effect of seawater aerosol on leaves of six plant species potentially useful for ornamental purposes...

Journal Identification = HORTI Article Identification = 3829 Date: March 5, 2011 Time: 1:40 pm

Eo

AGa

b

c

a

ARRA

KACCEFSS

1

aofieTdd2

ags

0d

Scientia Horticulturae 128 (2011) 332–341

Contents lists available at ScienceDirect

Scientia Horticulturae

journa l homepage: www.e lsev ier .com/ locate /sc ihor t i

ffect of seawater aerosol on leaves of six plant species potentially useful forrnamental purposes in coastal areas

ntonio Ferrantea, Alice Trivellini a,∗, Fernando Malorgiob, Giulia Carmassib, Paolo Vernierib,iovanni Serrac

Dept. Produzione Vegetale, Università degli Studi di Milano, via Celoria 2, 20133 Milan, ItalyDept. Biologia delle Piante Agrarie, University of Pisa, viale delle Piagge 23, 56124 Pisa, ItalyScuola Superiore Sant’Anna Pisa, Piazza Martiri della Libertà 33, 56100 Pisa, Italy

r t i c l e i n f o

rticle history:eceived 22 July 2010eceived in revised form 1 January 2011ccepted 7 January 2011

eywords:BAhlorophylloastalthyleneluorescencealt

a b s t r a c t

Aerosol marine strongly affects the growth and development of urban, garden and landscape plants. Fewstudies have focused on the effects of sodium chloride on plant growth, which is usually applied throughirrigation water or substrate media. Even less information is available on the eco-physiological responsesof plants to marine aerosol. The aim of this work was to evaluate the physiological responses of differentplant species to seawater nebulisation treatments. Plant species that are commonly used along sea frontswere selected as being potentially useful for the study. Plants were bought from a local nursery andincluded: Acacia cultriformis, Callistemon citrinus, Carissa edulis microphylla, Gaura lindheimeri, Jasminumsambac, Westringia fruticosa. Plants were placed in a randomised block design in a greenhouse and treatedfor 5 s with seawater or irrigation water (control) using a nebulisation system once every day for 77 days.

In the seawater aerosol-treated plants, the following parameters were monitored: leaf area damageusing an image analysis tool, chlorophyll a fluorescence, hormonal changes and cation concentrations.

pray We found that seawater aerosol treatment leads to: (i) leaf necrosis; (ii) chlorophyll loss; and (iii) adecrease in chlorophyll a fluorescence. The ion exclusion mechanism might have played a key role in thetolerance mechanism. Ethylene production increased in all species as a good biomarker and the strongincrease in ABA content in the sensitive species may play a role in the plant’s adaptation to stress.

The chlorophyll a fluorescence parameters such as Fv/Fm and the performance index were the mostaffected by treatment and led to a screening sensitive (A. cultriformis and G. lindheimeri) and tolerant (W.
fruticosa) species.
. Introduction

Plant growth along sea fronts is affected by dust depositionnd seawater aerosol (SWA), which induce severe abiotic stressn vegetation. These effects are enhanced by the increased sur-actant contamination of seawater, which reduces plant survivaln coastal areas (McCune, 1991; Bussotti et al., 1997). The influ-nce of salt spray declines with the distance from the shoreline.
he most common physiological disorders are visible through leafamage (necrosis), chlorophyll loss and growth reduction inducingwarf stature (Cheplick and Demetri, 1999; Griffiths and Orians,003). Salt spray and edaphic effects have been widely studied in
∗ Corresponding author. Tel.: +39 050 2216506; fax: +39 050 2216525.E-mail addresses: [email protected] (A. Ferrante),

[email protected] (A. Trivellini), [email protected] (F. Malorgio),[email protected] (G. Carmassi), [email protected] (P. Vernieri),[email protected] (G. Serra).

304-4238/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.scienta.2011.01.008

© 2011 Elsevier B.V. All rights reserved.

other coastal communities. Results showed that spray limits thegrowth of heathland plants, which vary in their response to saltspray (Griffiths and Orians, 2004). Plant responses to salt sprayare species dependent however common symptoms include leafappearance and morphology alteration. At an ultrastructural level,rock-rose exposed to salt spray showed changes in chloroplastmorphology associated with a reduction in starch grains (Sánchez-Blanco et al., 2004). Physiologically, plants exposed to high levelsof salt in the soil respond with an increase in osmotic potentialthrough osmolyte accumulation, protectants and antioxidants. Theorganic osmolytes that usually contribute to counteracting saltstress are amino acids, soluble sugars, proline and low-molecularweight compounds (Ashraf and Foolad, 2007). Of the plant hor-mones, ethylene and abscisic acid (ABA) are strongly induced in
salt stressed plants. Ethylene is induced by a generic stress includ-ing salt, while ABA is specifically accumulated in drought and saltstress conditions (Sha Valli Khan et al., 2007).
Plants grown along sea fronts undergo severe genetic selectionin order to gain an equilibrium in the ecosystem. One study on

dx.doi.org/10.1016/j.scienta.2011.01.008

http://www.sciencedirect.com/science/journal/03044238

http://www.elsevier.com/locate/scihorti

mailto:[email protected]






dx.doi.org/10.1016/j.scienta.2011.01.008


A. Ferrante et al. / Scientia Horticulturae 128 (2011) 332–341 333

Table 1Major ion composition in seawater used for spray application treatment (SWA), compared with seawater from different areas (Cotruvo, 2005).

Elements SWA used (mg/L) Typical seawater (mg/L) Eastern Mediterranean (mg/L)

Chloride (Cl−) 21,000 18,980 21,200Sodium (Na+) 11,300 10,556 11,800Sulfate (SO4

2−) 2800 2649 2950Magnesium (Mg2+) 746 1262 1403Calcium (Ca2+) 490 400 423Potassium (K+) 400 380 463Bicarbonate (HCO3

−) – 140 –Strontium (Sr2+) – 13 –Bromide (Br−) 7.7 65 155Borate (BO3

3−) – 26 72Fluoride (F−) –Silicate (SiO3

2−) –Iodide (I−) –Others –

Aca

cia

Car

issa

Jasm

inu

m

Cal

liste

mo

n

Wes

trin

gia

Gau

ra

0

50

100

150

a

b

b

b

a

c

Lea

f d

amag

e (%

)

Fig. 1. Leaf damage percentage in different ornamental species exposed to seawa-tsT

pttiisl

irSmasa

The following species bought from a local nursery in the Pisa

TCe

er aerosol after 77 days. Values are means with standard errors (n = 5). Data wereubjected to one-way ANOVA analysis. Differences in means were determined usingukey’s test. Different letters indicate statistical differences for P < 0.05.

lant salt resistance carried out using 29 different species showedhat the most tolerant were those naturally grown in coastal habi-ats (Sykes and Wilson, 1988). In addition to the eco-physiologicalmportance, identifying the resistance of ornamental plants to SWAs of great interest for improving the attractiveness of many touristites and ameliorating the green areas of towns and private housesocated in coastal areas.

The turnover of ornamental species available in flower marketss very high. A rapid tool is therefore important for screening plantesistance to marine aerosol and growth in coastal environments.ince leaves are the first target of SWA, chlorophyll a fluorescence
ight be a useful tool for identifying tolerant species. Chlorophyllfluorescence is a non-destructive, fast and easy to perform mea-
urement (Maxwell and Johnson, 2000). Chlorophyll a fluorescencend the parameters derived from it have often been used for study-

able 2hlorophyll content (a.u.) measured by a chlorophyll meter (Hansatech, UK) determined arrors (n = 5). Differences between controls and SWA were determined using the t-test. A

Acacia cultriformis Callistemon citrinus Carissa edulis m

Dark green

Control 22.6 ± 3.98 53.4 ± 3.57 155.7 ± 31.25SWA – 41.7 ± 6.34 52.3 ± 21.22Reduction (−) or increase (+) −20% −66P – ns *

* Young leaves.** P < 0.01.

*** P < 0.001.

1 –1 –

<1 2– –

ing plant stress in a wide range of stressful conditions (Strasser etal., 1995). The measurement of the functional status of photosys-tem II determined by chlorophyll a fluorescence has been used tolead the genetic improvement of beans for heat resistance (Petkovaet al., 2007).

This research was carried out to investigate the effect of seawa-ter aerosol on the heath of the leaves of six ornamental species. Theaim was to investigate the effects of saltwater on the leaf area ofornamental species characterized by different levels of resistance tosalt stress. Plants with resistance to seawater aerosol are required intourist areas and private gardens located in coastal areas. Ornamen-tal plants are usually used for decoration along roads, roundabouts,around hotels or houses. In all these situations irrigation systemsare used, thus the roots of these plants are not usually exposedto salty water. Consequently we focused on the effects of seawa-ter on leaves of plants that were no subject to salt stress at theroots. The species used in this work were selected considering theirdifferent levels of salt tolerance. Gaura lindheimeri and Acacia cul-triformis were the most sensitive and Westringia fruticosa the mosttolerant, while the others had an intermediate level of salt sensitiv-ity. Chlorophyll a fluorescence was used to test its application as arapid screening tool for identifying f ornamental shrub tolerance orsensitivity to salt spray. Mineral content and plant hormones weresimultaneously determined in treated plants in order to understandthe physiological changes that plants adopt in response to seawateraerosol.

2. Materials and methods

2.1. Plant material

(Italy) area for use in the study: A. cultriformis Cunn. ex G. Don,Callistemon citrinus (Curtis) Skeels, Carissa edulis var. microphyllaPichon, G. lindheimeri Engelm. and A. Gray, Jasminum sambac (L.)Ait., W. fruticosa (Willd.) Druce. Plants were grown in 3 L plastic pots

fter 77 days of exposure to seawater aerosol. Data are reported as means ± standardsterisks represent statistical differences.

icrophylla Gaura lindheimeri Jasminum sambac Westringia fruticosa

Light green Blue Green

49.5 ± 4.13 4.0 ± 0.26 10.1 ± 0.9 82.3 ± 6.13 5.4 ± 0.4518.7 ± 3.72 12.6 ± 2.35* – 27.7 ± 11.17 3.2 ± 1.02−60 +70* −66 −40*** ** – ** ns


334 A. Ferrante et al. / Scientia Horticulturae 128 (2011) 332–341

0.0

0.2

0.4

0.6

0.8

ControlSWA

Acacia

*

Fv/

Fm

0.0

0.2

0.4

0.6

0.8

1.0Carissa

Fv/F

m

0.0

0.2

0.4

0.6

0.8 Gaura

**

Fv/

Fm

0 20 40 60 800.0

0.2

0.4

0.6

0.8Jasminum

* **

Time (d)

Fv/

Fm

0 20 40 60 80 1000.0

0.2

0.4

0.6

0.8

1.0Westringia

Time (d)

Fv/F

m

0.0

0.2

0.4

0.6

0.8

1.0Callistemon

Fv/F

m

F entalD A wer*

ct(wtaf

atpwt(wc0w

2

uplP(b(

ig. 2. Maximum quantum efficiency of photosystem II (Fv/FM) in different ornamata are means with standard errors (n = 5). Differences between controls and SW

*P < 0.01.

ontaining peat/pumice (1:1, v/v) in an open greenhouse belongingo the Department of Biologia delle Piante Agrarie, University of Pisa43◦42.343′N and 10◦25.280′E). During the spring of 2009, plantsere placed in a randomised block design with five plants for each

reatment in an open greenhouse with only the roof covered withlong life PE film 80 �m for 18 months in order to prevent any rain

rom washing the plants.Treatments with seawater or irrigation water (control) were

pplied once every day for 5 s using a nebulisation system, for aotal of 77 days. The seawater composition was analysed after itsreparation and the chemical composition was compared (Table 1)ith the mineral content of natural seawater (Cotruvo, 2005). Fer-

ilization was performed using a slow release 3 g L−1 Osmocote12:12:30) 8-month release. All plants were watered twice a dayith a drip irrigation that provided 120 g per plant−1. The mineral

omposition of the irrigation water was mM: Na 9.5, Ca 1.52, Mg.74, Cl 9.8, S 0.59; �M: Fe 37.4, B 20.4, Zn 2.1, Mn 2.7, HCO3

− 5.3ith pH 7.2 and EC (dS/m−1) 1.2.

.2. Chlorophyll content and chlorophyll a fluorescence

The chlorophyll content was colorimetrically measured in vivosing a non-destructive instrument (CL-01, Hansatech, UK). Chloro-hyll a fluorescence transients were determined on dark adapted

eaves kept for 30 min at room temperature, using a portable HandyEA (Hansatech, UK). Measurements were taken of the leaf surface4 mm diameter) exposed to an excitation light intensity (ultra-right red LEDs with a peak at 650 nm) of 3000 �mol m−2 s−1

600 W m−2) emitted by three diodes. Leaf fluorescence detec-

species after 77 days of treatment with water (control) or seawater aerosol (SWA).e determined using the t-test. Asterisks represent statistical differences, *P < 0.05,

tion was measured by a fast response PIN photodiode with a RG9long pass filter (Hansatech, technical manual). The parametersmeasured were Fo, Fm and Fv/Fm. JIP analysis was performed todetermine the following indices: performance index (PI), trappedenergy flux per CS (TRo/CS), electron transport flux per CS (ETo/CS),dissipation of energy per CS (DIo/CS), and density of reaction cen-tres at P stage (RC/CSm).

2.3. Abscisic acid and ethylene measurements

ABA was determined by an indirect ELISA based on the useof DBPA1 monoclonal antibody, raised against S(+)-ABA (Vernieriet al., 1989). The ELISA was performed following Walker-Simmons(1987), with minor modifications. Leaf samples (100 mg FW) werecollected, weighed, frozen in liquid nitrogen, then stored at −80 ◦Cuntil the analysis. ABA was measured after extraction in distilledwater (water:tissue ratio = 10:1, v:w) overnight at 4 ◦C. Plates werecoated with 200 �L per well ABA-4′-BSA conjugate and incubatedovernight at 4 ◦C, then washed three times with 75 mM PBS buffer,pH 7.0, containing 1 g L−1 BSA and 1 mL L−1 Tween 20, keeping thethird washing solution for 30 min at 37 ◦C. Next 100 �L ABA stan-dard solution or sample and, subsequently, 100 �l DBPA1 solution(lyophilized cell culture medium diluted in PBS buffer containing10 g L−1 BSA and 0.5 mL L−1 Tween 20, at a final concentration of
50 �g/mL) were added to each well and competition was allowedto occur at 37 ◦C for 30 min. The plates were then washed againas described above and 200 �l per well of a secondary antibody(alkaline phosphatase-conjugated rabbit anti-mouse (Sigma, Italy)in a PBS buffer containing 10 g L−1 BSA and 0.5 mL L−1 Tween 20, at



F t orna( and S*

aan36p

tftsPcdc

2

awis

2

uT

ig. 3. Trapped energy per cross-section (TRo/CS) derived from JIP tests in differenSWA). Data are means with standard errors (n = 5). Differences between controlsP < 0.05, **P < 0.01, and ***P < 0.001.

final dilution of 1:2000) were added and incubated for 30 mint 37 ◦C. The plates were washed again and 200 �L per well p-itrophenyl phosphate were added and incubated for 30 min at7 ◦C. Absorbance readings at 415 nm were obtained using an MDL80 Perkin–Elmer microplate reader. For each treatment, four inde-endent samples were assayed in triplicate.

Ethylene production was measured by enclosing leaves in air-ight containers (250 mL). Two millilitres gas samples were takenrom the headspace of the containers after 1 h incubation at roomemperature. The ethylene concentration in the sample was mea-ured by a gas chromatograph (HP5890, Hewlett-Packard, Menloark, CA) using a flame ionization detector (FID), a stainless steelolumn (150 cm × 0.4 cm ∅ packed with Hysep T), column andetector temperatures of 70 and 350 ◦C, respectively, and nitrogenarrier gas at a flow rate of 30 mL min−1.

.4. Leaf mineral content determination

The dry weight of samples (10 g) was determined and miner-lised (60 min at 220 ◦C) using nitric and perchloric acids. Cationsere determined using an atomic absorption spectrometer (Var-

an AA 24FS, Australia). The following cations were determined:odium (Na), potassium (K), calcium (Ca), and magnesium (Mg).

.5. Leaf damage analysis

Leaf damage was measured as a percentage of the total areasing the image processing and analysis program UTHSCSA Image-ool ver. 3 (http://ddsdx.uthscsa.edu/dig/itdesc.html).

mental species after 77 days of treatment with water (control) or seawater aerosolWA were determined using the t-test. Asterisks represent statistical differences,

2.6. Statistical analysis

The data in the tables and figures are means and standarderrors (n = 5). Differences between controls and treatments withseawater were determined by t-test analysis. The data presented inFig. 1 were subjected to one-way ANOVA analysis. The differencesbetween means were determined using Tukey’s post-test.

3. Results

The salt treatment affected the physiology and biochemicalparameters of plants differently. The first visible symptom of saltstress was a reduction in chlorophyll and leaf necrosis (Table 2).

3.1. Leaf damage, chlorophyll and chlorophyll a fluorescence

A. cultriformis and G. lindheimeri showed 100% leaf necrosis(Fig. 1) while W. fruticosa was not affected by SWA throughout theentire experimental period. C. edulis and C. citrinus showed severeleaf damage ranging from 46% to 50% of the total leaf area. On theother hand, J. sambac showed 34% leaf damage.

Chlorophyll declined in all the species but a higher reductionwas observed in C. edulis var. microphylla and J. sambac. These werespecies with a higher chlorophyll content. W. fruticosa showed anintermediate level of loss of chlorophyll (Table 2).

The chlorophyll a fluorescence parameters measured in dark-adapted leaves were affected differently according to the species.The maximum yield of PSII (Fv/Fm ratio) decreased after 2–3 weeksin G. lindheimeri and in A. cultriformis to 0.4–0.5, before leaf necrosis.In the sensitive species, Fv/Fm rapidly declined under SWA. In W.

http://ddsdx.uthscsa.edu/dig/itdesc.html



0

400

800

1200

1600 AcaciaSWAControl

RC

/CS

m (

a.u

.)

0

500

1000

1500

2000Carissa

*

RC

/CS

m (a.u

.)

0

500

1000

1500Gaura

***

RC

/CS

m (

a.u

.)

0

500

1000

1500Callistemon

*

RC

/CS

m (a.u

.)

0 20 40 60 800

400

800

1200

1600 Jasminum

******

**

Time (d)

RC

/CS

m (

a.u

.)

0 20 40 60 80 1000

500

1000

1500

2000

2500Westringia

**

Time (d)

RC

/CS

m (a.u

.)

F sts ins tweend

ft(

SaietttcGwrt

im2

lo(tad

ca

ig. 4. Density of reaction centres per cross-section (RC/CSm) derived from JIP teeawater aerosol (SWA). Data are means with standard errors (n = 5). Differences beifferences, *P < 0.05, **P < 0.01, and ***P < 0.001.

ruticosa Fv/Fm was not influenced by salt stress and after 77 dayshe plants under treatment showed higher values than the controlsFig. 2).

Trapped energy per cross-section (TRo/CS) was lower in theWA treated plants. The initial TRo/CS values were between 400nd 600. Higher differences between controls and SWA were foundn C. edulis, and TRo/CS progressively declined after 30 days ofxposure (Fig. 3). TRo/CS increased only in W. fruticosa with bothreatments and values were over 600. The density of active reac-ion centres per cross-section (RC/CSm) was lower in salt sprayreated plants at the beginning of the experiment, except for A.ultriformis and W. fruticosa that did not show any differences. In. lindheimeri RC/CSm drastically declined during the first threeeeks of SWA exposure. After two months in J. sambac and C. cit-

inus, there were no statistically different RC/CSm levels in SWA orhe controls (Fig. 4).

The electron transport flux per cross-section (ETo/CS) decreasedn the stressed plants. ETo/CS was higher in A. Cultriformis, approxi-

ately 400, while in all other species the initial values ranged from00 to 300 at the beginning of the experiment (Fig. 5).

The dissipation energy per cross-section (DIo/CS) increased in G.indheimeri under the SWA treatment. Significant differences werebserved in J. sambac during the first month of the experimentFig. 6). The performance index (PI) declined drastically in the firsthree weeks in A. cultriformis and G. lindheimeri. The PI in C. citrinus
nd J. sambac decreased in the first month then recovered and noifferences were observed between the controls and SWA (Fig. 7).
At the end of the experimental period, chlorophyll a fluores-ence was also measured under modulated light conditions usingportable FMS2 instrument. In all species, the quantum yield of

different ornamental species after 77 days of treatment with water (control) orcontrols and SWA were determined using the t-test. Asterisks represent statistical

photosystem II (FPS2) in light adapted leaves was lower in saltstressed plants, but statistically significant differences were onlyobserved in J. sambac plants. The highest relative yield was observedin C. edulis leaves (Fig. 8A). The SWA treatments affected the non-photochemical quenching (NPQ) differently in the six species. Ahigher value (1.3) was found in J. sambac stressed plants (Fig. 8B).Photochemical quenching (qP) was lower in all species exposed toSWA treatments compared to controls, but statistical differenceswere only found in C. edulis (Fig. 8C). The electron transport rate(ETR) was variable in the salt stress plants as can be observed bythe error bars. Significant differences between controls and SWAtreated plants were found in C. citrinus (Fig. 8D).

3.2. ABA content and ethylene production

The endogenous ABA concentration was higher in SWA sen-sitive species and its accumulation in the leaves was more thandouble. In A. cultriformis stressed plants, leaf ABA levels were2.5-fold higher after 21 days than in the controls. G. lindheimerishowed a 4-fold increase compared with controls. Species withan intermediate tolerance accumulated less ABA in response tothe salt treatment. Higher ABA levels were observed in C. edulisand J. sambac control plants, showing 131 and 139 ng g−1 FW,respectively. Tolerant plants did not show any significant dif-ference in leaf ABA content (Fig. 9A). Ethylene production was
higher in the SWA treatment in all species except C. citrinus.Ethylene production in control plants was below 1000 pl h−1 g−1
FW in all species, with the exception of G. lindheimeri whichhad an average of 1555 pl h−1 g−1 FW (Fig. 9B). SWA treat-ment greatly enhanced ethylene production. Only in C. citrinus



0

100

200

300

400

500Acacia

ControlSWA

ETo

/CS

(a.

u.)

0

100

200

300

400Carissa

****

ETo

/CS

(a.u.)

0

100

200

300Gaura

***

ETo

/CS

(a.

u.)

100

150

200

250

300

350Callistemon

ETo

/CS

(a.u.)

0 20 40 60 800

100

200

Jasminum

*****

Time (d)

ETo

/CS

(a.

u.)

0 20 40 60 80 1000

100

200

300Westringia

Time (d)

ETo

/CS

(a.u.)

F ifferea rols a*

wp

3

ecarShslAb(

4

koarAhhcmMa

ig. 5. Electron transport flux per cross-section (ETo/CS) derived from JIP tests in derosol (SWA). Data are means with standard errors (n = 5). Differences between contP < 0.05, **P < 0.01, and ***P < 0.001.

ere no significant differences found with respect to the controllants.

.3. Leaf mineral content

The mineral analyses of dry leaves harvested at the end of thexperiments showed a high level of Na in all the treated plants. A.ultriformis and G. lindheimeri, which desiccated after three weeks,bsorbed the highest levels of Na, 48.5 g kg−1 and 64.9 g kg−1 DW,espectively. Other mineral elements were affected differently byWA depending on the species. The K content decreased in G. lind-eimeri and W. fruticosa, while it increased in C. edulis. In otherpecies no significant differences were observed. Ca content wasower in treated plants and significant differences were found in. cultriformis, G. lindheimeri and W. fruticosa. The Mg content dou-led in almost all salt-treated plants except C. edulis and W. fruticosaFig. 10).

. Discussion

The negative effect of salinity on photosynthesis has long beennown. However, only a few papers have provided measurementsf chlorophyll a fluorescence in plants exposed to SWA. In our studyll species exposed to SWA for 77 days showed a visible and drasticeduction in chlorophyll content ranging from 20% to 66%, except. cultriformis and G. lindheimeri (Table 1). The leaves of G. lind-eimeri were completely desiccated after three weeks of treatment,
owever after five weeks new sprouts developed, and were rich inhlorophyll and anthocyanins (data not shown). Anthocyanin accu-ulation induced by saline condition has previously been seen inorus alba and sugarcane leaves (Ramanjulu et al., 1993; Wahid
nd Chazanfar, 2006). Leaf necrosis and chlorophyll reduction are a

nt ornamental species after 77 days of treatment with water (control) or seawaternd SWA were determined using the t-test. Asterisks represent statistical differences,

common result of severe salt stress (Ashraf, 2009), and older leavesare more susceptible in salt sensitive plants (Parvaiz and Satyawati,2008).

SWA affected the maximal photochemical activity of PSII(expressed as Fv/Fm), measured after dark adaptation in Acacia andGaura plants, indicating that these are the most sensitive speciesin which the salt residues on leaves led to sustained photo-damageand phytotoxicity. Similar results have been obtained through saltywater (200 mM NaCl) irrigation in C. citrinus showing a signifi-cant reduction in Fm and Fv/Fm especially when air temperatureincreased (Mugnai et al., 2009). In contrast, W. fruticosa plantsunder SWA were clearly much more resistant to salinity stress.This resistance was due to the photochemical efficiency (Fv/Fm) ofPSII in dark-adapted leaves, which did not change after 77 days ofstress application. Moreover, SWA spray treatments in C. citrinus, C.edulis and J. Sambac, which had an intermediate tolerance, showedslightly lower chlorophyll a fluorescence efficiency, although thevalues were not statistically significant.

In general, in treated plants showing a high (A. cultriformis andG. lindheimeri) and intermediate sensitivity (C. citrinus, C. edulis andJ. sambac) to SWA, the chlorophyll content and the Fv/Fm ratioboth decreased. These results suggest that reduced plant pigmentmay represent a possible mechanism to protect PSII against pho-toinhibition through a reduction in the number of light-harvestingantennae and thus affecting the photochemical efficiency of PSII.

Of the various approaches to the analysis of chlorophyll a fluo-rescence values, the JIP-test (Strasser and Strasser, 1995; Strasser
et al., 2000, 2010), has been frequently employed to understand theresponses of the photosynthetic apparatus to different physiologi-cal, genetic and environmental conditions (Ferrante and Maggiore,2007; Mugnai et al., 2009). In this study we used the followingderived parameters: trapping (TR) of excitation energy, electron



F nt orn( and S*

ts(httaaR

edntsastbeloya(es

A

ig. 6. Dissipation energy per cross-section (DIo/CS) derived from JIP tests in differeSWA). Data are means with standard errors (n = 5). Differences between controlsP < 0.05.

ransport (ETR), dissipation energy (DI), reaction center (RC) perample area called cross-section (CS), and the performance indexPI). It seems that the higher susceptibility, especially of G. lind-eimeri plants, to SWA may be related to a decrease in the electronransport per excited cross-section (ETo/CS), due to an inactiva-ion of the reaction center (Mehta et al., 2010). The decrease in themount of active reaction centres per excited cross-section (RC/CS)lso supported the idea that active RCs are converted into inactiveCs.

We believe that the decrease in active reaction centres perxcited cross-section (RC/CS) may be associated with an increasedissipation efficiency of absorbed light as heat, as shown by the sig-ificantly higher value of this parameter (DIo/CS) which increasedhe sensitivity of G. lindheimeri. In contrast, the most tolerantpecies W. fruticosa showed an increase in the number of active RCsnd a decrease in energy dissipation. These results taken togetheruggest that physiological parameters, associated with the func-ioning of the photosynthetic apparatus, may be used as reliableiomarkers to discriminate between species with a different tol-rance to SWA. The chlorophyll fluorescence measured from theight adapted leaves of Lycium nodosum subjected to saline sprayr saline irrigation, revealed a reduction in the relative quantumield of PSII, electron transport rate, photochemical quenching andn increase in the non-photochemical quenching of fluorescence
Tezara et al., 2003). Our results are in agreement with the lit-rature, and in most cases the differences were not statisticallyignificant.
An analysis of the endogenous levels of plant hormones such asBA and ethylene revealed a general pattern of hormonal changes

amental species after 77 days of treatment with water (control) or seawater aerosolWA were determined using the t-test. Asterisks represent statistical differences,

in salt-stressed plants (Gomez-Cadenas et al., 1998; Zapata et al.,2007). Therefore, ABA and ethylene are involved as modulators ofsome of the responses to salt stress (Gomez-Cadenas et al., 1998).In our study the ABA content and ethylene production in the mostsensitive species to salt spray, such as Acacia and Gaura plants,increased about two fold and one fold or less respectively. Jasminumand Carissa, which showed an intermediate tolerance to SWA, hada significantly higher increase in ethylene production (about fourfold). On the other hand, the ABA contents decreased significantly(one fold or less) under salt spray. No changes in ABA content, anda slight increase in ethylene evolution were found in Westringiaplants, which have previously shown the highest tolerance to SWA.

ABA is well known to be normally involved in plant droughtstress and to regulate stomata opening, thus playing a crucial role inplant adaptation to stress (Chaves et al., 2009). Salt stressed plantsaccumulate ABA in relation to their salt tolerance (Xiong and Zhu,2003; Perales et al., 2005) and ABA may ameliorate plant toleranceto salinity by its enhancement (Zhu, 2001; Ahmad et al., 2009).Moreover, the application of exogenous ABA appears to enhanceresistance in bean and wheat plants (Khadri et al., 2006; Gurmaniet al., 2009). Thus, the higher fold induction in ABA content in thesensitive species to SWA in our study, suggests a role in plant adap-tation to stress by developing a sort of tolerance in these plants.

On the other hand, the highest tolerant species to SWA, such
as Westringia, did not accumulate ABA, and this may result in itsintrinsic adaptability to the SWA environment. In the species withan intermediate salt spray tolerance, the hormonal balance seemsto operate in two distinct ways. In Callistemon, ABA and ethylenewere not involved in the adaptation to stress, in fact neither ABA



0

1

2

3Acacia

ControlSWA

PI (

a.u

.)

0

1

2

3Carissa

PI (a.u

.)

0.0

0.5

1.0

1.5

2.0

2.5Gaura

***PI (

a.u

.)

0.0

0.5

1.0

1.5

2.0

2.5

Callistemon

PI (a.u

.)

0 20 40 60 800.0

0.2

0.4

0.6

0.8Jasminum

******

*

Time (d)

PI (

a.u

.)

0 20 40 60 80 1000

1

2

3

4

Westringia

Time (d)

PI (a.u

.)

F es aftm termia

nCic

Fq(

ig. 7. Performance index (PI) derived from JIP tests in different ornamental specieans with standard errors (n = 5). Differences between controls and SWA were de

nd ***P < 0.001.

or ethylene showed any differences compared to the control. Inarissa and Jasminum, it seems that the dramatic enhancement

n ethylene production might have been antagonized by the ABAontent. The antagonism between ABA and ethylene, which recip-

0.0

0.2

0.4

0.6

0.8

1.0

**

A

ΦP

S2

(a.u

.)

C

Car

issa

Cal

liste

mo

n

Jasm

inu

m

Wes

trin

gia

0.0

0.5

1.0

1.5

2.0 ***ControlSWA

B

NP

Q (

a.u

.)

D

ig. 8. Chlorophyll a fluorescence parameters under modulate light conditions in ornamuantum yield of photosystem II, (B) non-photochemical quenching, (C) photochemicaln = 5). Differences between controls and SWA were determined using the t-test. Asterisk

er 77 days of treatment with water (control) or seawater aerosol (SWA). Data arened using the t-test. Asterisks represent statistical differences, *P < 0.05, **P < 0.01,

rocally regulate each other’s metabolism and signaling pathway,has been reported in a study on the antithetic crosstalk of thesetwo hormones upon seed germination and early seedling growthin Arabidopsis (Cheng et al., 2009).

0.0

0.2

0.4

0.6

0.8

1.0

***

qP

(a.u.)

Car

issa

Cal

liste

mo

n

Jasm

inu

m

Wes

trin

gia

0

20

40

60

*** ET

R (a.u

.)

ental plants after 77 days of treatment with seawater aerosol (SWA). (A) Relativequenching, and (D) electron transport rate. Data are means with standard errors

s represent statistical differences, **P < 0.01 and ***P < 0.001.


340 A. Ferrante et al. / Scientia Horticu

0

50

100

150

200

**

n.s.***

**

*

n.s.

AA

BA

(n

g g

-1 F

W)

Aca

cia

Cal

liste

mo

n

Car

issa

Gau

ra

Jasm

inu

m

Wes

trin

gia

0

1000

2000

3000

4000

5000

***n.s.

***

**

***

***

ControlSWA

B

Eth

ylen

e (p

l h-1

g-1

FW

)

Fig. 9. (A) Abscisic acid (ABA) content in leaves and (B) ethylene production from dif-ferent ornamental plants after 77 days of treatment with water (control) or seawateraerosol (SWA). Data are means with standard errors (n = 5). Differences betweencontrols and SWA were determined using the t-test. Asterisks represent statisticaldifferences, *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 10. Concentration of cations (Na, K, Ca and Mg) in leaves of different ornamental pData are means with standard errors (n = 5). Differences between controls and SWA wer**P < 0.01, and ***P < 0.001.

lturae 128 (2011) 332–341

A potential biomarker of salinity tolerance is the adsorption andaccumulation of ions in the leaves of plants, which can be affectedby the selection pressure of the surrounding environment. In thisstudy, cations were monitored in order to study the interactionsbetween Na and other macroelements that are essential for normalplant growth. Na content significantly increased in all the speciesstudied, but less in Westringia, whereas the other macroelementsshowed a species-specific behaviour.

In general salt accumulation on leaves decreased the Ca lev-els, with the exception of Jasminum. K levels increased in Carissaand Acacia, and decreased in Gaura and Westringia. The Mg con-tent increased in sensitive or intermediate tolerant plants, while alower concentration was found in tolerant species. Previous stud-ies have shown that Ca and K uptake are negatively affected by asaline environment. Wheat plants grown in media containing NaClconcentrations showed a reduction in Ca (Gurmani et al., 2009),and cucumber grown under salt conditions manifested a decreasein K (Zhu, 2003; Huang et al., 2009). Under salt conditions the Mgconcentration in plant tissues may increase or decrease, dependingon the organs or on the species (Marosz and Nowak, 2008; Keutgenand Pawelzik, 2008).

In our study, the species considered most tolerant (Westringia)to SWA showed a lower increase in Na content compared to the sen-sitive and intermediate species. Therefore, considering that othermacroelements have shown species-specific behaviors, it is possi-ble that an ion exclusion mechanism might play a key role in the
tolerance strategy to SWA in these six different species.
lants after 77 days of treatment with water (control) or seawater aerosol (SWA).e determined using the t-test. Asterisks represent statistical differences, *P < 0.05,


orticu

5

msOberaapwp

A

I

R

A

A

A

B

C

C

C

C

F

G

G

G

G

H

A. Ferrante et al. / Scientia H

. Conclusions

The differences in six ornamental species exposed to SWA wereore evident over time. Our results showed that the most sensitive

pecies to seawater aerosol were A. cultriformis and G. lindheimeri.n the other hand, W. fruticosa was the most tolerant, while J. sam-ac, C. edulis and C. citrinus showed an intermediate behaviour. Ourxperiments enabled us to demonstrate how chlorophyll a fluo-escence parameters significantly changed under SWA stress. Inddition, the results from the JIP test contributed to the evalu-tion of SWA tolerance in the species studied. The fluorescencearameters that best described the physiological plant behaviourere the Fv/Fm that measure the maximum quantum yield of PSIIhotochemistry, and the performance index (PI).

cknowledgement

This work was part of the EcoIdriFlor project supported by thetalian Ministry of Agriculture and Forestry (MIPAF).

eferences

hmad, M.S.A., Ali, Q., Ashraf, M., Haider, M.Z., Abbas, Q., 2009. Involvement ofpolyamines, abscisic acid and anti-oxidative enzymes in adaptation of Blue Pan-icgrass (Panicum antidotale Retz.) to saline environments. Environ. Exp. Bot. 66,409–417.

shraf, M., Foolad, M.R., 2007. Roles of glycine betaine and proline in improvingplant abiotic stress resistance. Environ. Exp. Bot. 59, 206–216.

shraf, M.Y., 2009. Salt tolerance mechanisms in some halophytes from Saudi Arabiaand Egypt. Res. J. Agric. Biol. Sci. 5 (3), 191–206.

ussotti, F.A., Bottacci, P., Grossoni, B., Mori, C.T., 1997. Cytological and structuralchanges in Pinus pinea L. needles following the application of anionic surfactant.Plant Cell Environ. 20, 513–520.

haves, M.M., Flexas, J., Pinheiro, C., 2009. Photosynthesis under drought and saltstress: regulation mechanisms from whole plant to cell. Ann. Bot. 103, 551–560.

heng, W., Chiang, M.H., Hwang, S.G., Lin, P.C., 2009. Antagonism between abscisicacid and ethylene in Arabidopsis acts in parallel with the reciprocal regulationof their metabolism and signaling pathways. Plant Mol. Biol. 71, 61–80.

heplick, G.P., Demetri, H., 1999. Impact of saltwater spray and sand deposition onthe coastal annual Triplasis purpurea (Poaceae). Am. J. Bot. 86 (5), 703–710.

otruvo, J.A., 2005. Water desalinization processes and associated health and envi-ronmental issues. Water Cond. Purif. 1, 13–17.

errante, A., Maggiore, T., 2007. Chlorophyll a fluorescence measurements to evalu-ate storage time and temperature of Valeriana leafy vegetables. Postharvest Biol.Technol. 45, 73–80.

omez-Cadenas, A., Tadeo, F.R., Primo-Millo, E., Talon, M., 1998. Involvement ofabscisic acid and ethylene in the responses of citrus seedlings to salt shock.Physiol. Plant. 103, 475–484.

riffiths, M.E., Orians, C.M., 2003. Salt spray differentially affects water status, necro-sis, and growth in coastal sandplain heathland species. Am. J. Bot. 90, 1188–1196.

riffiths, M.E., Orians, C.M., 2004. Salt spray effects on forest succession in rarecoastal sandplain heathlands: evidence from field surveys and Pinus rigida trans-plant experiments. J. Torrey Bot. Soc. 131, 23–31.

urmani, A.R., Bano, A., Din, J., Khan, S.U., Hussain, I., 2009. Effect of phytohormoneson growth and ion accumulation of wheat under salinity stress. Afr. J. Biotechnol.8 (9), 1887–1894.

uang, Y., Tang, R., Cao, Q., Bie, Z., 2009. Improving the fruit yield and quality ofcucumber by grafting onto the salt tolerant rootstock under NaCl stress. Sci.Hortic. 122, 26–31.

lturae 128 (2011) 332–341 341

Keutgen, A.J., Pawelzik, E., 2008. Quality and nutritional value of strawberry fruitunder long term salt stress. Food Chem. 107, 1413–1420.

Khadri, M., Tejera, N.A., Lluch, C., 2006. Alleviation of salt stress in common bean(Phaseolus vulgaris) by exogenous abscisic acid supply. J. Plant Growth Regul. 25(2), 110–119.

Marosz, A., Nowak, J.S., 2008. Effect of salinity stress on growth and macroelementsuptake of four tree species. Dendrobiology 59, 23–29.

Maxwell, K., Johnson, G.N., 2000. Chlorophyll fluorescence—a practical guide. J. Exp.Bot. 51, 659–668.

McCune, D.C., 1991. Effects of airborne saline particles on vegetation in relation tovariables of exposure and other factors. Environ. Pollut. 74, 176–203.

Mehta, P., Jajoo, A., Mathur, S., Bharti, S., 2010. Chlorophyll a fluorescence studyrevealing effects of high salt stress on Photosystem II in wheat leaves. PlantPhysiol. Biochem. 48, 16–20.

Mugnai, S., Ferrante, A., Petrognani, L., Serra, G., Vernieri, P., 2009. Stress-inducedvariation in leaf gas exchange and chlorophyll a fluorescence in Callistemonplants. Res. J. Biol. Sci. 4, 913–921.

Parvaiz, A., Satyawati, S., 2008. Salt stress and phyto-biochemical responses ofplants—a review. Plant Soil Environ. 54, 89–99.

Perales, L., Arbona, V., Gomez-Cadenas, A., Cornejo, M.J., Sanz, A., 2005. A relationshipbetween tolerance to dehydration of rice cell lines and ability for ABA synthesisunder stress. Plant Physiol. Biochem. 43, 779–786.

Petkova, V., Denev, I.D., Cholakov, D., Porjazov, I., 2007. Field screening for heat tol-erant common bean cultivars (Phaseolus vulgaris L.) by measuring of chlorophyllfluorescence induction parameters. Sci. Hortic. 111, 101–106.

Ramanjulu, S., Veeranjaneyulu, K., Sudhakar, C., 1993. Physiological changes inducedby NaCl in mulberry var. Mysore local. Ind. J. Plant Physiol. 36, 273–275.

Sánchez-Blanco, M.J., Rodrìguez, P., Olmos, E., Morales, M.A., Torrecillas, A., 2004. Dif-ferences in the effects of simulated sea aerosol on water relations, salt content,and leaf ultrastructure of rock-rose plants. J. Environ. Qual. 33, 1369–1375.

Sha Valli Khan, P.S., Hoffmann, L., Renaut, J., Hausman, J.F., 2007. Current initiativesin proteomics for the analysis of plant salt tolerance. Curr. Sci. 93, 807–817.

Strasser, B.J., Strasser, R.J., 1995. Measuring fast fluorescence transients to addressenvironmental questions: the JIP-test. In: Mathis, P. (Ed.), Photosynthesis: FromLight to Biosphere. Kluwer Academic Publishers, Dordrecht, pp. 977–980.

Strasser, R.J., Srivastava, A., Govindjee, 1995. Polyphasic chlorophyll a fluorescencetransient in plants and cyanobacteria. Photochem. Photobiol. 61, 32–42.

Strasser, R.J., Srivastava, A., Tsimilli-Michael, M., 2000. The fluorescence transient asa tool to characterize and screen photosynthetic samples. In: Yunus, M., Pathre,U., Mohanty, P. (Eds.), Probing Photosynthesis: Mechanisms, Regulation andAdaptation. Taylor & Francis, London, pp. 445–483.

Strasser, R.J., Tsimilli-Michael, M., Srivastava, A., 2010. Analysis of the chlorophylla fluorescence transient. In: Papageorgiou, G., Govindjee (Eds.), Chloro-phyll a Fluorescence: A Signature of Photosynthesis. Springer, Dordrecht, pp.321–362.

Sykes, M.T., Wilson, J.B., 1988. An experimental investigation into the response ofsome New Zealand sand dune species to salt spray. Ann. Bot. 62, 159–166.

Tezara, W., Martínez, D., Rengifo, E., Herrera, A., 2003. Photosynthetic responses ofthe tropical spiny shrub Lycium nodosum (Solanaceae) to drought, soil salinityand saline spray. Ann. Bot. 92, 757–765.

Vernieri, P., Perata, P., Armellini, D., Bugnoli, M., Presentini, R., Lorenzi, R., Ceccarelli,N., Alpi, A., Tognoni, F., 1989. Solid phase radioimmunoassay for the quantitationof abscisic acid in plant crude extracts using a new monoclonal antibody. J. PlantPhysiol. 134, 441–446.

Wahid, A., Chazanfar, A., 2006. Possible involvement of some secondary metabolitesin salt tolerance of sugarcane. J. Plant Physiol. 163, 723–730.

Walker-Simmons, M., 1987. ABA levels and sensitivity in developing wheat embryosof sprouting resistant and susceptible cultivars. Plant Physiol. 84, 61–66.

Xiong, L., Zhu, J.K., 2003. Regulation of abscisic acid biosynthesis. Plant Physiol. 133,29–36.

Zapata, P.J., Botella, M.A., Pretel, M.T., Serrano, M., 2007. Responses of ethylenebiosynthesis to saline stress in seedlings of eight plant species. Plant GrowthRegul. 53, 97–106.

Zhu, J.K., 2001. Plant salt tolerance. Trends Plant Sci. 6, 66–71.Zhu, J.K., 2003. Regulation of ion homeostasis under salt stress. Curr. Opin. Plant Biol.

6, 441–445.

effect of seawater aerosol on leaves of six plant species potentially useful for ornamental purposes...

Documents