ROS andoxidative damage are universal events in PCD in plant cells[28] In this paper we show that the content of sdotO

2

minus increasesin parallel with cell death in GA-treated layers (Figure 5(c))In non-GA-treated layers the burst of sdotO

2

minus and H2O2and

10 Oxidative Medicine and Cellular Longevity

the accumulation of MDA are also accompanied by PCD(Figure 2)These results suggest that ROS play a key role bothin GA-treated [29] and in non-GA-treated aleurone layers(Figures 2(a) 2(b) and 5(c))

Aleurone cells contain a suite of ROS-metabolizingenzymes GA-induced PCD in layers is accompanied by adecline in activity of ROS metabolizing enzymes which leadsto increased susceptibility of aleurone cells to ROS [29] Anovel aspect of our work is that NaHS treatment effectivelyreduces the accumulation of ROS in barley aleurone layersregardless the presence of GA (Figures 2 and 5) therebydelaying PCD process in these cells We propose that theH2S donor reduces ROS accumulation in layers by increasing

the activity of ROS-scavenging enzymes The data in presentstudy show that H

2S treatment maintains significantly higher

POD activity in GA-treated layers (Figure 5(d)) and higherSOD POD CAT and APX activity in non-GA-treatedlayers (Figure 3) The increased activity of ROS-scavengingenzymes in NaHS-treated likely promotes the cellrsquos abilityto metabolize ROS In addition LOX activities which areresponsible for lipid peroxidation are downregulated inNaHS-treated aleurone layers at early stage of treatment(Figures 3(e) and 5(e)) Meanwhile quantitative-PCR anal-ysis shows that expression of HvSOD1 HvAPX HvCAT1and HvCAT2 genes in non-GA-treated layers is maintainedat higher levels in NaHS treatment compared with watercontrols (Figure 4) Consistent with lower LOX activity inNaHS-treated aleurone layers the accumulation of LOXtranscripts is also reduced (Figure 4) In summary H

2S slows

down ROS-induced PCD in barley aleurone layers probablyby enhancing the activity and expression of ROS-scavengingenzymes and reducing the peroxidation of membrane lipids

Consistently Xie et al [18] found that H2S delayed GA-

triggered PCD in wheat aleurone layers by increasing GSHcontent and heme oxygenase-1 gene expression Here weprovide evidence thatH

2S can alleviate both natural PCD and

GA-triggered PCD through the modulation of antioxidantenzyme activities and their expression Compared with theslow natural PCDprocess the present study also confirms thepivotal role of GA in triggering PCD (Figure 5)

The role of ROS in GA and ABA signaling in barleyaleurone cells is recently clarified [30] in which they foundthat the production of H

2O2 a type of ROS was induced by

GA in aleurone cells but suppressed by ABA Furthermoreexogenous H

2O2appeared to promote the induction of 120572-

amylases by GA by promoting the expression of GAMyb and120572-amylase genes whereas antioxidants suppressed the induc-tion of 120572-amylase Unexpectedly we found that H

2S reduces

ROS accumulation and delays PCD process in barley aleu-rone layers in the presence or absence of GA and meanwhilepromotes the secretion of 120572-amylase suggesting that theantioxidant H

2S works through an unknown way to regulate

120572-amylase secretion and antioxidants do not always suppressthe induction of 120572-amylase Besides the activation of 120572-amylase by H

2S in the absence of GA implies that 120572-amylase

can be secreted independent of GA signaling pathwayTherefore the present findings advance our knowledge onthe relations between PCD process and 120572-amylase secretion

and the independence of 120572-amylase secretion and GA path-way

The activation of cysteine proteases was instrumental inthe PCD of soybean cells while cystatin an endogenouscysteine protease inhibitor gene inhibited cysteine proteaseactivity and blocked PCD in these cells [7] In this paperwe show that H

2S downregulates the transcriptions of two

barley cysteine proteinases HvEPA and HvCP3-31 in non-GA-treated barley aleurone layers thereby delaying cellcomponent degradation and PCD process

5 Conclusion

In summary we report the role of H2S in delaying PCD in

barley aleurone layers regardless of the presence or absence ofGA without repressing 120572-amylase induction suggesting thatthe function ofH

2Smay be universal in regulating plant PCD

PCD in plant cells is regulated by many internal and externalfactors such as the hormones (GA andABA) Ca2+ ROS andNO [6] It will be interesting to knowwhether H

2S is involved

in other signals and how 120572-amylase is induced by H2S in the

presence or absence of GA in cereal aleurone cells

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ying-Xin Zhang and Kang-Di Hu contributed equally to thiswork

Acknowledgments

Theauthors acknowledge Russell Jones University of Califor-nia at Berkeley for editing the paperThisworkwas supportedby the Natural Science Foundation of China (nos 3127180331301820 31300133 and 31470013) the Scientific ResearchFoundation for Returned Overseas Chinese Scholars (SRFfor ROCS MOE) the Natural Science Foundations of AnhuiProvince (11040606M85) and the Anhui Provincial Educa-tion Department (nos 2012AJZR0028 ZD200910)

References

[1] T Jabs ldquoReactive oxygen intermediates as mediators of pro-grammed cell death in plants and animalsrdquo Biochemical Phar-macology vol 57 no 3 pp 231ndash245 1999

[2] A Fath P Bethke V Beligni and R Jones ldquoActive oxygenand cell death in cereal aleurone cellsrdquo Journal of ExperimentalBotany vol 53 no 372 pp 1273ndash1282 2002

[3] A Fath P Bethke J Lonsdale R Meza-Romero and R JonesldquoProgrammed cell death in cereal aleuronerdquo Plant MolecularBiology vol 44 no 3 pp 255ndash266 2000

[4] P C Bethke J E Lonsdale A Fath and R L Jones ldquoHormon-ally regulated programmed cell death in barley aleurone cellsrdquoThe Plant Cell vol 11 no 16 pp 1033ndash1045 1999

Oxidative Medicine and Cellular Longevity 11

[5] P EHockberger T A Skimina V E Centonze et al ldquoActivationof flavin-containing oxidases underlies light-induced produc-tion of H

2O2in mammalian cellsrdquo Proceedings of the National

Academy of Sciences of the United States of America vol 96 no11 pp 6255ndash6260 1999

[6] P C Bethke and R L Jones ldquoCell death of barley aleuroneprotoplasts is mediated by reactive oxygen speciesrdquo The PlantJournal vol 25 no 1 pp 19ndash29 2001

[7] M Solomon B Belenghi M Delledonne E Menachem andA Levine ldquoThe involvement of cysteine proteases and proteaseinhibitor genes in the regulation of programmed cell death inplantsrdquoThe Plant Cell vol 11 no 3 pp 431ndash443 1999

[8] R Wang ldquoTworsquos company threersquos a crowd can H2S be the third

endogenous gaseous transmitterrdquo The FASEB Journal vol 16no 13 pp 1792ndash1798 2002

[9] E Bloem A Riemenschneider J Volker et al ldquoSulphur supplyand infection with Pyrenopeziza brassicae influence L-cysteinedesulphydrase activity in Brassica napus Lrdquo Journal of Experi-mental Botany vol 55 no 406 pp 2305ndash2312 2004

[10] H Zhang L-Y Hu K-D Hu Y-D He S-H Wang and J-P Luo ldquoHydrogen sulfide promotes wheat seed germinationand alleviates oxidative damage against copper stressrdquo Journalof Integrative Plant Biology vol 50 no 12 pp 1518ndash1529 2008

[11] H Zhang J Tang X-P Liu et al ldquoHydrogen sulfide promotesroot organogenesis in Ipomoea batatas Salix matsudana andGlycine maxrdquo Journal of Integrative Plant Biology vol 51 no 12pp 1086ndash1094 2009

[12] Z P Jin J J Shen Z J Qiao G D Yang R Wang andY X Pei ldquoHydrogen sulfide improves drought resistance inArabidopsis thalianardquo Biochemical and Biophysical ResearchCommunications vol 414 no 3 pp 481ndash486 2011

[13] Z P Jin SWXue YN Luo et al ldquoHydrogen sulfide interactingwith abscisic acid in stomatal regulation responses to droughtstress inArabidopsisrdquoPlant Physiology and Biochemistry vol 62pp 41ndash46 2013

[14] C Garcıa-Mata and L Lamattina ldquoHydrogen sulphide anovel gasotransmitter involved in guard cell signallingrdquo NewPhytologist vol 188 no 4 pp 977ndash984 2010

[15] L-Y Hu S-L Hu J Wu et al ldquoHydrogen sulfide prolongspostharvest shelf life of strawberry and plays an antioxidativerole in fruitsrdquo Journal of Agricultural and Food Chemistry vol60 no 35 pp 8684ndash8693 2012

[16] S-P Gao K-D Hu L-Y Hu et al ldquoHydrogen sulfide delayspostharvest senescence and plays an antioxidative role in fresh-cut kiwifruitrdquo HortScience vol 49 no 10 pp 1385ndash1392 2013

[17] S-P Li K-D Hu L-Y Hu et al ldquoHydrogen sulfide alleviatespostharvest senescence of broccoli by modulating antioxidantdefense and senescence-related gene expressionrdquo Journal ofAgricultural and Food Chemistry vol 62 no 5 pp 1119ndash11292014

[18] Y Xie C Zhang D Lai et al ldquoHydrogen sulfide delays GA-triggered programmed cell death inwheat aleurone layers by themodulation of glutathione homeostasis and heme oxygenase-1expressionrdquo Journal of Plant Physiology vol 171 no 2 pp 53ndash622014

[19] M J Chrispeels and J E Varner ldquoGibberellic acid enhancedsynthesis and release of 120572-amylase and ribonuclease by isolatedbarley and aleurone layersrdquo Plant Physiology vol 42 no 3 pp398ndash406 1967

[20] J R Tennant ldquoEvaluation of the trypan blue technique fordetermination of cell viabilityrdquo Transplantation vol 2 pp 685ndash694 1964

[21] M Tsuchiya M Suematsu and H Suzuki ldquoIn vivo visualiza-tion of oxygen radical-dependent photoemissionrdquo Methods inEnzymology vol 233 pp 128ndash140 1994

[22] R F del Maestro J Bjork and K-E Arfors ldquoIncrease inmicrovascular permeability induced by enzymatically gener-ated free radicals I In vivo studyrdquoMicrovascular Research vol22 no 3 pp 239ndash254 1981

[23] C Garcıa-Limones A Hervas J A Navas-Cortes R MJimenez-Dıaz and M Tena ldquoInduction of an antioxidantenzyme system and other oxidative stress markers associatedwith compatible and incompatible interactions between chick-pea (Cicer arietinum L) and Fusarium oxysporum f sp cicerisrdquoPhysiological and Molecular Plant Pathology vol 61 no 6 pp325ndash337 2002

[24] K Surrey ldquoSpectrophotometric method for determination oflipoxidase activityrdquo Plant Physiology vol 39 no 1 pp 65ndash701964

[25] G G Collins C F Jenner and L G Paleg ldquoThe metabolismof soluble nucleotides in wheat aleurone layers treated withgibberellic acidrdquo Plant P hysiology vol 49 no 3 pp 404ndash4101972

[26] G Noelting and P Bernfeld ldquoSur les enzymes amylolytiqucsIII La 120573-amylase dosage drsquoactivite et controle de lrsquoabsence drsquo120572-amylaserdquo Helvetica Chimica Acta vol 31 no 1 pp 286ndash2901948

[27] M Lisjak T Teklic I D Wilson M Whiteman and J THancock ldquoHydrogen sulfide environmental factor or signallingmoleculerdquo Plant Cell and Environment vol 36 no 9 pp 1607ndash1616 2013

[28] M C de Pinto A Paradiso P Leonetti and L de Gara ldquoHydro-gen peroxide nitric oxide and cytosolic ascorbate peroxidaseat the crossroad between defence and cell deathrdquo The PlantJournal vol 48 no 5 pp 784ndash795 2006

[29] A Fath P C Bethke and R L Jones ldquoEnzymes that scavengereactive oxygen species are down-regulated prior to gibberellicacid-induced programmed cell death in barley aleuronerdquo PlantPhysiology vol 126 no 1 pp 156ndash166 2001

[30] Y Ishibashi T Tawaratsumida K Kondo et al ldquoReactive oxy-gen species are involved in gibberellinabscisic acid signaling inbarley aleurone cellsrdquo Plant Physiology vol 158 no 4 pp 1705ndash1714 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 7

0

2

4

6

8

10

12

14

C0 C1 T1 C5 T5

ab

d

b

c

020406080

100120140160180

C0 C1 T1 C5 T5

a a

c

b

d

05

10152025303540

C0 C1 T1 C5 T5

a a

bc

d

0102030405060708090

C0 C1 T1 C5 T5

T5

a

b

c

d

e

00005

0010015

0020025

0030035

0040045

C0 C1 T1 C5 T5

ab

b

cd

0

50

100

150

200

250

C0 C1 T1 C5

aa

b

cc

0

200

400

600

C0 C1 T1 C5 T5a ab

c

d

0

4

8

12

C0 C1 T1 C5 T5

aa

b

cd

Relat

ive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

Rela

tive e

xpre

ssio

nRe

lativ

e exp

ress

ion

HvSOD1 HvAPX

HvCAT1 HvCAT2

HvLOX

HvEPA HvCP3-31

HvICY

Figure 4 Effects of H2S on the expression of HvSOD1 HvCAT1 HvCAT2 HvLOX cystatin (HvICY) and the cysteine proteases HvCP3-31

andHvEPA in barley aleurone layers Aleurone layers are incubated in NaHS (T) or water (C) and total RNA is obtained at 0 1 and 5 dMeansand SD values are calculated from three independent experiments Within each identified gene bars with different letters are significantlydifferent in comparison with the corresponding control at 119875 lt 001 according to Fisherrsquos least significant differences (LSD)

a characteristic response of aleurone cells toGAWe thereforetest whether the ameliorating effect of H

2S on PCD affects

the release of 120572-amylase As shown in Figure 6(a) NaHS pro-motes 120572-amylase release in water-treated aleurone layers Inthe presence ofGANaHS treatment also enhances120572-amylaserelease frombarley aleurone layers (Figure 6(b)) with 05mMNaHS exhibiting optimal effect Figure 6(c) shows the time

changes in 120572-amylase secretion in water control GA-treatedaleurone layers and GA plus 025mM NaHS treatment Theaccumulation of 120572-amylase in incubation medium in GA-treated layers increases and peaks on day 3 followed by aplateau but addition of 025mM NaHS brings about a morerapid increase till day 4 followed by a decrease on day 5(Figure 6(c)) The activity of 120572-amylase released following

8 Oxidative Medicine and Cellular Longevity

Con

trol

GA

GA+

H2S

24h 48h 72h 96h

(a)

0

20

40

60

80

100

120

24 72 96

Dea

d ce

lls (

)

Treatment time (d)

GA

ControlGA + H2S

(b)

0

50

100

150

200

250

0 1 2 3 4Treatment time (d)

GA

ControlGA + H2S

Con

tent

of

(120583gmiddot

gminus1

FW)

middotO2minus

(c)

40

60

80

100

120

140

160

180

0 1 2 3 4Treatment time (d)

Activ

ity o

f PO

D (U

middotgminus1

FW)

GA

ControlGA + H2S

(d)

GA

0

50

100

150

200

250

300

0 1 2 3 4Treatment time (d)

ControlGA + H2S

Activ

ity o

f LO

X (U

middotgminus1

FW)

(e)

Figure 5 NaHS delays PCD in GA-treated barley aleurone layers Aleurone layers are incubated in water GA or GA + NaHS (GA + H2S)

and after being stained with trypan blue images are obtained by lightmicroscopy (a) and the percentage of dead cells is shown in (b) Contentof sdotO2

minus (c) activity of POD (d) and LOX (e) are measured on 0 1 2 3 and 4 d Bar 100 120583m Data in (c) and (d) are expressed as means plusmn SDof three independent experiments with three replicates of 15 grains per treatment

Oxidative Medicine and Cellular Longevity 9

0 0005 005 025 05

(mM)

(a)

0 0005 005 025 05

(mM)

(b)

0

50

100

150

200

0 1 2 3 4 5Treatment time (d)

GA

ControlGA + H2S

Activ

ity o

f120572-a

myl

ase (

Umiddotgminus1

FW)

(c)

Figure 6 NaHS increases the activity of 120572-amylase in incubation medium of barley aleurone layers in the absence (a) and presence ((b)(c)) of GA Barley aleurone layers isolated from treated embryoless half grains are incubated in 0 0005 005 025 or 05mMNaHS for 24 h((a) (b)) After incubation aleurone layers are placed on agar-starch medium (containing 4 agar and 01 starch) for 16 h Agar plates arestained with I

2-KI solution to detect the activity of 120572-amylase (a) (b) shows native PAGE analysis of 120572-amylase activity in incubationmedium

surrounding the aleurone layers (c) indicates secreted 120572-amylase activity in incubation medium surrounding barley aleurone layers treatedwith water (control) GA or GA + NaHS (GA + H

2S) at different times of incubation Data in (c) are expressed as means plusmn SD of three

independent experiments with three replicates of 20 embryoless half grains per treatment

GA + NaHS treatment is significantly higher than that oflayers incubated only in GA during the whole treatmenttime As expected much lower 120572-amylase activity was onlyobserved inwater control after 3 days of incubation Togetherthis result indicates that H

2S delays PCD in barley aleurone

layers and meanwhile promotes 120572-amylase release regardlessof the presence of GA

4 Discussion

H2S participates in multiple processes in plants [27] In

this paper we show that H2S delays PCD in barley aleu-

rone layers regardless of the presence or absence of GAIn the absence of GA PCD in barley aleurone layers isevident on day 3 of incubation while H

2S delays PCD at

an optimal concentration of 025mM (Figure 1) Consistentwith the reports that GA accelerates PCD process in barley

aleurone layers GA treatment triggers cell death in about90 aleurone cells at 72 h (Figure 5(a)) GA-induced celldeath is slowed by the addition of NaHS and this H

2S

donor also prolongs the phase of 120572-amylase productionin GA-treated layers The promoting effect of NaHS on120572-amylase synthesis and its prolongation of cell survivalindicate that 25mMNaHS does not affect aleurone cell func-tion

ROS such as sdotO2

minus andH2O2 are inducers of PCD in plant

and animal cells [1] It is reported that the peroxidation ofmembrane lipids and damage to the plasma membrane canoccur when the rate of ROS production overcomes the cellsrsquoability for scavenging ROS [27] Overproduction of ROS andoxidative damage are universal events in PCD in plant cells[28] In this paper we show that the content of sdotO

2

minus increasesin parallel with cell death in GA-treated layers (Figure 5(c))In non-GA-treated layers the burst of sdotO

2

minus and H2O2and

10 Oxidative Medicine and Cellular Longevity

the accumulation of MDA are also accompanied by PCD(Figure 2)These results suggest that ROS play a key role bothin GA-treated [29] and in non-GA-treated aleurone layers(Figures 2(a) 2(b) and 5(c))

Aleurone cells contain a suite of ROS-metabolizingenzymes GA-induced PCD in layers is accompanied by adecline in activity of ROS metabolizing enzymes which leadsto increased susceptibility of aleurone cells to ROS [29] Anovel aspect of our work is that NaHS treatment effectivelyreduces the accumulation of ROS in barley aleurone layersregardless the presence of GA (Figures 2 and 5) therebydelaying PCD process in these cells We propose that theH2S donor reduces ROS accumulation in layers by increasing

the activity of ROS-scavenging enzymes The data in presentstudy show that H

2S treatment maintains significantly higher

POD activity in GA-treated layers (Figure 5(d)) and higherSOD POD CAT and APX activity in non-GA-treatedlayers (Figure 3) The increased activity of ROS-scavengingenzymes in NaHS-treated likely promotes the cellrsquos abilityto metabolize ROS In addition LOX activities which areresponsible for lipid peroxidation are downregulated inNaHS-treated aleurone layers at early stage of treatment(Figures 3(e) and 5(e)) Meanwhile quantitative-PCR anal-ysis shows that expression of HvSOD1 HvAPX HvCAT1and HvCAT2 genes in non-GA-treated layers is maintainedat higher levels in NaHS treatment compared with watercontrols (Figure 4) Consistent with lower LOX activity inNaHS-treated aleurone layers the accumulation of LOXtranscripts is also reduced (Figure 4) In summary H

2S slows

down ROS-induced PCD in barley aleurone layers probablyby enhancing the activity and expression of ROS-scavengingenzymes and reducing the peroxidation of membrane lipids

Consistently Xie et al [18] found that H2S delayed GA-

triggered PCD in wheat aleurone layers by increasing GSHcontent and heme oxygenase-1 gene expression Here weprovide evidence thatH

2S can alleviate both natural PCD and

GA-triggered PCD through the modulation of antioxidantenzyme activities and their expression Compared with theslow natural PCDprocess the present study also confirms thepivotal role of GA in triggering PCD (Figure 5)

The role of ROS in GA and ABA signaling in barleyaleurone cells is recently clarified [30] in which they foundthat the production of H

2O2 a type of ROS was induced by

GA in aleurone cells but suppressed by ABA Furthermoreexogenous H

2O2appeared to promote the induction of 120572-

amylases by GA by promoting the expression of GAMyb and120572-amylase genes whereas antioxidants suppressed the induc-tion of 120572-amylase Unexpectedly we found that H

2S reduces

ROS accumulation and delays PCD process in barley aleu-rone layers in the presence or absence of GA and meanwhilepromotes the secretion of 120572-amylase suggesting that theantioxidant H

2S works through an unknown way to regulate

120572-amylase secretion and antioxidants do not always suppressthe induction of 120572-amylase Besides the activation of 120572-amylase by H

2S in the absence of GA implies that 120572-amylase

can be secreted independent of GA signaling pathwayTherefore the present findings advance our knowledge onthe relations between PCD process and 120572-amylase secretion

and the independence of 120572-amylase secretion and GA path-way

The activation of cysteine proteases was instrumental inthe PCD of soybean cells while cystatin an endogenouscysteine protease inhibitor gene inhibited cysteine proteaseactivity and blocked PCD in these cells [7] In this paperwe show that H

2S downregulates the transcriptions of two

barley cysteine proteinases HvEPA and HvCP3-31 in non-GA-treated barley aleurone layers thereby delaying cellcomponent degradation and PCD process

5 Conclusion

In summary we report the role of H2S in delaying PCD in

barley aleurone layers regardless of the presence or absence ofGA without repressing 120572-amylase induction suggesting thatthe function ofH

2Smay be universal in regulating plant PCD

PCD in plant cells is regulated by many internal and externalfactors such as the hormones (GA andABA) Ca2+ ROS andNO [6] It will be interesting to knowwhether H

2S is involved

in other signals and how 120572-amylase is induced by H2S in the

presence or absence of GA in cereal aleurone cells

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ying-Xin Zhang and Kang-Di Hu contributed equally to thiswork

Acknowledgments

Theauthors acknowledge Russell Jones University of Califor-nia at Berkeley for editing the paperThisworkwas supportedby the Natural Science Foundation of China (nos 3127180331301820 31300133 and 31470013) the Scientific ResearchFoundation for Returned Overseas Chinese Scholars (SRFfor ROCS MOE) the Natural Science Foundations of AnhuiProvince (11040606M85) and the Anhui Provincial Educa-tion Department (nos 2012AJZR0028 ZD200910)

References

[1] T Jabs ldquoReactive oxygen intermediates as mediators of pro-grammed cell death in plants and animalsrdquo Biochemical Phar-macology vol 57 no 3 pp 231ndash245 1999

[2] A Fath P Bethke V Beligni and R Jones ldquoActive oxygenand cell death in cereal aleurone cellsrdquo Journal of ExperimentalBotany vol 53 no 372 pp 1273ndash1282 2002

[3] A Fath P Bethke J Lonsdale R Meza-Romero and R JonesldquoProgrammed cell death in cereal aleuronerdquo Plant MolecularBiology vol 44 no 3 pp 255ndash266 2000

[4] P C Bethke J E Lonsdale A Fath and R L Jones ldquoHormon-ally regulated programmed cell death in barley aleurone cellsrdquoThe Plant Cell vol 11 no 16 pp 1033ndash1045 1999

Oxidative Medicine and Cellular Longevity 11

[5] P EHockberger T A Skimina V E Centonze et al ldquoActivationof flavin-containing oxidases underlies light-induced produc-tion of H

2O2in mammalian cellsrdquo Proceedings of the National

Academy of Sciences of the United States of America vol 96 no11 pp 6255ndash6260 1999

[6] P C Bethke and R L Jones ldquoCell death of barley aleuroneprotoplasts is mediated by reactive oxygen speciesrdquo The PlantJournal vol 25 no 1 pp 19ndash29 2001

[7] M Solomon B Belenghi M Delledonne E Menachem andA Levine ldquoThe involvement of cysteine proteases and proteaseinhibitor genes in the regulation of programmed cell death inplantsrdquoThe Plant Cell vol 11 no 3 pp 431ndash443 1999

[8] R Wang ldquoTworsquos company threersquos a crowd can H2S be the third

endogenous gaseous transmitterrdquo The FASEB Journal vol 16no 13 pp 1792ndash1798 2002

[9] E Bloem A Riemenschneider J Volker et al ldquoSulphur supplyand infection with Pyrenopeziza brassicae influence L-cysteinedesulphydrase activity in Brassica napus Lrdquo Journal of Experi-mental Botany vol 55 no 406 pp 2305ndash2312 2004

[10] H Zhang L-Y Hu K-D Hu Y-D He S-H Wang and J-P Luo ldquoHydrogen sulfide promotes wheat seed germinationand alleviates oxidative damage against copper stressrdquo Journalof Integrative Plant Biology vol 50 no 12 pp 1518ndash1529 2008

[11] H Zhang J Tang X-P Liu et al ldquoHydrogen sulfide promotesroot organogenesis in Ipomoea batatas Salix matsudana andGlycine maxrdquo Journal of Integrative Plant Biology vol 51 no 12pp 1086ndash1094 2009

[12] Z P Jin J J Shen Z J Qiao G D Yang R Wang andY X Pei ldquoHydrogen sulfide improves drought resistance inArabidopsis thalianardquo Biochemical and Biophysical ResearchCommunications vol 414 no 3 pp 481ndash486 2011

[13] Z P Jin SWXue YN Luo et al ldquoHydrogen sulfide interactingwith abscisic acid in stomatal regulation responses to droughtstress inArabidopsisrdquoPlant Physiology and Biochemistry vol 62pp 41ndash46 2013

[14] C Garcıa-Mata and L Lamattina ldquoHydrogen sulphide anovel gasotransmitter involved in guard cell signallingrdquo NewPhytologist vol 188 no 4 pp 977ndash984 2010

[15] L-Y Hu S-L Hu J Wu et al ldquoHydrogen sulfide prolongspostharvest shelf life of strawberry and plays an antioxidativerole in fruitsrdquo Journal of Agricultural and Food Chemistry vol60 no 35 pp 8684ndash8693 2012

[16] S-P Gao K-D Hu L-Y Hu et al ldquoHydrogen sulfide delayspostharvest senescence and plays an antioxidative role in fresh-cut kiwifruitrdquo HortScience vol 49 no 10 pp 1385ndash1392 2013

[17] S-P Li K-D Hu L-Y Hu et al ldquoHydrogen sulfide alleviatespostharvest senescence of broccoli by modulating antioxidantdefense and senescence-related gene expressionrdquo Journal ofAgricultural and Food Chemistry vol 62 no 5 pp 1119ndash11292014

[18] Y Xie C Zhang D Lai et al ldquoHydrogen sulfide delays GA-triggered programmed cell death inwheat aleurone layers by themodulation of glutathione homeostasis and heme oxygenase-1expressionrdquo Journal of Plant Physiology vol 171 no 2 pp 53ndash622014

[19] M J Chrispeels and J E Varner ldquoGibberellic acid enhancedsynthesis and release of 120572-amylase and ribonuclease by isolatedbarley and aleurone layersrdquo Plant Physiology vol 42 no 3 pp398ndash406 1967

[20] J R Tennant ldquoEvaluation of the trypan blue technique fordetermination of cell viabilityrdquo Transplantation vol 2 pp 685ndash694 1964

[21] M Tsuchiya M Suematsu and H Suzuki ldquoIn vivo visualiza-tion of oxygen radical-dependent photoemissionrdquo Methods inEnzymology vol 233 pp 128ndash140 1994

[22] R F del Maestro J Bjork and K-E Arfors ldquoIncrease inmicrovascular permeability induced by enzymatically gener-ated free radicals I In vivo studyrdquoMicrovascular Research vol22 no 3 pp 239ndash254 1981

[23] C Garcıa-Limones A Hervas J A Navas-Cortes R MJimenez-Dıaz and M Tena ldquoInduction of an antioxidantenzyme system and other oxidative stress markers associatedwith compatible and incompatible interactions between chick-pea (Cicer arietinum L) and Fusarium oxysporum f sp cicerisrdquoPhysiological and Molecular Plant Pathology vol 61 no 6 pp325ndash337 2002

[24] K Surrey ldquoSpectrophotometric method for determination oflipoxidase activityrdquo Plant Physiology vol 39 no 1 pp 65ndash701964

[25] G G Collins C F Jenner and L G Paleg ldquoThe metabolismof soluble nucleotides in wheat aleurone layers treated withgibberellic acidrdquo Plant P hysiology vol 49 no 3 pp 404ndash4101972

[26] G Noelting and P Bernfeld ldquoSur les enzymes amylolytiqucsIII La 120573-amylase dosage drsquoactivite et controle de lrsquoabsence drsquo120572-amylaserdquo Helvetica Chimica Acta vol 31 no 1 pp 286ndash2901948

[27] M Lisjak T Teklic I D Wilson M Whiteman and J THancock ldquoHydrogen sulfide environmental factor or signallingmoleculerdquo Plant Cell and Environment vol 36 no 9 pp 1607ndash1616 2013

[28] M C de Pinto A Paradiso P Leonetti and L de Gara ldquoHydro-gen peroxide nitric oxide and cytosolic ascorbate peroxidaseat the crossroad between defence and cell deathrdquo The PlantJournal vol 48 no 5 pp 784ndash795 2006

[29] A Fath P C Bethke and R L Jones ldquoEnzymes that scavengereactive oxygen species are down-regulated prior to gibberellicacid-induced programmed cell death in barley aleuronerdquo PlantPhysiology vol 126 no 1 pp 156ndash166 2001

[30] Y Ishibashi T Tawaratsumida K Kondo et al ldquoReactive oxy-gen species are involved in gibberellinabscisic acid signaling inbarley aleurone cellsrdquo Plant Physiology vol 158 no 4 pp 1705ndash1714 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Oxidative Medicine and Cellular Longevity

Con

trol

GA

GA+

H2S

24h 48h 72h 96h

(a)

0

20

40

60

80

100

120

24 72 96

Dea

d ce

lls (

)

Treatment time (d)

GA

ControlGA + H2S

(b)

0

50

100

150

200

250

0 1 2 3 4Treatment time (d)

GA

ControlGA + H2S

Con

tent

of

(120583gmiddot

gminus1

FW)

middotO2minus

(c)

40

60

80

100

120

140

160

180

0 1 2 3 4Treatment time (d)

Activ

ity o

f PO

D (U

middotgminus1

FW)

GA

ControlGA + H2S

(d)

GA

0

50

100

150

200

250

300

0 1 2 3 4Treatment time (d)

ControlGA + H2S

Activ

ity o

f LO

X (U

middotgminus1

FW)

(e)

Figure 5 NaHS delays PCD in GA-treated barley aleurone layers Aleurone layers are incubated in water GA or GA + NaHS (GA + H2S)

and after being stained with trypan blue images are obtained by lightmicroscopy (a) and the percentage of dead cells is shown in (b) Contentof sdotO2

minus (c) activity of POD (d) and LOX (e) are measured on 0 1 2 3 and 4 d Bar 100 120583m Data in (c) and (d) are expressed as means plusmn SDof three independent experiments with three replicates of 15 grains per treatment

Oxidative Medicine and Cellular Longevity 9

0 0005 005 025 05

(mM)

(a)

0 0005 005 025 05

(mM)

(b)

0

50

100

150

200

0 1 2 3 4 5Treatment time (d)

GA

ControlGA + H2S

Activ

ity o

f120572-a

myl

ase (

Umiddotgminus1

FW)

(c)

Figure 6 NaHS increases the activity of 120572-amylase in incubation medium of barley aleurone layers in the absence (a) and presence ((b)(c)) of GA Barley aleurone layers isolated from treated embryoless half grains are incubated in 0 0005 005 025 or 05mMNaHS for 24 h((a) (b)) After incubation aleurone layers are placed on agar-starch medium (containing 4 agar and 01 starch) for 16 h Agar plates arestained with I

2-KI solution to detect the activity of 120572-amylase (a) (b) shows native PAGE analysis of 120572-amylase activity in incubationmedium

surrounding the aleurone layers (c) indicates secreted 120572-amylase activity in incubation medium surrounding barley aleurone layers treatedwith water (control) GA or GA + NaHS (GA + H

2S) at different times of incubation Data in (c) are expressed as means plusmn SD of three

independent experiments with three replicates of 20 embryoless half grains per treatment

GA + NaHS treatment is significantly higher than that oflayers incubated only in GA during the whole treatmenttime As expected much lower 120572-amylase activity was onlyobserved inwater control after 3 days of incubation Togetherthis result indicates that H

2S delays PCD in barley aleurone

layers and meanwhile promotes 120572-amylase release regardlessof the presence of GA

4 Discussion

H2S participates in multiple processes in plants [27] In

this paper we show that H2S delays PCD in barley aleu-

rone layers regardless of the presence or absence of GAIn the absence of GA PCD in barley aleurone layers isevident on day 3 of incubation while H

2S delays PCD at

an optimal concentration of 025mM (Figure 1) Consistentwith the reports that GA accelerates PCD process in barley

aleurone layers GA treatment triggers cell death in about90 aleurone cells at 72 h (Figure 5(a)) GA-induced celldeath is slowed by the addition of NaHS and this H

2S

donor also prolongs the phase of 120572-amylase productionin GA-treated layers The promoting effect of NaHS on120572-amylase synthesis and its prolongation of cell survivalindicate that 25mMNaHS does not affect aleurone cell func-tion

ROS such as sdotO2

minus andH2O2 are inducers of PCD in plant

and animal cells [1] It is reported that the peroxidation ofmembrane lipids and damage to the plasma membrane canoccur when the rate of ROS production overcomes the cellsrsquoability for scavenging ROS [27] Overproduction of ROS andoxidative damage are universal events in PCD in plant cells[28] In this paper we show that the content of sdotO

2

minus increasesin parallel with cell death in GA-treated layers (Figure 5(c))In non-GA-treated layers the burst of sdotO

2

minus and H2O2and

10 Oxidative Medicine and Cellular Longevity

the accumulation of MDA are also accompanied by PCD(Figure 2)These results suggest that ROS play a key role bothin GA-treated [29] and in non-GA-treated aleurone layers(Figures 2(a) 2(b) and 5(c))

Aleurone cells contain a suite of ROS-metabolizingenzymes GA-induced PCD in layers is accompanied by adecline in activity of ROS metabolizing enzymes which leadsto increased susceptibility of aleurone cells to ROS [29] Anovel aspect of our work is that NaHS treatment effectivelyreduces the accumulation of ROS in barley aleurone layersregardless the presence of GA (Figures 2 and 5) therebydelaying PCD process in these cells We propose that theH2S donor reduces ROS accumulation in layers by increasing

the activity of ROS-scavenging enzymes The data in presentstudy show that H

2S treatment maintains significantly higher

POD activity in GA-treated layers (Figure 5(d)) and higherSOD POD CAT and APX activity in non-GA-treatedlayers (Figure 3) The increased activity of ROS-scavengingenzymes in NaHS-treated likely promotes the cellrsquos abilityto metabolize ROS In addition LOX activities which areresponsible for lipid peroxidation are downregulated inNaHS-treated aleurone layers at early stage of treatment(Figures 3(e) and 5(e)) Meanwhile quantitative-PCR anal-ysis shows that expression of HvSOD1 HvAPX HvCAT1and HvCAT2 genes in non-GA-treated layers is maintainedat higher levels in NaHS treatment compared with watercontrols (Figure 4) Consistent with lower LOX activity inNaHS-treated aleurone layers the accumulation of LOXtranscripts is also reduced (Figure 4) In summary H

2S slows

down ROS-induced PCD in barley aleurone layers probablyby enhancing the activity and expression of ROS-scavengingenzymes and reducing the peroxidation of membrane lipids

Consistently Xie et al [18] found that H2S delayed GA-

triggered PCD in wheat aleurone layers by increasing GSHcontent and heme oxygenase-1 gene expression Here weprovide evidence thatH

2S can alleviate both natural PCD and

GA-triggered PCD through the modulation of antioxidantenzyme activities and their expression Compared with theslow natural PCDprocess the present study also confirms thepivotal role of GA in triggering PCD (Figure 5)

The role of ROS in GA and ABA signaling in barleyaleurone cells is recently clarified [30] in which they foundthat the production of H

2O2 a type of ROS was induced by

GA in aleurone cells but suppressed by ABA Furthermoreexogenous H

2O2appeared to promote the induction of 120572-

amylases by GA by promoting the expression of GAMyb and120572-amylase genes whereas antioxidants suppressed the induc-tion of 120572-amylase Unexpectedly we found that H

2S reduces

ROS accumulation and delays PCD process in barley aleu-rone layers in the presence or absence of GA and meanwhilepromotes the secretion of 120572-amylase suggesting that theantioxidant H

2S works through an unknown way to regulate

120572-amylase secretion and antioxidants do not always suppressthe induction of 120572-amylase Besides the activation of 120572-amylase by H

2S in the absence of GA implies that 120572-amylase

can be secreted independent of GA signaling pathwayTherefore the present findings advance our knowledge onthe relations between PCD process and 120572-amylase secretion

and the independence of 120572-amylase secretion and GA path-way

The activation of cysteine proteases was instrumental inthe PCD of soybean cells while cystatin an endogenouscysteine protease inhibitor gene inhibited cysteine proteaseactivity and blocked PCD in these cells [7] In this paperwe show that H

2S downregulates the transcriptions of two

barley cysteine proteinases HvEPA and HvCP3-31 in non-GA-treated barley aleurone layers thereby delaying cellcomponent degradation and PCD process

5 Conclusion

In summary we report the role of H2S in delaying PCD in

barley aleurone layers regardless of the presence or absence ofGA without repressing 120572-amylase induction suggesting thatthe function ofH

2Smay be universal in regulating plant PCD

PCD in plant cells is regulated by many internal and externalfactors such as the hormones (GA andABA) Ca2+ ROS andNO [6] It will be interesting to knowwhether H

2S is involved

in other signals and how 120572-amylase is induced by H2S in the

presence or absence of GA in cereal aleurone cells

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ying-Xin Zhang and Kang-Di Hu contributed equally to thiswork

Acknowledgments

Theauthors acknowledge Russell Jones University of Califor-nia at Berkeley for editing the paperThisworkwas supportedby the Natural Science Foundation of China (nos 3127180331301820 31300133 and 31470013) the Scientific ResearchFoundation for Returned Overseas Chinese Scholars (SRFfor ROCS MOE) the Natural Science Foundations of AnhuiProvince (11040606M85) and the Anhui Provincial Educa-tion Department (nos 2012AJZR0028 ZD200910)

References

[1] T Jabs ldquoReactive oxygen intermediates as mediators of pro-grammed cell death in plants and animalsrdquo Biochemical Phar-macology vol 57 no 3 pp 231ndash245 1999

[2] A Fath P Bethke V Beligni and R Jones ldquoActive oxygenand cell death in cereal aleurone cellsrdquo Journal of ExperimentalBotany vol 53 no 372 pp 1273ndash1282 2002

[3] A Fath P Bethke J Lonsdale R Meza-Romero and R JonesldquoProgrammed cell death in cereal aleuronerdquo Plant MolecularBiology vol 44 no 3 pp 255ndash266 2000

[4] P C Bethke J E Lonsdale A Fath and R L Jones ldquoHormon-ally regulated programmed cell death in barley aleurone cellsrdquoThe Plant Cell vol 11 no 16 pp 1033ndash1045 1999

Oxidative Medicine and Cellular Longevity 11

[5] P EHockberger T A Skimina V E Centonze et al ldquoActivationof flavin-containing oxidases underlies light-induced produc-tion of H

2O2in mammalian cellsrdquo Proceedings of the National

Academy of Sciences of the United States of America vol 96 no11 pp 6255ndash6260 1999

[6] P C Bethke and R L Jones ldquoCell death of barley aleuroneprotoplasts is mediated by reactive oxygen speciesrdquo The PlantJournal vol 25 no 1 pp 19ndash29 2001

[7] M Solomon B Belenghi M Delledonne E Menachem andA Levine ldquoThe involvement of cysteine proteases and proteaseinhibitor genes in the regulation of programmed cell death inplantsrdquoThe Plant Cell vol 11 no 3 pp 431ndash443 1999

[8] R Wang ldquoTworsquos company threersquos a crowd can H2S be the third

endogenous gaseous transmitterrdquo The FASEB Journal vol 16no 13 pp 1792ndash1798 2002

[9] E Bloem A Riemenschneider J Volker et al ldquoSulphur supplyand infection with Pyrenopeziza brassicae influence L-cysteinedesulphydrase activity in Brassica napus Lrdquo Journal of Experi-mental Botany vol 55 no 406 pp 2305ndash2312 2004

[10] H Zhang L-Y Hu K-D Hu Y-D He S-H Wang and J-P Luo ldquoHydrogen sulfide promotes wheat seed germinationand alleviates oxidative damage against copper stressrdquo Journalof Integrative Plant Biology vol 50 no 12 pp 1518ndash1529 2008

[11] H Zhang J Tang X-P Liu et al ldquoHydrogen sulfide promotesroot organogenesis in Ipomoea batatas Salix matsudana andGlycine maxrdquo Journal of Integrative Plant Biology vol 51 no 12pp 1086ndash1094 2009

[12] Z P Jin J J Shen Z J Qiao G D Yang R Wang andY X Pei ldquoHydrogen sulfide improves drought resistance inArabidopsis thalianardquo Biochemical and Biophysical ResearchCommunications vol 414 no 3 pp 481ndash486 2011

[13] Z P Jin SWXue YN Luo et al ldquoHydrogen sulfide interactingwith abscisic acid in stomatal regulation responses to droughtstress inArabidopsisrdquoPlant Physiology and Biochemistry vol 62pp 41ndash46 2013

[14] C Garcıa-Mata and L Lamattina ldquoHydrogen sulphide anovel gasotransmitter involved in guard cell signallingrdquo NewPhytologist vol 188 no 4 pp 977ndash984 2010

[15] L-Y Hu S-L Hu J Wu et al ldquoHydrogen sulfide prolongspostharvest shelf life of strawberry and plays an antioxidativerole in fruitsrdquo Journal of Agricultural and Food Chemistry vol60 no 35 pp 8684ndash8693 2012

[16] S-P Gao K-D Hu L-Y Hu et al ldquoHydrogen sulfide delayspostharvest senescence and plays an antioxidative role in fresh-cut kiwifruitrdquo HortScience vol 49 no 10 pp 1385ndash1392 2013

[17] S-P Li K-D Hu L-Y Hu et al ldquoHydrogen sulfide alleviatespostharvest senescence of broccoli by modulating antioxidantdefense and senescence-related gene expressionrdquo Journal ofAgricultural and Food Chemistry vol 62 no 5 pp 1119ndash11292014

[18] Y Xie C Zhang D Lai et al ldquoHydrogen sulfide delays GA-triggered programmed cell death inwheat aleurone layers by themodulation of glutathione homeostasis and heme oxygenase-1expressionrdquo Journal of Plant Physiology vol 171 no 2 pp 53ndash622014

[19] M J Chrispeels and J E Varner ldquoGibberellic acid enhancedsynthesis and release of 120572-amylase and ribonuclease by isolatedbarley and aleurone layersrdquo Plant Physiology vol 42 no 3 pp398ndash406 1967

[20] J R Tennant ldquoEvaluation of the trypan blue technique fordetermination of cell viabilityrdquo Transplantation vol 2 pp 685ndash694 1964

[21] M Tsuchiya M Suematsu and H Suzuki ldquoIn vivo visualiza-tion of oxygen radical-dependent photoemissionrdquo Methods inEnzymology vol 233 pp 128ndash140 1994

[22] R F del Maestro J Bjork and K-E Arfors ldquoIncrease inmicrovascular permeability induced by enzymatically gener-ated free radicals I In vivo studyrdquoMicrovascular Research vol22 no 3 pp 239ndash254 1981

[23] C Garcıa-Limones A Hervas J A Navas-Cortes R MJimenez-Dıaz and M Tena ldquoInduction of an antioxidantenzyme system and other oxidative stress markers associatedwith compatible and incompatible interactions between chick-pea (Cicer arietinum L) and Fusarium oxysporum f sp cicerisrdquoPhysiological and Molecular Plant Pathology vol 61 no 6 pp325ndash337 2002

[24] K Surrey ldquoSpectrophotometric method for determination oflipoxidase activityrdquo Plant Physiology vol 39 no 1 pp 65ndash701964

[25] G G Collins C F Jenner and L G Paleg ldquoThe metabolismof soluble nucleotides in wheat aleurone layers treated withgibberellic acidrdquo Plant P hysiology vol 49 no 3 pp 404ndash4101972

[26] G Noelting and P Bernfeld ldquoSur les enzymes amylolytiqucsIII La 120573-amylase dosage drsquoactivite et controle de lrsquoabsence drsquo120572-amylaserdquo Helvetica Chimica Acta vol 31 no 1 pp 286ndash2901948

[27] M Lisjak T Teklic I D Wilson M Whiteman and J THancock ldquoHydrogen sulfide environmental factor or signallingmoleculerdquo Plant Cell and Environment vol 36 no 9 pp 1607ndash1616 2013

[28] M C de Pinto A Paradiso P Leonetti and L de Gara ldquoHydro-gen peroxide nitric oxide and cytosolic ascorbate peroxidaseat the crossroad between defence and cell deathrdquo The PlantJournal vol 48 no 5 pp 784ndash795 2006

[29] A Fath P C Bethke and R L Jones ldquoEnzymes that scavengereactive oxygen species are down-regulated prior to gibberellicacid-induced programmed cell death in barley aleuronerdquo PlantPhysiology vol 126 no 1 pp 156ndash166 2001

[30] Y Ishibashi T Tawaratsumida K Kondo et al ldquoReactive oxy-gen species are involved in gibberellinabscisic acid signaling inbarley aleurone cellsrdquo Plant Physiology vol 158 no 4 pp 1705ndash1714 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 9

0 0005 005 025 05

(mM)

(a)

0 0005 005 025 05

(mM)

(b)

0

50

100

150

200

0 1 2 3 4 5Treatment time (d)

GA

ControlGA + H2S

Activ

ity o

f120572-a

myl

ase (

Umiddotgminus1

FW)

(c)

Figure 6 NaHS increases the activity of 120572-amylase in incubation medium of barley aleurone layers in the absence (a) and presence ((b)(c)) of GA Barley aleurone layers isolated from treated embryoless half grains are incubated in 0 0005 005 025 or 05mMNaHS for 24 h((a) (b)) After incubation aleurone layers are placed on agar-starch medium (containing 4 agar and 01 starch) for 16 h Agar plates arestained with I

2-KI solution to detect the activity of 120572-amylase (a) (b) shows native PAGE analysis of 120572-amylase activity in incubationmedium

surrounding the aleurone layers (c) indicates secreted 120572-amylase activity in incubation medium surrounding barley aleurone layers treatedwith water (control) GA or GA + NaHS (GA + H

2S) at different times of incubation Data in (c) are expressed as means plusmn SD of three

independent experiments with three replicates of 20 embryoless half grains per treatment

GA + NaHS treatment is significantly higher than that oflayers incubated only in GA during the whole treatmenttime As expected much lower 120572-amylase activity was onlyobserved inwater control after 3 days of incubation Togetherthis result indicates that H

2S delays PCD in barley aleurone

layers and meanwhile promotes 120572-amylase release regardlessof the presence of GA

4 Discussion

H2S participates in multiple processes in plants [27] In

this paper we show that H2S delays PCD in barley aleu-

rone layers regardless of the presence or absence of GAIn the absence of GA PCD in barley aleurone layers isevident on day 3 of incubation while H

2S delays PCD at

an optimal concentration of 025mM (Figure 1) Consistentwith the reports that GA accelerates PCD process in barley

aleurone layers GA treatment triggers cell death in about90 aleurone cells at 72 h (Figure 5(a)) GA-induced celldeath is slowed by the addition of NaHS and this H

2S

donor also prolongs the phase of 120572-amylase productionin GA-treated layers The promoting effect of NaHS on120572-amylase synthesis and its prolongation of cell survivalindicate that 25mMNaHS does not affect aleurone cell func-tion

ROS such as sdotO2

minus andH2O2 are inducers of PCD in plant

and animal cells [1] It is reported that the peroxidation ofmembrane lipids and damage to the plasma membrane canoccur when the rate of ROS production overcomes the cellsrsquoability for scavenging ROS [27] Overproduction of ROS andoxidative damage are universal events in PCD in plant cells[28] In this paper we show that the content of sdotO

2

minus increasesin parallel with cell death in GA-treated layers (Figure 5(c))In non-GA-treated layers the burst of sdotO

2

minus and H2O2and

10 Oxidative Medicine and Cellular Longevity

the accumulation of MDA are also accompanied by PCD(Figure 2)These results suggest that ROS play a key role bothin GA-treated [29] and in non-GA-treated aleurone layers(Figures 2(a) 2(b) and 5(c))

Aleurone cells contain a suite of ROS-metabolizingenzymes GA-induced PCD in layers is accompanied by adecline in activity of ROS metabolizing enzymes which leadsto increased susceptibility of aleurone cells to ROS [29] Anovel aspect of our work is that NaHS treatment effectivelyreduces the accumulation of ROS in barley aleurone layersregardless the presence of GA (Figures 2 and 5) therebydelaying PCD process in these cells We propose that theH2S donor reduces ROS accumulation in layers by increasing

the activity of ROS-scavenging enzymes The data in presentstudy show that H

2S treatment maintains significantly higher

POD activity in GA-treated layers (Figure 5(d)) and higherSOD POD CAT and APX activity in non-GA-treatedlayers (Figure 3) The increased activity of ROS-scavengingenzymes in NaHS-treated likely promotes the cellrsquos abilityto metabolize ROS In addition LOX activities which areresponsible for lipid peroxidation are downregulated inNaHS-treated aleurone layers at early stage of treatment(Figures 3(e) and 5(e)) Meanwhile quantitative-PCR anal-ysis shows that expression of HvSOD1 HvAPX HvCAT1and HvCAT2 genes in non-GA-treated layers is maintainedat higher levels in NaHS treatment compared with watercontrols (Figure 4) Consistent with lower LOX activity inNaHS-treated aleurone layers the accumulation of LOXtranscripts is also reduced (Figure 4) In summary H

2S slows

down ROS-induced PCD in barley aleurone layers probablyby enhancing the activity and expression of ROS-scavengingenzymes and reducing the peroxidation of membrane lipids

Consistently Xie et al [18] found that H2S delayed GA-

triggered PCD in wheat aleurone layers by increasing GSHcontent and heme oxygenase-1 gene expression Here weprovide evidence thatH

2S can alleviate both natural PCD and

GA-triggered PCD through the modulation of antioxidantenzyme activities and their expression Compared with theslow natural PCDprocess the present study also confirms thepivotal role of GA in triggering PCD (Figure 5)

The role of ROS in GA and ABA signaling in barleyaleurone cells is recently clarified [30] in which they foundthat the production of H

2O2 a type of ROS was induced by

GA in aleurone cells but suppressed by ABA Furthermoreexogenous H

2O2appeared to promote the induction of 120572-

amylases by GA by promoting the expression of GAMyb and120572-amylase genes whereas antioxidants suppressed the induc-tion of 120572-amylase Unexpectedly we found that H

2S reduces

ROS accumulation and delays PCD process in barley aleu-rone layers in the presence or absence of GA and meanwhilepromotes the secretion of 120572-amylase suggesting that theantioxidant H

2S works through an unknown way to regulate

120572-amylase secretion and antioxidants do not always suppressthe induction of 120572-amylase Besides the activation of 120572-amylase by H

2S in the absence of GA implies that 120572-amylase

can be secreted independent of GA signaling pathwayTherefore the present findings advance our knowledge onthe relations between PCD process and 120572-amylase secretion

and the independence of 120572-amylase secretion and GA path-way

The activation of cysteine proteases was instrumental inthe PCD of soybean cells while cystatin an endogenouscysteine protease inhibitor gene inhibited cysteine proteaseactivity and blocked PCD in these cells [7] In this paperwe show that H

2S downregulates the transcriptions of two

barley cysteine proteinases HvEPA and HvCP3-31 in non-GA-treated barley aleurone layers thereby delaying cellcomponent degradation and PCD process

5 Conclusion

In summary we report the role of H2S in delaying PCD in

barley aleurone layers regardless of the presence or absence ofGA without repressing 120572-amylase induction suggesting thatthe function ofH

2Smay be universal in regulating plant PCD

PCD in plant cells is regulated by many internal and externalfactors such as the hormones (GA andABA) Ca2+ ROS andNO [6] It will be interesting to knowwhether H

2S is involved

in other signals and how 120572-amylase is induced by H2S in the

presence or absence of GA in cereal aleurone cells

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ying-Xin Zhang and Kang-Di Hu contributed equally to thiswork

Acknowledgments

Theauthors acknowledge Russell Jones University of Califor-nia at Berkeley for editing the paperThisworkwas supportedby the Natural Science Foundation of China (nos 3127180331301820 31300133 and 31470013) the Scientific ResearchFoundation for Returned Overseas Chinese Scholars (SRFfor ROCS MOE) the Natural Science Foundations of AnhuiProvince (11040606M85) and the Anhui Provincial Educa-tion Department (nos 2012AJZR0028 ZD200910)

References

[1] T Jabs ldquoReactive oxygen intermediates as mediators of pro-grammed cell death in plants and animalsrdquo Biochemical Phar-macology vol 57 no 3 pp 231ndash245 1999

[2] A Fath P Bethke V Beligni and R Jones ldquoActive oxygenand cell death in cereal aleurone cellsrdquo Journal of ExperimentalBotany vol 53 no 372 pp 1273ndash1282 2002

[3] A Fath P Bethke J Lonsdale R Meza-Romero and R JonesldquoProgrammed cell death in cereal aleuronerdquo Plant MolecularBiology vol 44 no 3 pp 255ndash266 2000

[4] P C Bethke J E Lonsdale A Fath and R L Jones ldquoHormon-ally regulated programmed cell death in barley aleurone cellsrdquoThe Plant Cell vol 11 no 16 pp 1033ndash1045 1999

Oxidative Medicine and Cellular Longevity 11

[5] P EHockberger T A Skimina V E Centonze et al ldquoActivationof flavin-containing oxidases underlies light-induced produc-tion of H

2O2in mammalian cellsrdquo Proceedings of the National

Academy of Sciences of the United States of America vol 96 no11 pp 6255ndash6260 1999

[6] P C Bethke and R L Jones ldquoCell death of barley aleuroneprotoplasts is mediated by reactive oxygen speciesrdquo The PlantJournal vol 25 no 1 pp 19ndash29 2001

[7] M Solomon B Belenghi M Delledonne E Menachem andA Levine ldquoThe involvement of cysteine proteases and proteaseinhibitor genes in the regulation of programmed cell death inplantsrdquoThe Plant Cell vol 11 no 3 pp 431ndash443 1999

[8] R Wang ldquoTworsquos company threersquos a crowd can H2S be the third

endogenous gaseous transmitterrdquo The FASEB Journal vol 16no 13 pp 1792ndash1798 2002

[9] E Bloem A Riemenschneider J Volker et al ldquoSulphur supplyand infection with Pyrenopeziza brassicae influence L-cysteinedesulphydrase activity in Brassica napus Lrdquo Journal of Experi-mental Botany vol 55 no 406 pp 2305ndash2312 2004

[10] H Zhang L-Y Hu K-D Hu Y-D He S-H Wang and J-P Luo ldquoHydrogen sulfide promotes wheat seed germinationand alleviates oxidative damage against copper stressrdquo Journalof Integrative Plant Biology vol 50 no 12 pp 1518ndash1529 2008

[11] H Zhang J Tang X-P Liu et al ldquoHydrogen sulfide promotesroot organogenesis in Ipomoea batatas Salix matsudana andGlycine maxrdquo Journal of Integrative Plant Biology vol 51 no 12pp 1086ndash1094 2009

[12] Z P Jin J J Shen Z J Qiao G D Yang R Wang andY X Pei ldquoHydrogen sulfide improves drought resistance inArabidopsis thalianardquo Biochemical and Biophysical ResearchCommunications vol 414 no 3 pp 481ndash486 2011

[13] Z P Jin SWXue YN Luo et al ldquoHydrogen sulfide interactingwith abscisic acid in stomatal regulation responses to droughtstress inArabidopsisrdquoPlant Physiology and Biochemistry vol 62pp 41ndash46 2013

[14] C Garcıa-Mata and L Lamattina ldquoHydrogen sulphide anovel gasotransmitter involved in guard cell signallingrdquo NewPhytologist vol 188 no 4 pp 977ndash984 2010

[15] L-Y Hu S-L Hu J Wu et al ldquoHydrogen sulfide prolongspostharvest shelf life of strawberry and plays an antioxidativerole in fruitsrdquo Journal of Agricultural and Food Chemistry vol60 no 35 pp 8684ndash8693 2012

[16] S-P Gao K-D Hu L-Y Hu et al ldquoHydrogen sulfide delayspostharvest senescence and plays an antioxidative role in fresh-cut kiwifruitrdquo HortScience vol 49 no 10 pp 1385ndash1392 2013

[17] S-P Li K-D Hu L-Y Hu et al ldquoHydrogen sulfide alleviatespostharvest senescence of broccoli by modulating antioxidantdefense and senescence-related gene expressionrdquo Journal ofAgricultural and Food Chemistry vol 62 no 5 pp 1119ndash11292014

[18] Y Xie C Zhang D Lai et al ldquoHydrogen sulfide delays GA-triggered programmed cell death inwheat aleurone layers by themodulation of glutathione homeostasis and heme oxygenase-1expressionrdquo Journal of Plant Physiology vol 171 no 2 pp 53ndash622014

[19] M J Chrispeels and J E Varner ldquoGibberellic acid enhancedsynthesis and release of 120572-amylase and ribonuclease by isolatedbarley and aleurone layersrdquo Plant Physiology vol 42 no 3 pp398ndash406 1967

[20] J R Tennant ldquoEvaluation of the trypan blue technique fordetermination of cell viabilityrdquo Transplantation vol 2 pp 685ndash694 1964

[21] M Tsuchiya M Suematsu and H Suzuki ldquoIn vivo visualiza-tion of oxygen radical-dependent photoemissionrdquo Methods inEnzymology vol 233 pp 128ndash140 1994

[22] R F del Maestro J Bjork and K-E Arfors ldquoIncrease inmicrovascular permeability induced by enzymatically gener-ated free radicals I In vivo studyrdquoMicrovascular Research vol22 no 3 pp 239ndash254 1981

[23] C Garcıa-Limones A Hervas J A Navas-Cortes R MJimenez-Dıaz and M Tena ldquoInduction of an antioxidantenzyme system and other oxidative stress markers associatedwith compatible and incompatible interactions between chick-pea (Cicer arietinum L) and Fusarium oxysporum f sp cicerisrdquoPhysiological and Molecular Plant Pathology vol 61 no 6 pp325ndash337 2002

[24] K Surrey ldquoSpectrophotometric method for determination oflipoxidase activityrdquo Plant Physiology vol 39 no 1 pp 65ndash701964

[25] G G Collins C F Jenner and L G Paleg ldquoThe metabolismof soluble nucleotides in wheat aleurone layers treated withgibberellic acidrdquo Plant P hysiology vol 49 no 3 pp 404ndash4101972

[26] G Noelting and P Bernfeld ldquoSur les enzymes amylolytiqucsIII La 120573-amylase dosage drsquoactivite et controle de lrsquoabsence drsquo120572-amylaserdquo Helvetica Chimica Acta vol 31 no 1 pp 286ndash2901948

[27] M Lisjak T Teklic I D Wilson M Whiteman and J THancock ldquoHydrogen sulfide environmental factor or signallingmoleculerdquo Plant Cell and Environment vol 36 no 9 pp 1607ndash1616 2013

[28] M C de Pinto A Paradiso P Leonetti and L de Gara ldquoHydro-gen peroxide nitric oxide and cytosolic ascorbate peroxidaseat the crossroad between defence and cell deathrdquo The PlantJournal vol 48 no 5 pp 784ndash795 2006

[29] A Fath P C Bethke and R L Jones ldquoEnzymes that scavengereactive oxygen species are down-regulated prior to gibberellicacid-induced programmed cell death in barley aleuronerdquo PlantPhysiology vol 126 no 1 pp 156ndash166 2001

[30] Y Ishibashi T Tawaratsumida K Kondo et al ldquoReactive oxy-gen species are involved in gibberellinabscisic acid signaling inbarley aleurone cellsrdquo Plant Physiology vol 158 no 4 pp 1705ndash1714 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

10 Oxidative Medicine and Cellular Longevity

the accumulation of MDA are also accompanied by PCD(Figure 2)These results suggest that ROS play a key role bothin GA-treated [29] and in non-GA-treated aleurone layers(Figures 2(a) 2(b) and 5(c))

Aleurone cells contain a suite of ROS-metabolizingenzymes GA-induced PCD in layers is accompanied by adecline in activity of ROS metabolizing enzymes which leadsto increased susceptibility of aleurone cells to ROS [29] Anovel aspect of our work is that NaHS treatment effectivelyreduces the accumulation of ROS in barley aleurone layersregardless the presence of GA (Figures 2 and 5) therebydelaying PCD process in these cells We propose that theH2S donor reduces ROS accumulation in layers by increasing

the activity of ROS-scavenging enzymes The data in presentstudy show that H

2S treatment maintains significantly higher

POD activity in GA-treated layers (Figure 5(d)) and higherSOD POD CAT and APX activity in non-GA-treatedlayers (Figure 3) The increased activity of ROS-scavengingenzymes in NaHS-treated likely promotes the cellrsquos abilityto metabolize ROS In addition LOX activities which areresponsible for lipid peroxidation are downregulated inNaHS-treated aleurone layers at early stage of treatment(Figures 3(e) and 5(e)) Meanwhile quantitative-PCR anal-ysis shows that expression of HvSOD1 HvAPX HvCAT1and HvCAT2 genes in non-GA-treated layers is maintainedat higher levels in NaHS treatment compared with watercontrols (Figure 4) Consistent with lower LOX activity inNaHS-treated aleurone layers the accumulation of LOXtranscripts is also reduced (Figure 4) In summary H

2S slows

down ROS-induced PCD in barley aleurone layers probablyby enhancing the activity and expression of ROS-scavengingenzymes and reducing the peroxidation of membrane lipids

Consistently Xie et al [18] found that H2S delayed GA-

triggered PCD in wheat aleurone layers by increasing GSHcontent and heme oxygenase-1 gene expression Here weprovide evidence thatH

2S can alleviate both natural PCD and

GA-triggered PCD through the modulation of antioxidantenzyme activities and their expression Compared with theslow natural PCDprocess the present study also confirms thepivotal role of GA in triggering PCD (Figure 5)

The role of ROS in GA and ABA signaling in barleyaleurone cells is recently clarified [30] in which they foundthat the production of H

2O2 a type of ROS was induced by

GA in aleurone cells but suppressed by ABA Furthermoreexogenous H

2O2appeared to promote the induction of 120572-

amylases by GA by promoting the expression of GAMyb and120572-amylase genes whereas antioxidants suppressed the induc-tion of 120572-amylase Unexpectedly we found that H

2S reduces

ROS accumulation and delays PCD process in barley aleu-rone layers in the presence or absence of GA and meanwhilepromotes the secretion of 120572-amylase suggesting that theantioxidant H

2S works through an unknown way to regulate

120572-amylase secretion and antioxidants do not always suppressthe induction of 120572-amylase Besides the activation of 120572-amylase by H

2S in the absence of GA implies that 120572-amylase

can be secreted independent of GA signaling pathwayTherefore the present findings advance our knowledge onthe relations between PCD process and 120572-amylase secretion

and the independence of 120572-amylase secretion and GA path-way

The activation of cysteine proteases was instrumental inthe PCD of soybean cells while cystatin an endogenouscysteine protease inhibitor gene inhibited cysteine proteaseactivity and blocked PCD in these cells [7] In this paperwe show that H

2S downregulates the transcriptions of two

barley cysteine proteinases HvEPA and HvCP3-31 in non-GA-treated barley aleurone layers thereby delaying cellcomponent degradation and PCD process

5 Conclusion

In summary we report the role of H2S in delaying PCD in

barley aleurone layers regardless of the presence or absence ofGA without repressing 120572-amylase induction suggesting thatthe function ofH

2Smay be universal in regulating plant PCD

PCD in plant cells is regulated by many internal and externalfactors such as the hormones (GA andABA) Ca2+ ROS andNO [6] It will be interesting to knowwhether H

2S is involved

in other signals and how 120572-amylase is induced by H2S in the

presence or absence of GA in cereal aleurone cells

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Ying-Xin Zhang and Kang-Di Hu contributed equally to thiswork

Acknowledgments

Theauthors acknowledge Russell Jones University of Califor-nia at Berkeley for editing the paperThisworkwas supportedby the Natural Science Foundation of China (nos 3127180331301820 31300133 and 31470013) the Scientific ResearchFoundation for Returned Overseas Chinese Scholars (SRFfor ROCS MOE) the Natural Science Foundations of AnhuiProvince (11040606M85) and the Anhui Provincial Educa-tion Department (nos 2012AJZR0028 ZD200910)

References

[1] T Jabs ldquoReactive oxygen intermediates as mediators of pro-grammed cell death in plants and animalsrdquo Biochemical Phar-macology vol 57 no 3 pp 231ndash245 1999

[2] A Fath P Bethke V Beligni and R Jones ldquoActive oxygenand cell death in cereal aleurone cellsrdquo Journal of ExperimentalBotany vol 53 no 372 pp 1273ndash1282 2002

[3] A Fath P Bethke J Lonsdale R Meza-Romero and R JonesldquoProgrammed cell death in cereal aleuronerdquo Plant MolecularBiology vol 44 no 3 pp 255ndash266 2000

[4] P C Bethke J E Lonsdale A Fath and R L Jones ldquoHormon-ally regulated programmed cell death in barley aleurone cellsrdquoThe Plant Cell vol 11 no 16 pp 1033ndash1045 1999

Oxidative Medicine and Cellular Longevity 11

[5] P EHockberger T A Skimina V E Centonze et al ldquoActivationof flavin-containing oxidases underlies light-induced produc-tion of H

2O2in mammalian cellsrdquo Proceedings of the National

Academy of Sciences of the United States of America vol 96 no11 pp 6255ndash6260 1999

[6] P C Bethke and R L Jones ldquoCell death of barley aleuroneprotoplasts is mediated by reactive oxygen speciesrdquo The PlantJournal vol 25 no 1 pp 19ndash29 2001

[7] M Solomon B Belenghi M Delledonne E Menachem andA Levine ldquoThe involvement of cysteine proteases and proteaseinhibitor genes in the regulation of programmed cell death inplantsrdquoThe Plant Cell vol 11 no 3 pp 431ndash443 1999

[8] R Wang ldquoTworsquos company threersquos a crowd can H2S be the third

endogenous gaseous transmitterrdquo The FASEB Journal vol 16no 13 pp 1792ndash1798 2002

[9] E Bloem A Riemenschneider J Volker et al ldquoSulphur supplyand infection with Pyrenopeziza brassicae influence L-cysteinedesulphydrase activity in Brassica napus Lrdquo Journal of Experi-mental Botany vol 55 no 406 pp 2305ndash2312 2004

[10] H Zhang L-Y Hu K-D Hu Y-D He S-H Wang and J-P Luo ldquoHydrogen sulfide promotes wheat seed germinationand alleviates oxidative damage against copper stressrdquo Journalof Integrative Plant Biology vol 50 no 12 pp 1518ndash1529 2008

[11] H Zhang J Tang X-P Liu et al ldquoHydrogen sulfide promotesroot organogenesis in Ipomoea batatas Salix matsudana andGlycine maxrdquo Journal of Integrative Plant Biology vol 51 no 12pp 1086ndash1094 2009

[12] Z P Jin J J Shen Z J Qiao G D Yang R Wang andY X Pei ldquoHydrogen sulfide improves drought resistance inArabidopsis thalianardquo Biochemical and Biophysical ResearchCommunications vol 414 no 3 pp 481ndash486 2011

[13] Z P Jin SWXue YN Luo et al ldquoHydrogen sulfide interactingwith abscisic acid in stomatal regulation responses to droughtstress inArabidopsisrdquoPlant Physiology and Biochemistry vol 62pp 41ndash46 2013

[14] C Garcıa-Mata and L Lamattina ldquoHydrogen sulphide anovel gasotransmitter involved in guard cell signallingrdquo NewPhytologist vol 188 no 4 pp 977ndash984 2010

[15] L-Y Hu S-L Hu J Wu et al ldquoHydrogen sulfide prolongspostharvest shelf life of strawberry and plays an antioxidativerole in fruitsrdquo Journal of Agricultural and Food Chemistry vol60 no 35 pp 8684ndash8693 2012

[16] S-P Gao K-D Hu L-Y Hu et al ldquoHydrogen sulfide delayspostharvest senescence and plays an antioxidative role in fresh-cut kiwifruitrdquo HortScience vol 49 no 10 pp 1385ndash1392 2013

[17] S-P Li K-D Hu L-Y Hu et al ldquoHydrogen sulfide alleviatespostharvest senescence of broccoli by modulating antioxidantdefense and senescence-related gene expressionrdquo Journal ofAgricultural and Food Chemistry vol 62 no 5 pp 1119ndash11292014

[18] Y Xie C Zhang D Lai et al ldquoHydrogen sulfide delays GA-triggered programmed cell death inwheat aleurone layers by themodulation of glutathione homeostasis and heme oxygenase-1expressionrdquo Journal of Plant Physiology vol 171 no 2 pp 53ndash622014

[19] M J Chrispeels and J E Varner ldquoGibberellic acid enhancedsynthesis and release of 120572-amylase and ribonuclease by isolatedbarley and aleurone layersrdquo Plant Physiology vol 42 no 3 pp398ndash406 1967

[20] J R Tennant ldquoEvaluation of the trypan blue technique fordetermination of cell viabilityrdquo Transplantation vol 2 pp 685ndash694 1964

[21] M Tsuchiya M Suematsu and H Suzuki ldquoIn vivo visualiza-tion of oxygen radical-dependent photoemissionrdquo Methods inEnzymology vol 233 pp 128ndash140 1994

[22] R F del Maestro J Bjork and K-E Arfors ldquoIncrease inmicrovascular permeability induced by enzymatically gener-ated free radicals I In vivo studyrdquoMicrovascular Research vol22 no 3 pp 239ndash254 1981

[23] C Garcıa-Limones A Hervas J A Navas-Cortes R MJimenez-Dıaz and M Tena ldquoInduction of an antioxidantenzyme system and other oxidative stress markers associatedwith compatible and incompatible interactions between chick-pea (Cicer arietinum L) and Fusarium oxysporum f sp cicerisrdquoPhysiological and Molecular Plant Pathology vol 61 no 6 pp325ndash337 2002

[24] K Surrey ldquoSpectrophotometric method for determination oflipoxidase activityrdquo Plant Physiology vol 39 no 1 pp 65ndash701964

[25] G G Collins C F Jenner and L G Paleg ldquoThe metabolismof soluble nucleotides in wheat aleurone layers treated withgibberellic acidrdquo Plant P hysiology vol 49 no 3 pp 404ndash4101972

[26] G Noelting and P Bernfeld ldquoSur les enzymes amylolytiqucsIII La 120573-amylase dosage drsquoactivite et controle de lrsquoabsence drsquo120572-amylaserdquo Helvetica Chimica Acta vol 31 no 1 pp 286ndash2901948

[27] M Lisjak T Teklic I D Wilson M Whiteman and J THancock ldquoHydrogen sulfide environmental factor or signallingmoleculerdquo Plant Cell and Environment vol 36 no 9 pp 1607ndash1616 2013

[28] M C de Pinto A Paradiso P Leonetti and L de Gara ldquoHydro-gen peroxide nitric oxide and cytosolic ascorbate peroxidaseat the crossroad between defence and cell deathrdquo The PlantJournal vol 48 no 5 pp 784ndash795 2006

[29] A Fath P C Bethke and R L Jones ldquoEnzymes that scavengereactive oxygen species are down-regulated prior to gibberellicacid-induced programmed cell death in barley aleuronerdquo PlantPhysiology vol 126 no 1 pp 156ndash166 2001

[30] Y Ishibashi T Tawaratsumida K Kondo et al ldquoReactive oxy-gen species are involved in gibberellinabscisic acid signaling inbarley aleurone cellsrdquo Plant Physiology vol 158 no 4 pp 1705ndash1714 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Oxidative Medicine and Cellular Longevity 11

[5] P EHockberger T A Skimina V E Centonze et al ldquoActivationof flavin-containing oxidases underlies light-induced produc-tion of H

2O2in mammalian cellsrdquo Proceedings of the National

Academy of Sciences of the United States of America vol 96 no11 pp 6255ndash6260 1999

[6] P C Bethke and R L Jones ldquoCell death of barley aleuroneprotoplasts is mediated by reactive oxygen speciesrdquo The PlantJournal vol 25 no 1 pp 19ndash29 2001

[7] M Solomon B Belenghi M Delledonne E Menachem andA Levine ldquoThe involvement of cysteine proteases and proteaseinhibitor genes in the regulation of programmed cell death inplantsrdquoThe Plant Cell vol 11 no 3 pp 431ndash443 1999

[8] R Wang ldquoTworsquos company threersquos a crowd can H2S be the third

endogenous gaseous transmitterrdquo The FASEB Journal vol 16no 13 pp 1792ndash1798 2002

[9] E Bloem A Riemenschneider J Volker et al ldquoSulphur supplyand infection with Pyrenopeziza brassicae influence L-cysteinedesulphydrase activity in Brassica napus Lrdquo Journal of Experi-mental Botany vol 55 no 406 pp 2305ndash2312 2004

[10] H Zhang L-Y Hu K-D Hu Y-D He S-H Wang and J-P Luo ldquoHydrogen sulfide promotes wheat seed germinationand alleviates oxidative damage against copper stressrdquo Journalof Integrative Plant Biology vol 50 no 12 pp 1518ndash1529 2008

[11] H Zhang J Tang X-P Liu et al ldquoHydrogen sulfide promotesroot organogenesis in Ipomoea batatas Salix matsudana andGlycine maxrdquo Journal of Integrative Plant Biology vol 51 no 12pp 1086ndash1094 2009

[12] Z P Jin J J Shen Z J Qiao G D Yang R Wang andY X Pei ldquoHydrogen sulfide improves drought resistance inArabidopsis thalianardquo Biochemical and Biophysical ResearchCommunications vol 414 no 3 pp 481ndash486 2011

[13] Z P Jin SWXue YN Luo et al ldquoHydrogen sulfide interactingwith abscisic acid in stomatal regulation responses to droughtstress inArabidopsisrdquoPlant Physiology and Biochemistry vol 62pp 41ndash46 2013

[14] C Garcıa-Mata and L Lamattina ldquoHydrogen sulphide anovel gasotransmitter involved in guard cell signallingrdquo NewPhytologist vol 188 no 4 pp 977ndash984 2010

[15] L-Y Hu S-L Hu J Wu et al ldquoHydrogen sulfide prolongspostharvest shelf life of strawberry and plays an antioxidativerole in fruitsrdquo Journal of Agricultural and Food Chemistry vol60 no 35 pp 8684ndash8693 2012

[16] S-P Gao K-D Hu L-Y Hu et al ldquoHydrogen sulfide delayspostharvest senescence and plays an antioxidative role in fresh-cut kiwifruitrdquo HortScience vol 49 no 10 pp 1385ndash1392 2013

[17] S-P Li K-D Hu L-Y Hu et al ldquoHydrogen sulfide alleviatespostharvest senescence of broccoli by modulating antioxidantdefense and senescence-related gene expressionrdquo Journal ofAgricultural and Food Chemistry vol 62 no 5 pp 1119ndash11292014

[18] Y Xie C Zhang D Lai et al ldquoHydrogen sulfide delays GA-triggered programmed cell death inwheat aleurone layers by themodulation of glutathione homeostasis and heme oxygenase-1expressionrdquo Journal of Plant Physiology vol 171 no 2 pp 53ndash622014

[19] M J Chrispeels and J E Varner ldquoGibberellic acid enhancedsynthesis and release of 120572-amylase and ribonuclease by isolatedbarley and aleurone layersrdquo Plant Physiology vol 42 no 3 pp398ndash406 1967

[20] J R Tennant ldquoEvaluation of the trypan blue technique fordetermination of cell viabilityrdquo Transplantation vol 2 pp 685ndash694 1964

[21] M Tsuchiya M Suematsu and H Suzuki ldquoIn vivo visualiza-tion of oxygen radical-dependent photoemissionrdquo Methods inEnzymology vol 233 pp 128ndash140 1994

[22] R F del Maestro J Bjork and K-E Arfors ldquoIncrease inmicrovascular permeability induced by enzymatically gener-ated free radicals I In vivo studyrdquoMicrovascular Research vol22 no 3 pp 239ndash254 1981

[23] C Garcıa-Limones A Hervas J A Navas-Cortes R MJimenez-Dıaz and M Tena ldquoInduction of an antioxidantenzyme system and other oxidative stress markers associatedwith compatible and incompatible interactions between chick-pea (Cicer arietinum L) and Fusarium oxysporum f sp cicerisrdquoPhysiological and Molecular Plant Pathology vol 61 no 6 pp325ndash337 2002

[24] K Surrey ldquoSpectrophotometric method for determination oflipoxidase activityrdquo Plant Physiology vol 39 no 1 pp 65ndash701964

[25] G G Collins C F Jenner and L G Paleg ldquoThe metabolismof soluble nucleotides in wheat aleurone layers treated withgibberellic acidrdquo Plant P hysiology vol 49 no 3 pp 404ndash4101972

[26] G Noelting and P Bernfeld ldquoSur les enzymes amylolytiqucsIII La 120573-amylase dosage drsquoactivite et controle de lrsquoabsence drsquo120572-amylaserdquo Helvetica Chimica Acta vol 31 no 1 pp 286ndash2901948

[27] M Lisjak T Teklic I D Wilson M Whiteman and J THancock ldquoHydrogen sulfide environmental factor or signallingmoleculerdquo Plant Cell and Environment vol 36 no 9 pp 1607ndash1616 2013

[28] M C de Pinto A Paradiso P Leonetti and L de Gara ldquoHydro-gen peroxide nitric oxide and cytosolic ascorbate peroxidaseat the crossroad between defence and cell deathrdquo The PlantJournal vol 48 no 5 pp 784ndash795 2006

[29] A Fath P C Bethke and R L Jones ldquoEnzymes that scavengereactive oxygen species are down-regulated prior to gibberellicacid-induced programmed cell death in barley aleuronerdquo PlantPhysiology vol 126 no 1 pp 156ndash166 2001

[30] Y Ishibashi T Tawaratsumida K Kondo et al ldquoReactive oxy-gen species are involved in gibberellinabscisic acid signaling inbarley aleurone cellsrdquo Plant Physiology vol 158 no 4 pp 1705ndash1714 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom