antioxidant enzyme responses to chilling stress in differentially

Journal of Experimental Botany, Vol. 48, No. 310, pp. 1105-1113, May 1997Journal ofExperimentalBotany

Antioxidant enzyme responses to chilling stress indifferentially sensitive inbred maize lines

D. Mark Hodges1'4, Christopher J. Andrews2'5, Douglas A. Johnson3 and Robert I. Hamilton2'5

1 Department of Chemistry, University of Lethbridge, 4401 University Drive, Lethbridge, Alberta,Canada T1K 3M42 Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, Central Experimental Farm,Ottawa, Ontario, Canada K1A 0C63 Department of Biology, University of Ottawa, 30 Marie Curie, PO Box 450, STN A, Ottawa, Ontario,Canada K1N 6N5

Received 17 December 1996; Accepted 28 January 1997

Abstract

Antioxidant enzyme activities were determined at thefirst, third and fifth leaf stages of four inbred lines ofmaize (£ea mays L.) exhibiting differential sensitivity tochilling. Plants were exposed to a photoperiod of 16:8L: D for one of three treatments: (a) control (25 °C), (b)control treatment plus an exposure to a short-termchilling shock (11 °C 1 d prior to harvesting), and (c)long-term (11 C constant) chilling exposure. Catalase(CAT; EC 1.11.1.6), ascorbate peroxidase (ASPX; EC1.11.1.11), superoxide dismutase (SOD; EC 1.15.1.1),glutathione reductase (GR; EC 1.6.4.2), and mono-dehydroascorbate reductase (MDHAR; EC 1.6.5.4)activities were assessed. Reducing and non-reducingsugars and starch concentrations were determined asgeneral metabolic indicators of stress. Reduced activit-ies of CAT, ASPX, and MDHAR may contribute tolimiting chilling tolerance at the early stages of devel-opment in maize. Changes in levels of sugar and starchindicated a more rapid disruption of carbohydrate util-ization in comparison to photosynthetic rates in thechilling-sensitive line under short-term chilling shocksand suggested a greater degree of acclimation in thetolerant lines over longer periods of chilling.

Key words: Antioxidant enzymes, differential chillingsensitivity, maize, soluble carbohydrates, Zea mays.

Introduction

Plants, as well as other organisms, have evolved antioxid-ant systems in order to protect against toxic species of

oxygen. Superoxide dismutases (SOD; EC 1.15.1.1) are agroup of enzymes which accelerate the conversion ofO^ to H2O2 (Salin, 1988). In turn, both catalases (CAT;EC 1.11.1.6), located mainly in the glyoxysomes, peroxi-somes, and mitochondria (CAT-3, maize) (Prasad et al.,1994), and various forms of peroxidases are responsiblefor the removal of H2O2 from biological systems (Gossettet al., 1994). In the chloroplasts, ascorbate is oxidized tothe monodehydroascorbate radical in the presence ofH2O2 by ascorbate peroxidase (ASPX; EC 1.11.1.11)(Hossain et al., 1984). This radical can then either bereduced back to ascorbate by NADPH-requiring mono-dehydroascorbate reductase (MDHAR; EC 1.6.5.4), ornon-enzymatically to dehydroascorbate, which is thenreduced to ascorbate by glutathione and the action ofdehydroascorbate reductase (DHAR; EC 1.8.5.1) (Jahnkeet al., 1991). Subsequent glutathione reduction occurs byglutathione reductase (GR; EC 1.6.4.2) in anotherNADPH-requiring reaction (Halliwell and Foyer, 1978).Ultimately, this cycle results in H2O2 being reduced byphotosynthetically generated NADPH (Salin, 1988).

Chilling conditions may lead to an increase in theamounts of toxic oxygen compounds present in plantsystems (Wise and Naylor, 1987; Hodgson and Raison,1991; Tsang et al., 1991). Chilling of sensitive plants,such as maize {Zea mays L.), in light is more damagingto the photosynthetic apparatus than chilling in darkness(Krause, 1988; Somersalo and Krause, 1989) due to theprocess of chilling-induced photoinhibition. This processis characterized by an over-energization of the photosys-tem reaction centres generally resulting from an inad-equate supply of the natural electron acceptor NADP+.

4 To whom correspondence should be addressed: Fax: +1 403 329 2057. E-mail: [email protected]" ECORC Contribution No. 971163.

O Oxford University Press 1997

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1106 Hodges et al.

The NADP+ pool can be limited by a reduction in theCO2 fixation rate (Cakmak and Marschner, 1992; Elstnerand Osswald, 1994), restricted carbon metabolism beinga symptom of low temperature stress (Schoner andKrause, 1990). Molecular oxygen may then becomereduced instead of NADP+ at the Fe-S centres or byferredoxin, producing O~ (Long, 1983).

The duration of chilling stress can have importanteffects on chilling-sensitive plants. Physiological dysfunc-tions resulting from molecular changes induced at lowtemperature can be reversed if the tissue is returned tonon-chilling temperatures before the dysfunction becomespersistent (Lyons et al., 1979). Prolonged chilling canlead to metabolic damage which may, however, be allevi-ated to an extent due to hardening or conditioning(Stamp, 1984). Comparisons between short- and long-term chilling treatments on antioxidant responses havenot appeared in the literature. Similarly, very little workhas been reported on chilling sensitivity in relation to theage of plants subjected to chilling stress.

Short-term (<3d) chilling conditions often result inthe accumulation of sugars and starch in leaves (Mitchelland Madore, 1992). Longer term chilling treatments inspecies such as tomato (Lycopersicon esculentum L.)(Bruggemann et al., 1992), alfalfa (Medicago sativa L.)(Castonguay et al., 1995), sunflower (Helianthus annuusL.), and rape (Brassica napus L.) (Paul et al., 1992),however, have resulted in the impairment of starch forma-tion. The accumulation of solutes such as proteins (Guy,1990) and sugars (Rhodes, 1987; Olien and Clark, 1993)has been linked to acclimation effects.

Several studies comparing different species havereported that chilling-resistant species or strains have agreater antioxidant capacity than other species which aresensitive (Jahnke et al., 1991; Walker and McKersie,1993, Gossett et al., 1994). In the current study, thedifferences in antioxidant enzyme capacities within inbredmaize lines exhibiting differential chilling sensitivity wereexamined. Reduced activities of CAT, ASPX, andMDHAR may contribute to limiting chilling tolerance atthe early stages of development in maize.

Materials and methods

Plant material

The four inbred lines of maize (Zea mays L.) selected for studyexhibited differential sensitivity to chilling based upon germina-tion and emergence (Hodges et al., 1994) and early growth drymass parameters (Hodges et al., 1995) (Table 1) in both thelaboratory and the field. One line, CO251, was shown throughall tests to be the most chilling-sensitive of eight original inbredlines tested. The other three lines, CO255, CO304, and CO308,depending upon the growth parameter assayed, were demon-strated to be either the most, or one of the more, chillingtolerant tested. These elite inbreds were produced in the 1993Agriculture Canada nursery, Ottawa, Canada.

Material was germinated for 5 d in the dark at 25 °C, untilthe coleoptile was approximately 2 cm long, and then pinnedinto rectangular styrofoam rafts and floated on trays containingHoaglands solution (Hodges et al., 1995) in a growth chamber(Conviron E-15). The photocycle was 16:8 L:D with a photonflux rate of 450-500 /xmol m" 2 s"1. The tissue was harvested atthe first, third and fifth leaf stages under three experimentalgrowth conditions: 25°C constant (control), 25°C with a 1 d11 °C short-term chilling shock, and 11 °C constant (long-termchilling exposure). For the 25 °C control experiments, thematerial reached these stages 2, 5, and 10 d from pinning. Forthe 11 °C 1 d treatment, the material was harvested on the sameschedule as the controls, except for the exposure to 11 °C I dprior to harvesting. Leaves were harvested from the 11 °C long-term chilling treatment at 8, 19, and 29 d from pinning for thefirst, third and fifth leaf stages, respectively. Growth chambertemperatures were closely monitored at the plant level with aYSI 44TE tele-thermometer complete with hooded thermo-couples. For the third and fifth leaf stages, the two mostrecently expanded leaves were harvested. The main midrib wasremoved from all leaf tissue.

Extract preparation and protein determination

Leaves (1.5 g fresh material) were extracted by grindingapproximately 30 s in a mortar and pestle with 0.5 g inert sand,0.25 g polyvinylpolypyrrolidone, and 20 ml chilled 50 mMphosphate buffer (pH 7.8). The extraction buffer used for theASPX assay contained 0.2 mM ascorbate. Extracts were thencentrifuged (Dupont Instruments Sorvall RC-5B) at 12000gfor 20 min at 2°C. For catalase (CAT), ascorbate peroxidase(ASPX), and glutathione reductase (GR) assays, the extractswere immediately desalted by passage through a Sephadex G-25column pre-equilibrated with 50 mM phosphate buffer (pH 7.8).

All enzymes were assessed spectrophotometrically on aMilton Roy Spectronic 1001 Plus equipped with a Haake F3

Table 1. Control, chill and chill: control ratios of the percentage germination and of the fourth-leaf stage total dry masses of the fourinbred maize lines

Germination and dry mass data are from Hodges et al. (1994) and Hodges et ah (1995), respectively. For control and chill data, means followedby the same letter are not significantly different at the 0.05 level according to Fisher's LSD.

Line

CO251CO255CO304CO308

Percentage

Control

94.8 ab95.2 ab

100.0 b92.7 a

germination

Chill

65.4 a94.2 c

100.0 d84.4 b

Chill/control

0.690.991.000.91

Total dry mass

Control

0.157 b0.150 b0.146 b0.215 a

Chill

0.117c0.196 b0.127 c0.236 a

Chill/control

0.751.320.881.09

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digital water bath for temperature control. The cuvette assaytemperature (25 °C) had been previously calibrated with a YS144TE tele-thermometer.

CAT activity was assayed in a reaction mixture containing0.85 ml 50 raM phosphate buffer (pH 7.8), 0.5 ml 30 mM H2O2,and 0.15 ml of the extract in a method following Aebi (1983).Activity was determined by following the decomposition ofH2O2 at 240 nm.

ASPX activity was determined in a method modified fromGossett et al. (1994). The reaction mixture contained 0.5 mllOOmM phosphate buffer (pH 7.8), 0.1ml l.OmM EDTA,0.1ml 1.5 mM ascorbate, 0.1ml 1.0 mM H2O2, and 0.2 mlextract. Activity was determined by following the H2O2-dependent decomposition of ascorbate at 265 nm.

Superoxide dismutase (SOD) activity was assayed by follow-ing the reduction of cytochrome c at 550 nm in a proceduremodified from McCord and Fridovich (1969). The reactionmixture contained 0.95 ml of solution A consisting of 25 ml50 mM phosphate buffer (pH 7.8), 0.5 mM xanthine in 0.001N NaOH, 0.08 mM cytochrome c, and 0.02 ml solution Bconsisting of 0.06 ml 0.2 U ml"1 xanthine oxidase (units asdefined by Sigma Chemical Co.) in 5 ml 0.1 mM EDTAdisodium salt alone or with 0.06 ml extract. Activity wasdetermined by monitoring at 550 nm the inhibition of thereduction rate of cytochrome between mixtures with andwithout the enzyme extract.

GR activity was determined following Goldberg and Spooner(1983). The reaction mixture contained 0.8 ml 100 mM phos-phate buffer (pH7.8), 0.1ml 100 mM oxidized glutathione,0.1 ml 15 mM EDTA disodium salt, 0.02 ml 10 mM NADPHin 1% (w/v) NaHCO3, and 0.3 ml extract. Activity wasdetermined by following the oxidation of NADPH at 340 nm.

Monodehydroascorbate reductase (MDHAR) was assayedaccording to a method following Hossain et al. (1984). Eachreaction mixture contained 0.5 ml 50 mM TRIS-HC1 buffer(pH 7.8), 0.05 ml 0.125% Triton X-100, 0.1 ml 0.2 mM NADH,0.1ml 2.5 mM ascorbate, 0.05 ml of 12.5 U ml"1 ascorbateoxidase (units as defined by Sigma Chemical Co.) in 10 mlH2O, and 0.2 ml extract. The reaction was followed bymeasuring the decrease in absorbance at 340 nm due to NADHoxidation.

Protein concentration was determined spectrophotometricallyat 595 nm using the Bio-Rad Protein Assay Dye ReagentConcentrate (catalogue number 500-0006) in a method basedon Bradford (1976). Bovine gamma-globulin (0.25-1.4 mg ml"1)was used as a standard reference.

Carbohydrate assays

Reducing and non-reducing sugars were extracted by grinding0.5 g of frozen tissue with 0.25 g inert sand and 5 ml ofdeionized water in a mortar and pestle. The extract wastransferred with a further 20 ml deionized water into anErlenmeyer flask and then boiled for 20 min. After cooling, theextracts were filtered (Whatman No. 4) and the final volumeadjusted to 25 ml. Reducing sugars were determined followingNelson (1944). Total sugars were determined after hydrolysingsucrose with invertase (^-D-fructofuransoside fructohydro-lase EC 3.2.1.36) (10 units ml"1) according to Westhaferet al. (1982). Non-reducing sugars were assessed by sub-tracting reducing sugars from total sugars. Glucose(0.045-0.18 mg ml"1) was used as the standard reference.

Starch was extracted by grinding 0.75-1.0 g frozen tissuewith 2.5 ml 8 M HO and 0.2 g of inert sand in a mortar andpestle. The extract was transferred with 20 ml DMSO into anErlenmeyer flask and then incubated at 60 °C in a controlled

Antioxidant enzyme responses to chilling stress 1107

water bath (Thelco 83) for I h. After rapid cooling, the additionof 2.5 ml 8 M NaOH was followed by diluting to 50 ml with0.112 M citrate buffer (pH 4.0). For each test, 0.1 ml of theextract was used. Starch concentration was determined spectro-photometrically using a coupled enzyme system (Boehringer-Mannheim test combination, catalogue number 207 748). Inthis system, starch is hydrolysed to D-glucose by amyloglucosid-ase (EC 3.2.1.3). The D-glucose formed is then converted byhexokinase (EC 2.7.1.1) to glucose-6-phosphate. Glucose-6-phosphate then becomes oxidized by NADP+ and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) to form gluconate-6-phosphate. The resulting formation of NADPH is monitoredat 340 nm.

Statistical analysis

All enzyme and metabolite assays were based on at least tworeadings from four independent replicates. The effects of inbredline, temperature regime, and growth stage on enzymaticactivities and carbohydrate concentrations were analysed by athree-factor completely randomized ANOVA. Enzyme andcarbohydrate data from control treatments are given asmeans ±SE. Data from both short- and long-term chillingtreatments are expressed as the percentage of control and areshown as means ±SE (n = 4) averaged over the four lines.Significance of the percentage germination (Hodges et al., 1994)and total dry weight (Hodges et al., 1995) data (Table 1) werecalculated using Fisher's LSD (P<0.05).

Results

Analysis of variance

Results of the ANOVA indicated that all main effects ofline, growth temperature, and growth stage were highlysignificant (Table 2). The interaction of growth temper-ature, growth stage, and inbred line, of great interesthere, was significant for all parameters except superoxidedismutase (SOD) activity.

Control antioxidant enzyme activities and carbohydrateconcentrations

Activities of catalase (CAT), ascorbate peroxidase(ASPX), and monodehydroascorbate reductase(MDHAR) in the 25 °C control material were generallyhighest in all three leaf stages in the chilling-sensitiveCO251 maize line (Tables 2, 3). Unlike the other antioxid-ant enzymes, control levels of ASPX and MDHARdecreased to a lower level as the plants aged. Duringgrowth at control temperatures, total sugars increasedfrom being significantly lowest at the first leaf stage tosignificantly highest at the fifth leaf stage in the chilling-sensitive line (Tables 2, 4). Levels of non-reducing sugarswere low at the first leaf stage in all lines, but increasedthrough the growth stages. Non-reducing sugars generallyremained substantially lower than reducing sugars. Starchconcentrations increased approximately 15-fold from firstto fifth leaf stage when grown under control conditions,but were generally lower in the chilling-sensitive line(Table 5).

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1108 Hodges et al.

Table 2. Levels of significance for a three factor AN OVA for activities of CAT, ASPX, SOD, GR, and MDHAR concentrations oftotal sugars (TS), reducing sugars (RS), non-reducing sugars (NRS), and starch of the four inbred maize lines

Source

LineTemperature regime (TR)Growth stage (GS)Line x TRLine x GST R x G SLine x TR x GS

CAT ASPX

* • • *

* • • **•+ *

N.S. *NS. *

SOD

***

• ••****N.S.***N.S.

GR MDHAR TS RS NRS Starch

* • • *n

* * * *t

• * + **

N.S. •'* *

* *** *

** **** **¥ * **

¥ * • •

k * * *

N • *

K * • •

• ** •

• •

> = P^0.O0\, ** = , N.S. = not significant

Table 3 CAT (fjjnol H2O2 decomposedmin l mg ' protein),ASPX (mmol H2O2 decomposed min'1 mg'1

cytochrome c conserved min ' mgprotein), SOD

" ' protein), GR (\imolNADPH oxidized min 1 mg 1 protein), and MDHAR (unto INADH oxidized min'1 mg'1 protein) activities in three leafstages of the chilling-sensitive CO251 and chilling-resistantCO255, CO304, and CO308 inbred lines of maize grown under25° C control treatment

CATCO251CO255CO304CO308

ASPXCO251CO255CO304CO308

SODCO251CO255CO304CO308

GRCO251CO255CO304CO308

MDHARCO251CO255CO304CO308

1st leaf stage

216.5±32.4120.9 ±9.383.5±13.286.7 ±13.2

3.2 + 0.12.3±0 11.6±0.01.8±0.1

182.0±9.6140.4 ±20.8144.8±16.4166.8±22.4

42.8 ±1.444.8 ±1.531.2±3.559.6 ±3.0

75.1 ±4.653.9±2.149.6 ±6.039.5 ±1-1

3rd leaf stage

217.2±12.3166.9±5.9206.1 ±27.2172.8±19.7

1.9±0.31.5±0.21.3±0.31.6±0.1

288.7±46.3285.4 ±20.1296.5 ±24.1245.3 ±14.0

28.3±2.538.7 ±1.835.0±7.659.1 ±10.9

19.1 ±3.612.1 ± 1 - 012.5±0.419.1 ±2.9

5th leaf stage

138.9± 11.644.8 ±11.2

108.0± 12.183.2± 10.8

2.6±0.l2.6±0.01.2 ±0.11.4±0.1

91.9±8.673.9±7.170.3 ±5.183.3±6.8

13.8±0.510.4± 1.58.2±0.7

13.1 ±0.8

19.1 ±1-111.9 ±0.66.9 ±0.9

17.3±2.9

Table5 Starch concentrations (mgg ' fr.wt.) of the three leafstages of the chilling-sensitive CO251 and chilling-resistantCO255, CO304, and CO308 inbred lines of maize grown under25 °C (control), 11 °C short-term chilling, and 11 °C long-termchilling treatments

Figures in parentheses indicate the changes in starch concentrationexpressed as a percentage of control.

Line Leaf stage Control(25 °C)

Short-term Long-termire

CO251

CO255

CO304

CO308

1

3

5

1

3

5

1

3

5

1

3

5

0.66±0.26

6.47 ±0.97

15.56±6.5O

2.25±0.28

7.60 ±3.02

20.83 ±2.43

2.91 ±0.30

5.80 ±1.00

26.83 ±0.94

2.11 ±0.09

7.83 ±0.46

30.06 ±2.99

2.72 ±0.09(415.7)30.64±2.51(473 6)34.45 ±1.99(221.4)3.84 ±1.37

(1704)16.46±3.13(216.6)37.05 ±4.63(177.9)4.80 ±1.06

(164.9)19.89±4.27(343.1)33.25±2.28(123.9)6.14± 1.91

(290.6)16.31 ±3.02(208.4)30.29 ±3.05(100.8)

0.04 ±0.01(5.6)0.64±0.18(9.9)0.39 ±0.14(2.5)0.89 ±0.09(39 4)1.16±0.38(15.2)6.74± 1.71(32.3)1.46 ±0.79(50.0)1.87±0.59(32.3)1.97 ±0.62(7.3)0.26 ±0.07(12.2)6 23± 1.41(79.6)2.23 ±0.86(7.4)

Table 4. Reducing, non-reducing, and total soluble sugars (mgg ' fr.wt.) extracted from the three leaf stages of the chilling-sensitiveCO251 and chilling-tolerant CO255, CO304, and CO308 inbred lines of maize grown under the 25 °C control treatment

Line

CO251CO255CO304CO308

Reducingsugars

1.17±0.141.75 ±0.041.69 ±0.231.70±0.16

1 st leaf stage

Non-reducingsugars

0.06±0.010.26 ±0.190.36±0.150.09 ±0.03

Totalsugars

1.23±0.141.75 ±0.082.05 ±0.151.79±0.13

Reducingsugars

1.04 ±0.200.38 ±0.031.25±0.300.92 ±0.08

3rd leaf stage

Non-reducingsugars

0.13±0.060.78±O.ll0.22 ±0.020.21 ±0.03

Totalsugars

1.17±0.191.16±0.101.18±0.351.12±0.07

5th leaf stage

Reducingsugars

1.35±0.160.83 ±0.041.08±0.361.07 ±0.04

Non-reducingsugars

0.77±0.110.29 ±0.070.36 ±0.050.42 ±0.12

Totalsugars

2.12±0.171.16±0.080.98±0.191.18±0.09

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Effects of short-term chilling on antioxidant enzymes

After short-term chilling for 1 d, the percentage of controlof activities of ASPX and MDHAR were significantlylower in the chilling-sensitive CO251 line (Figs 1-3).There were no substantial differences in SOD activitiesbetween the chilling-sensitive and chilling-tolerant lines(Figs 1-3). There was an increase in CAT and glutathionereductase (GR) activities in the chilling-sensitive line asthe plants aged, ultimately expressing activities signific-antly greater than those of the chilling-tolerant lines (Figs1-3). There were no significant differences between thechilling-sensitive and chilling-tolerant lines in terms of


700^

(a)

(b)

Fig. 1. Percentages of control of CAT, ASPX, SOD, GR, and MDHARof the first leaf developmental stage in the chilling-sensitive CO251 andchilling-tolerant CO255, CO304, and CO308 inbred maize lines for11 °C short-term (a) and 11 °C long-term chilling (b) periods. Verticalbars represent the mean±SE of four replications.

TttodLMf11*CShort-t«cTn25"A«a»y

_

-

-

-

7—,

jjrfU • I T Irfi

nnrai

SIi rm! UU3!i ffflI BES[

EmDi; rmI LUJ

• iim• im 1

1CAT ASPX SOO OR

CO308

(a)

(b)

Fig. 2. Percentages of control of CAT, ASPX, SOD, GR, and MDHARof the third leaf developmental stage in the chilling-sensitive CO251and chilling-tolerant CO255, CO304, and CO308 inbred maize lines for11 °C short-term (a) and 11 °C long-term chilling (b) periods. Verticalbars represent the mean ± SE of four replications.

activities of ASPX and MDHAR at the fifth leaf stage(Fig. 3).

Effect of long-term chilling on antioxidant enzymes

When the four maize lines were subjected to the long-term chilling regime, an increase relative to controls ofactivities of GR were noted for all three developmentalstages (Figs 1-3). Activities of SOD in the four linesincreased over that of the controls for the first twodevelopmental stages (Figs 1, 2), and MDHAR andASPX increased relative to the controls for the first andfifth leaf developmental stages, respectively (Figs 1, 3).

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1110 Hodges etal.

220

200

180

180

100

80

SO

40

20

0

H'CShort-tonn25'AtMy

ASPX 8OD| |CO251

E2

OR

CO306

(a)

ASPX SOO| |CO2S1

J g2

(b)

Fig. 3. Percentages of control of CAT, ASPX, SOD, GR, and MDHARof the fifth leaf developmental stage in the chilling-sensitive CO251 andchilling-tolerant CO255, CO304, and CO308 inbred maize lines for11 °C short-term (a) and 11 °C long-term chilling (b) periods. Verticalbars represent the mean ± SE of four replications.

After long-term chilling for 8 d, the percentage ofcontrol activities of MDHAR in the chilling-sensitiveCO251 line were significantly lower than those of thechilling tolerant lines (Fig. 1). This difference betweenthe lines was lost as chilling exposure increased (Figs 2,3). Activity of this enzyme was generally higher for thelong-term as compared to the short-term chilling treat-ments. The activity of ASPX was significantly mostreduced in the chilling-sensitive line relative to the tolerantlines for the first leaf stage under long-term chilling(Fig. 1). Activity of CAT was significantly lowest in thissensitive line for the first and third developmental stages(Figs 1, 2). There were essentially no differences inactivities of SOD between chilling-sensitive and chilling-tolerant lines under long-term chilling (Figs 1-3).

Activities of GR for the chilling-sensitive line were nodifferent from those of the chilling tolerant lines at thefirst leaf stage, but increased to be highest, though notsignificantly so, by the fifth leaf stage (Figs 1-3). Overallvalues of SOD were observed to decrease during chillingas the plants aged while those of GR were constant andwell in excess of control values.

Effects of short-term chilling on carbohydrates

After 1 d chilling, total sugars at all growth stagesincreased markedly over the controls in the chilling-susceptible line, whereas the increase was substantiallyless in the tolerant lines (Fig. 4). These increases resultedfrom changes in both reducing and non-reducing compon-

CO2S1

CO304I

(a)

)CO2S1Y r ' CO26S E?"! CO304 ^m CO308

(b)

Fig. 4. Percentages of control of reducing (RS), non-reducing (NRS),and total (TS) soluble sugars of the first, third, and fifth leafdevelopmental stages in the chilling-sensitive CO251 and chilling-tolerant CO255, CO304, and CO308 inbred maize lines for 11 °C short-term (a) and 11 °C long-term chilling (b) periods. Vertical bars representthe mean ± SE of four replications.

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ents, but significantly far more non-reducing sugars wereaccumulated relative to the controls in the chilling sus-ceptible CO251 (Fig. 4). After 1 d chilling, starch contentsignificantly increased relative to the controls at all growthstages and more so in the chilling-sensitive CO251(Table 5).

Effects of long-term chilling on carbohydrates

After 8 d of chilling (the first leaf stage), reducing andnon-reducing sugars were significantly highest relative tothe controls in the chilling-sensitive line CO251 (Fig. 4).However, with continued chilling this trend was reversed,and by the fifth leaf stage total sugars were significantlyhighest for the chilling-tolerant lines (Fig. 4). Similar tothe sugars, particularly the non-reducing sugars, starchcontents were significantly higher in the chilling-tolerantlines under long-term chilling exposure (Table 5).

Discussion

Chilling effects on enyzmatic activities

The percentage of control values are indicative of theline-specific antioxidant enzyme response. These valuescorrect for the inherent genetic diiferences that each lineexhibited under the 25 °C control temperature (Hodgeset al., 1994, 1995, 1996). Thus, actual effects of chillingon the antioxidant enzyme levels can be assessed.

Oxidative stress has been shown to induce or enhancelevels of superoxide dismutase (SOD), glutathione reduc-tase (GR), and ascorbate peroxidase (ASPX) (Foyeret al., 1994). The activity of the antioxidant enzymesfound in these differentially chilling-sensitive maize linesexhibited substantial differences in response to chilling. Itis noteworthy that the activities of some of the enzymes,such as GR, were elevated in all four chilled inbredsrelative to the controls, suggesting that all lines experi-enced some degree of chilling-induced oxidative stress.

Catalase (CAT) had lower relative percentage of con-trol activity in the chilling- susceptible line as opposed tothe tolerant lines when chilled continuously through thefirst two growth stages. However, there was little differ-ence in the percentage of control activity of this enzymebetween lines after short-term chilling. Various otherworkers have reported such chilling-related decreases inCAT activity (MacRae and Ferguson, 1985; Schoner andKrause, 1990), and there is evidence that it can becomephotoinactivated (Feierabend et al., 1992). The lack ofinbred response to short-term ( I d ) chilling is perhapsindicative of insufficient time for photoinactivation ofCAT to occur.

The lower percentage of control activity of monodehyd-roascorbate reductase (MDHAR) in the chilling-sensitiveline for the first leaf stage under both short-term andlong-term chilling exposure suggests that the activity of


this enzyme rapidly decreased due to cold at the earliergrowth stages, but then through either increased synthesisor production of more tolerant isozymes, eventuallyrecovered as the plants aged. Jahnke et al. (1991) founda transient higher activity of MDHAR in a 1-5 d chill-stressed Zea mays L. cultivar in comparison to the morechilling-tolerant Zea diploperennis L. However, Walkerand McKersie (1993) found no increase in the activity ofMDHAR of chilling-sensitive Lycopersicon esculentum L.and the more tolerant L. hirstum L., or differences inactivities between the two species, when exposed to coldfor 3 d. Thus there is conflicting evidence on the activityof this enzyme.

Activity of ASPX has been observed to increase whenchilled in leaves of such plants as spinach (Spinaceaoleracea L.) (SchSner and Krause, 1990) and wheat{Triticum aestivum L.) (Mishra et al. (1993). The lowerpercentage of control activity of ASPX in the chilling-sensitive maize line for both chilling treatments at thefirst leaf stage implies that, as with MDHAR, this enzymehas less ability to detoxifiy toxic oxygen compounds thanin the tolerant lines at the earlier growth stages.

These results strongly suggest that the sensitivity ofCO251 to chilling-induced oxidative stress was greater atthe first leaf stage. Reduced ascorbate cycling due todepressed ASPX and CAT and/or MDHAR activitieswould not have only led to reduced H2O2 detoxification,but also to reduced scavenging of O^, and 'C^. Lowerconcentrations of j3-carotene, which plays an importantrole in quenching both iO2 and excess chlorophyll excita-tion energy, were also noted at the first leaf stage ofCO251 (Hodges etai, 1996). As the plant aged, increasingpercentage of control activities of the antioxidant enzymesin the chilling-sensitive line relative to the tolerant plantsmay follow from the initially lower concentrations of j8-carotene (Hodges et al., 1996) and activities of ASPXand CAT and/or MDHAR in this line. Concentrationsof ascorbate, glutathione, and /3-carotene also increasedin this sensitive line as the chilling treatment progressedand as the plants developed until they ultimately becameeither significantly higher or no different relative to thetolerant lines (Hodges et al., 1996). Increased capacitiesof these antioxidant enzymes and compounds may repres-ent a mechanism to compensate for the initially lowerconcentrations of j3-carotene (Hodges et al., 1996) andH2O2-detoxifying and ascorbate-regenerating capacitiesof this line at the earlier stages of development.

Chilling effects on carbohydrate concentrations

Soluble sugars generally accumulate in plants such aswinter rye (Secale cereale L.) (Koster and Lynch, 1992),alfalfa (Medicago sativa L.) (Castonguay et al., 1995) andwheat (Perras and Sarhan, 1984) under chilling conditionsleading to acclimation. Chilling of 1-8 d in maize led to

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1112 Hodges etal.

accumulations of total sugars in all lines with the greatestincrease in the chilling-sensitive CO251. However, furtherchilling led to an accumulation of total sugars in thechilling-tolerant lines only. Although starch levelsincreased in response to short-term chilling, they werefound to fall below control levels upon long-term chillingfrom 8-29 d. Although Farrar (1988) states that starchcommonly accumulates at low temperatures, this is notthe case with maize chilled for extended periods.

Carbohydrates commonly increase in source leavesunder chilling conditions mainly as a result of the rate ofcarbohydrate utilization being depressed more than therate of photosynthesis (Farrar, 1988). Thus CO251, beingmore chilling-sensitive than the other lines, would experi-ence a greater decrease in its rate of utilization in theshort term, resulting in greater accumulation of sugarsand starch. In the long term, however, the more chilling-tolerant lines presumably became acclimated to a greaterdegree. Since one of the manifestations of acclimation isthe increase in solutes such as sugars (Perras and Sarhan,1984; Koster and Lynch, 1992), this would account forthe greater accumulation in the chilling-tolerant maizelines. Pollock and Lloyd (1987) demonstrated that theenzymes involved in starch formation were much moresensitive to low temperatures than those involved insucrose synthesis. This may explain starch levels beingmuch more reduced in relation to controls than weretotal sugars under the long-term chilling treatments.

Conclusions

Reduced capacities of CAT, ASPX, and MDHAR in thechilling-sensitive line may have limited its ability to detox-ify toxic oxygen compounds generated by low temperaturestress at early stages of development. As the maizeseedlings developed, the increasing activities of CAT,ASPX, MDHAR, and GR in the most chilling-sensitiveline relative to the tolerant lines suggest that it becameless sensitive to chilling-induced oxidation with age.Accompanying changes in levels of sugar and starchindicate a more rapid disruption of carbohydrate utiliza-tion in comparison to photosynthetic rates in the chilling-sensitive line under short-term chilling shocks and sug-gests a greater degree of acclimation in the tolerant linesover longer periods of chilling. These enzyme and meta-bolic indicator data, as well as the antioxidant compounddata from earlier work (Hodges el ai, 1996), point tothe differing abilities of these lines in responding to, andtolerating, chilling stress.

Acknowledgements

This work was funded in part by a grant from Natural Sciencesand Engineering Council of Canada to DAJ. The authorswould like to acknowledge Drs Hugh Hope and Keith Pomeroy,

Agriculture and Agri-Food Canada, Ottawa, Ontario, Canada,and Dr Randall J Weselake, University of Lethbridge, Alberta,Canada, for their helpful comments.

References

Aebi HE. 1983. Catalase. In: Bergmeyer HU, ed. Methods ofenzymatic analysis, Vol. 3. Weinheim: Verlag Chemie, 273-86.

Bradford MM. 1976. A rapid and sensitive method for thequantification of microgram quantities of protein using theprinciple of protein-dye binding. Analytical Biochemistry72, 248-54.

Bruggemann W, van der Kooj TAW, van Hasselt PR. 1992.Long-term chilling of young tomato plants under low lightand subsequent recovery. II. Chlorophyll fluorescence,carbon metabolism and activity of ribulose-l,5-biphosphatecarboxylase/oxygenase. Planta 186, 179-87.

Cakmak I, Marschner H. 1992. Magnesium deficiency and highlight intensity enhance activities of superoxide dismutase,ascorbate peroxidase, and glutathione reductase in beanleaves. Physiologia Plantarum 98, 1222-7.

Castooguay Y, Nadeau P, Lechasseur P, Chouinard L. 1995.Differential accumulation of carbohydrates in alfalfa cultivarsof contrasting winter-hardiness. Crop Science 35, 509-16.

Elstner EF, Osswald W. 1994. Mechanisms of oxygen activationduring plant stress. In: Crawford RMM, Hendry GAF,Goodman BA, eds. Oxygen and environmental stress in plants.Proceedings of the Royal Society of Edinburgh 102B, 131-54.

Farrar JF. 1988. Temperature and the partitioning and translo-cation of carbon. In: Long SP, Woodward FI, eds. Plantsand temperature. Cambridge: The Company of BiologistsLimited, 203-35.

Feierabend J, Schaan C, Hertwig B. 1992. Photoinactivation ofcatalase occurs under both high- and low-temperature stressconditions and accompanies photoinhibition of photosystemII. Plant Physiology 100, 1534-61.

Foyer CH, Lelandais M, Kunert KJ. 1994. Photooxidative stressin plants. Physiologia Plantarum 92, 696-717.

Goldberg DM, Spooner RJ. 1983. Glutathione reductase. In:Bergmeyer HU, ed. Methods of enzymatic analysis, Vol. 3.Weinheim: Verlag Chemie, 259-65.

Gossett DR, Millhollon EP, Lucas MC. 1994. Antioxidantresponse to NaCl stress in salt-tolerant and salt-sensitivecultivars of cotton. Crop Science 34, 706-14.

Guy CL. 1990. Cold acclimation and freezing stress tolerance:role of protein metabolism. Annual Review of Plant Physiology41, 187-223.

Halliwell B, Foyer CH. 1978. Properties and physiologicalfunction of a glutathione reductase purified from spinachleaves by affinity chromatography. Planta 139, 9-17.

Hodges DM, Hamilton RI, Charest C. 1994. A chilling resistancetest for inbred maize lines. Canadian Journal of Plant Science74,687-91.

Hodges DM, Hamilton RI, Charest C. 1995. A chilling responsetest for early growth phase maize. Agronomy Journal87, 970-4.

Hodges DM, Andrews CJ, Johnson DA, Hamilton RI. 1996.Antioxidant compound responses to chilling stress in differen-tially sensitive inbred maize lines. Physiologia Plantarum98, 685-92.

Hodgson RAJ, Raison JK. 1991. Superoxide production bythylakoids during chilling and its implication in the suscepti-bility of plants to chilling-induced photoinhibition. Planta183, 222-8.

Hossain MA, Nakano Y, Asada K. 1984. Monodehydroascorbate

Dow



reductase in spinach chloroplasts and its participation inregeneration of ascorbate for scavenging hydrogen peroxide.Plant Cell Physiology 25, 385-95.

Jahnke LS, Hull MR, Long SP. 1991. Chilling stress andoxygen metabolizing enzymes in Zea mays and Zea diploper-ennis. Plant, Cell and Environment 14, 97-104.

Koster KL, Lynch DV. 1992. Solute accumulation and compart-mentation during the cold acclimation of puma rye. PlantPhysiology 98, 108-13.

Krause GH. 1988. Photoinhibition of photosynthesis. Anevaluation of damaging and protective mechanisms.Physiologia Plantarum 74, 566—74.

Long SP. 1983. C4 photosynthesis at low temperatures. PlantCell and Environment 6, 345-63.

Lyons JM, Graham D, Steponkns PL. 1979. The plant membranein response to low temperature. In: Lyons JM, Graham D,Raison JK, eds. Low temperature stress in crop plants: Therole of the membrane. New York Academic Press, 1-24.

MacRae EA, Ferguson IB. 1985. Changes in catalase activityand hydrogen peroxide concentration in plants in responseto low temperature. Physiologia Plantarum 65, 51-6.

McCord JM, Fridovich I. 1969. Superoxide dismutase. Anenzymic function for erythrocuprin (hemocuprin). Journal ofBiological Chemistry 11A, 6049-55.

Mishra NP, Mishra RK, Singhal GS. 1993. Changes in theactivities of anti-oxidant enzymes during exposure of intactwheat leaves to strong visible light at different temperaturesin the presence of protein synthesis inhibitors. PlantPhysiology 102, 903-10.

Mitchell DE, Madore MA. 1992. Patterns of assimilate produc-tion and translocation in muskmelon (Cucumis melo L.).Plant Physiology 99, 966-71.

Nelson N. 1944. A photometric adaption of the Somogyimethod for the determination of D-glucose. Journal ofBiological Chemistry 153, 375-80.

Olien CR, Clark JL. 1993. Changes in soluble carbohydratecomposition of barley, wheat, and rye during winter.Agronomy Journal 85, 21-9.

Paul MJ, Driscoll SP, Lawlor DW. 1992. Sink-regulation of


photosynthesis in relation to temperature in sunflower andrape. Journal of Experimental Botany 43, 147-53.

Pen-as M, Sarhan F. 1984 Energy state of spring and winterwheat during cold hardening. Soluble sugars and adeninenucleotides. Physiologia Plantarum 60, 129-32

Pollock CJ, Lloyd EJ. 1987. The effect of low temperatureupon starch, sucrose and fructan synthesis in leaves. Annalsof Botany 60, 231-5.

Prasad TK, Anderson MD, Martin BA, Stewart CR. 1994.Evidence for chilling-induced oxidative stress in maizeseedlings and a regulatory role for hydrogen peroxide. ThePlant Cell 6, 65-74.

Rhodes D. 1987. Metabolic responses to stress. In: Thebiochemistry of plants, Vol. 12. New York: Academic PressInc., 201-41.

Salin ML. 1988. Toxic oxygen species and protective systemsof the chloroplast. Physiologia Plantarum 72, 681-9.

Schdner S, Krause GH. 1990. Protective systems against activeoxygen species in spinach: response to cold acclimation inexcess light. Planta 180, 383-9.

Somersalo S, Krause GH. 1989. Photoinhibition at chillingtemperature: fluorescence characteristics of unhardened andcold-acclimated spinach leaves. Planta 177, 409-16.

Stamp P. 1984. Chilling tolerance of young plants demonstratedon the example of maize (Zea mays L.). Advances in Agronomyand Crop Science 1.

Tsang EWT, Bowler C, Herouart D, van Camp W, Villarroel R,Genetello C, van Montagu M, Inze D. 1991. Differentialregulation of superoxide dismutases in plants exposed toenvironmental stress. The Plant Cell 3, 783-92.

Walker MA, McKersie BD. 1993. Role of the ascorbate-glutathione antioxidant system in chilling resistance oftomato. Journal of Plant Physiology 141, 234-9.

Westhafer MA, Law Jr JT, Duff DT. 1982. Carbohydratequantification and relationships with N nutrition in cool-season turfgrass. Agronomy Journal 74, 270-4.

Wise RR, Naylor AW. 1987. Chilling-enhanced photooxidation:evidence for the role of singlet oxygen and superoxide in thebreakdown of pigments and endogenous antioxidants. PlantPhysiology 83, 278-82.

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