Environmental and Experimental Botany 86 (2013) 76– 85
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Environmental and Experimental Botany
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ibberellic acid mediated induction of salt tolerance in wheat plants: Growth,onic partitioning, photosynthesis, yield and hormonal homeostasis
uhammad Iqbala,∗, Muhammad Ashrafb,c
Department of Botany, Government College University, Faisalabad 38000, PakistanDepartment of Botany, University of Agriculture, Faisalabad 38040, PakistanKing Saud University, Riyadh, Saudi Arabia
r t i c l e i n f o
rticle history:eceived 12 October 2009eceived in revised form 6 June 2010ccepted 16 June 2010
In order to elucidate the GA3-priming-induced physiochemical changes responsible for induction of salttolerance in wheat, the primed and non-primed seeds of two spring wheat (Triticum aestivum L.) cul-tivars, namely, MH-97 (salt intolerant) and Inqlab-91 (salt tolerant) were sown in a field treated with15 dS m−1 NaCl salinity. Although all the three concentrations (100, 150 and 200 mg L−1) of GA3 wereeffective in improving grain yield in both cultivars, the effect of 150 mg L−1 GA3 was much pronouncedparticularly in the salt intolerant cultivar when under salt stress. Seed priming with GA3 altered thepattern of accumulation of different ions between shoots and roots in the adult plants of wheat undersaline conditions. Treatment with GA3 (150 mg L−1) decreased Na+ concentrations both in the shootsand roots and increased Ca2+ and K+ concentrations in the roots of both wheat cultivars. GA3-primingdid not show consistent effect on gaseous exchange characteristics and the concentrations of auxins inthe salt stressed plants of both wheat cultivars. However, all concentrations of GA3 reduced leaf freeABA levels in the salt intolerant, while reverse was true in the salt tolerant cultivar under saline condi-tions. Priming with GA3 (150 mg L−1) was very effective in enhancing salicylic acid (SA) concentrationin both wheat cultivars when under salt stress. Treatment with GA3 (100–150 mg L−1) lowered leaf free
putrescine (Put) and spermidine (Spd) concentrations in the plants of both wheat cultivars. The decreasein polyamines (Put and Spd) and ABA concentrations in the salt stressed plants of the salt intolerant cul-tivar treated with GA3 suggested that these plants might have faced less stress compared with control.Thus, physiologically, GA3-priming-induced increase in grain yield was attributed to the GA3-priming-induced modulation of ions uptake and partitioning (within shoots and roots) and hormones homeostasis under saline conditions.
. Introduction
Soil salinity is a major environmental stress that drasticallyffects crop productivity all over the world (Zhu, 2001). It could beartially overcome through plant breeding or by producing trans-enic plants, but the progress to develop such salt resistant plantss very slow due to the complex nature of salt tolerance (multigenicontrol). Accordingly, plant breeding and biotechnological methodspplied to improve salt-tolerance of rice, wheat and corn were notuch successful (Dionisio-Sese and Tobita, 2000), even after gene
mprovement (Ottow et al., 2005). Thus, the use of some other lowost and low risk methods like seed priming could be the attractive
olution to overcome the salinity problem.
Seed priming is a controlled hydration process followed byedrying that allows pre-germinative metabolic activities (physi-
ological and chemical) to proceed but prevents radicle emergence(Bradford, 1986; Parera and Cantliffe, 1991; Pill, 1994). Generally,priming improves the rate and uniformity of seed emergence andgrowth particularly under stress conditions (Bradford, 1986; Ashrafand Foolad, 2005; Iqbal et al., 2006a; Iqbal and Ashraf, 2007a).The accelerated germination treatment (using various plant growthregulators as priming agents) has recently been used to counter-act the deleterious effects of salinity on germination and growthin spring wheat (Iqbal and Ashraf, 2005a,b, 2007b; Iqbal et al.,2006b; Pessarakli et al., 2006). However, the effectiveness of dif-ferent priming agents varies under different stresses as well asin different crop species. Of various priming agents used for seedpriming, plant growth regulators have gained much importancedue to their consistent effects on germination and growth of vari-ous plant species (Iqbal and Ashraf, 2005b, 2006, 2007b; Atia et al.,
2009; Gurmani et al., 2009; Pérez-García, 2009).
Gibberellins (GAs) are generally involved in growth and devel-opment; they control seed germination, leaf expansion, stemelongation and flowering (Magome et al., 2004). The biosynthesis of
As is regulated by both developmental and environmental stimuliYamaguchi and Kamiya, 2000; Olszewski et al., 2002). In addition,As interact with other hormones to regulate various metabolicrocesses in the plants. However, many conflicting theories haveeen put forward concerning their interactions (Yang et al., 1996;an Huizen et al., 1997). It has recently been discovered in differ-nt species that the auxin indole-3-acetic acid (IAA) promotes GAiosynthesis (Wolbang et al., 2004). On the other hand, GA applica-ion enhances the catabolism of ABA (Gonai et al., 2004). However,o our knowledge, the interaction of exogenously applied GA3 (GA-riming) on endogenous auxins is still not reported at least in salttressed wheat plants.
In the long-term field experiments, growth is usually relatedith the photosynthetic capacity of the plants. However, GA3-riming-induced changes in gas exchange characteristics had noteen reported in any species. Some conflicting reports are there inhe literature concerning foliar application of GA3-induced changesn some gas exchange characteristics of plants. For instance, in
heat (Triticum aestivum), foliar application of GA3 had no con-istent effect on stomatal conductance (gs) and transpiration rateE) in the non-stressed or NaCl-treated plants (Ashraf et al., 2000).
e hypothesized that the contrasting effects of GA3 applicationn gas exchange characteristics in above studies were due to theariations in existing pool of ABA in the selected leaves. There-ore, we collected the leaves of both salt stressed and non-stressedlants to determine the ABA contents shortly after they were usedo determine the gas exchange characteristics.
The beneficial effects of GA3 on germination are well knownAngrish et al., 2001; Radi et al., 2001; Khan et al., 2002). How-ver, only a few reports are there concerning GA3-priming-inducedffects on later growth and development of plants grown underaline conditions. Salinity usually perturbs ionic levels both inhoots and roots of the stressed plants. However, GA3 (100 mg L−1)pplied as pre-sowing treatment resulted in the highest K+ anda2+ content in the shoots of both faba beans (Vicia faba) and cot-on (Gossypium barbadense) crops (Harb, 1992). It is widely reportedhat the K+ concentration within a tissue is inversely related witholyamines (PAs) levels (Smith, 1985; Flores, 1991; Iqbal et al.,006c). Moreover, ionic rather than osmotic component of salttress is involved in the initial steps of PAs accumulation in riceLefevre and Lutts, 2000). Therefore, it is likely that GA3 interactsith polyamines to modulate the accumulation and partitioning of
ons in plant tissues under salt stress.Plant stress tolerance usually correlates with their capacity to
nhance the synthesis of polyamines (PAs) upon encountering thetress (Evans and Malmberg, 1989; Bouchereau et al., 1999; Mo andua, 2002; Kasinathan and Wingler, 2004; Kasukabe et al., 2004;qbal and Ashraf, 2006; Iqbal et al., 2006c). Although a correlationetween stress tolerance and polyamine levels has been demon-trated in a number of studies using a variety of plant materials, thehysiological rationale for stress-induced polyamine accumulationemains unknown. In this context, it is of interest to know whetherA3-priming could diminish the salt-induced accumulation of PAs
n wheat plants.The mechanisms by which GA3-priming could induce salt tol-
rance in pants are not yet clear. Salinity perturbs the hormonalalance in plants. The hormonal homeostasis under salt stressherefore might be the possible mechanism of GA3-induced plantalt tolerance. Accordingly we hypothesized that pre-sowing treat-ent with GA3 could modulate growth by interacting with other
ndogenous plant hormones. Thus, the primary objective of thetudy was to determine whether GA3 could alter the endogenous
ool of auxins, abscisic acid, salicylic acid and polyamines in thealt stressed wheat plants. In addition, we determined whetherre-sowing treatments with GA3 could alter ion partitioning and/oraseous exchange characteristics to modulate growth and subse-
perimental Botany 86 (2013) 76– 85 77
quent grain yield in salt stressed plants of genetically diverse wheatcultivars.
2. Materials and methods
Two spring wheat (T. aestivum L.) cultivars, MH-97 (salt intoler-ant) and Inqlab-91 (salt tolerant; Hollington, 2000) were used in thepresent studies. The seeds were obtained from the Wheat Section,Ayub Agricultural Research Institute, Faisalabad, Pakistan. Solu-tions of 100, 150 and 200 mg L−1 of GA3 were used for seed priming.Distilled water (DW) was used for hydropriming (control). Healthywheat seeds (17 g for each treatment) were primed separately in100 mL solutions of GA3 or DW for 12 h at room temperature. Afterpre-soaking treatments, the surface dried seeds were allowed toair-dry for 12 h at room temperature. The air-dried seeds (primedseeds) as well as untreated seeds (non-primed seeds) were used forfield experiment.
2.1. Field experiments
Field experiment was conducted at the Botanical Garden,University of Agriculture, Faisalabad, where the average photo-synthetically active radiation (PAR) of the entire growth periodwas 1098 �mol m−2 s−1, and maximum and minimum relativehumidity (RH) 79 and 32% and the average maximum and mini-mum temperature 28 ◦C and 12 ◦C, respectively. Eight plots havinga length, width and depth of 457, 137 and 46 cm, respectively,and lined with polythene sheets were filled with thoroughlymixed sandy loam soil (pH 7.56; ECe = 2.84 dS m−1; saturation per-centage = 25.5). In order to develop 15 dS m−1 NaCl salinity, therequired amount of NaCl was dissolved in water for complete sat-uration of the soil in four plots (replicates) to homogenize soilsalinity. The other four plots (non-saline) had ECe 2.84 dS m−1.When the soil moisture content (12–14%) were suitable for ger-mination, the primed and non-primed seeds of each treatmentof both wheat cultivars were sown randomly in rows in sep-arate subplots keeping row to row spacing at 15 cm. The ECe
was checked periodically and maintained. In the second year,before sowing, the ECe was again maintained by adding calcu-lated amount of NaCl in solution form to rectify the fluctuationsin salinity due to monsoon rains within subplots. The plants wereirrigated carefully with tap water (ECw = 0.65 dS m−1) using plas-tic pipes during entire experiments. The position of pipes changedat each irrigation so as to minimize dilution in salinity at irriga-tion points. The experiments were laid out in a split–split plotdesign.
2.2. Growth and grain yield
At the boot stage, 16 plants from each treatment were uprootedand washed with DW. After drying with filter paper, roots werecarefully removed. The samples were dried in an oven at 65 ◦Cfor two weeks and shoot dry weights recorded. At maturity, plantheight, number of fertile tillers per plant, grain yield componentsand grain yield was recorded.
2.3. Determination of ions in the shoots and roots
The oven-dried and finely ground shoots and roots (0.1 g) weredigested in a digestion mixture (sulphuric acid–hydrogen perox-ide) according to Wolf (1982). The Na+, K+ and Ca2+ contents in the
digests were determined with a flame photometer (Jenway modelPFP7, Gransmore Green, Dunmow, Essex, UK). For chloride determi-nation, the ground shoot and root material (0.1 g) was extracted in10 mL DW at 80 ◦C for 6 h. The Cl− contents were determined with a
7 nd Experimental Botany 86 (2013) 76– 85
cU
2
aappH
2
iww(Ah
KvpcIao
2
opHp(
2
uMm(fiv
3
3
hcGhptT
ciGf
Fig. 1. Growth attributes of two cultivars of spring wheat at 2.84 dS m−1 (non-saline)or 15 dS m−1 NaCl (saline) when the seeds were primed with solutions of gibberel-lic acid (GA3). Bars represent the means ± SE (n = 4). GA-1, GA-2, and GA-3 show
Measurements of net CO2 assimilation rate (A), transpiration (E)nd stomatal conductance (gs) were made from 10.00 to 15.00 ht the boot stage on a fully expanded 3rd leaf (from top) of eachlant (4 plants per treatment), using an open system LCA-4 ADCortable infrared gas analyzer (Analytical Development Company,oddesdon, England).
.5. Determination of auxins, abscisic acid and salicylic acid
Auxins and abscisic acid were extracted and purified follow-ng the procedure by Guinn et al. (1986) and Kusaba et al. (1998)
ith some modifications (Iqbal et al., 2006b). Salicylic acid (SA)as extracted and purified following the procedure Enyedi et al.
1992) and Seskar et al. (1998) with some modifications (Iqbal andshraf, 2006). Authentic standards were used for quantification oformones and were run through for the whole procedure.
Analysis of hormones was performed by HPLC (Sykam GmbH,leinostheim, Germany) equipped with S-1121 dual piston sol-ent delivery system and S-3210 UV/vis diode array detector. Theeak areas were recorded and calculated using SRI peak simplehromatography data acquisition and integration software (SRInstruments, Torrance, CA, USA). The recoveries were calculated forll hormones and the values of IAA, IBA, ABA and SA were correctedn internal standards (Iqbal et al., 2006a).
.6. Determination of polyamines
Two gram fresh leaves (3rd leaf from top), harvested from plantsf each treatment were finely chopped in 20 mL of cold 5% aqueouserchloric acid and stored at −70 ◦C. For polyamines extraction andPLC analysis, benzoylation method was performed as describedreviously (Flores and Galston, 1982), with some modificationsIqbal and Ashraf, 2006).
.7. Data analysis
Data from repeated experiments were pooled and analyzed bysing the GLM module of CoStat version 6.2, CoHort Software, 2003,onterey, CA, USA, using a split–split plot design (d.f. = 48). Theean values were compared using the least significant difference
LSD) test following Snedecor and Cochran (1980). The data in thegures are represented as means ± SE (n = 4) along with the LSDalues at 5% level of probability for each parameter.
. Results
.1. GA3-priming modulates growth and gain yield
Priming with GA3 significantly (P ≤ 0.001) improved planteight under saline conditions. Compared with control, all the threeoncentrations of GA3 in the salt tolerant whereas 200 mg L−1 ofA3 in the salt intolerant cultivar was effective in increasing planteight when under salt stress (Fig. 1). Overall, the improvement inlant height due to GA3 priming was more in the salt intoleranthan that in the salt tolerant cultivar under non-saline conditions.he reverse was true when under saline conditions.
Salinity decreased the number of fertile tillers per plant signifi-
antly (P < 0.05) in both wheat cultivars. All concentrations of GA3n the salt stressed plants of the salt intolerant whereas 200 mg L−1
A3 in the salt tolerant cultivar was very effective in increasingertile tillers per plant when compared with control (Fig. 1).
solutions of 100, 150 and 200 mg L of GA3, respectively; HP, hydropriming; UT,untreated seeds. LSD0.05, least significant difference at 5% level of probability.
A significant reduction was observed in dry weights of shoots ofplants of both cultivars (P ≤ 0.001) under salt stress. However, seedpriming in 150 mg L−1 of GA3 improved shoot dry weights in thesalt intolerant cultivar when compared with control (Fig. 1).
Although salinity decreased the number of grains per ear onmainstem in both cultivars, priming with GA3 was very effective inimproving this attribute irrespective of cultivars under both salineand non-saline conditions (Fig. 2). Overall, the increase in num-ber of grains per ear due to GA3 priming on mainstem was morein the salt tolerant than in the salt intolerant cultivar when undersalt stress. Grain weight (100-grain wt.) decreased in both cultivarswhen under salt stress (Fig. 2). When compared with control, how-
ever, lower concentrations of GA3 (100–150 mg L−1) were effectivein increasing grain weight in the salt intolerant cultivar when undersalt stress.
M. Iqbal, M. Ashraf / Environmental and Ex
Fig. 2. Grain yield and its components of two cultivars of spring wheat at 2.84 dS m−1
(non-saline) or 15 dS m−1 NaCl (saline) when the seeds were primed with solutionsof gibberellic acid (GA3). Bars represent the means ± SE (n = 4). GA-1, GA-2, and GA-3sU
(Gtibdo
3
uwv
how solutions of 100, 150 and 200 mg L−1 of GA3, respectively; HP, hydropriming;T, untreated seeds. LSD0.05, least significant difference at 5% level of probability.
The imposition of salt stress caused a significant reductionP ≤ 0.05) in grain yield of both wheat cultivars (Fig. 2). Priming withA3 caused a significant increase in grain yield irrespective of cul-
ivars under both saline and non-saline conditions. Seed treatmentn GA3 (150 mg L−1) was very effective in enhancing grain yield ofoth cultivars when under salt stress (Fig. 2). Overall, the effects ofifferent GA3 concentrations were more on the salt intolerant thann the salt tolerant cultivar under saline conditions.
A slight reduction in net CO2 assimilation rate (A) was foundnder salt stress (Fig. 3). The priming agents differed significantlyith respect to this attribute. The non-stressed plants of both culti-
ars whereas salt stressed plants of the salt tolerant cultivar raised
perimental Botany 86 (2013) 76– 85 79
from GA3-treated (150 mg L−1) seeds had greater A when comparedwith control (Fig. 3).
Stomatal conductance (gs) of both cultivars decreased signifi-cantly due to salt stress (P ≤ 0.01; Fig. 3). However, priming agentsdid not differ in altering gs in both cultivars when under salt stress(Fig. 3). Plants of the salt intolerant and the salt tolerant cultivarraised from lower and higher concentrations of GA3 (100–150 and200 mg L−1), respectively had higher gs when grown under non-saline conditions.
Salt stress decreased transpiration rate (E) in both cultivarsunder saline conditions (Fig. 3). Priming agents improved E sig-nificantly under saline conditions in both cultivars (P ≤ 0.001).Although 150 mg L−1 GA3 in the salt tolerant and 100 mg/L in thesalt intolerant cultivar improved E under saline conditions com-pared with untreated plants, the effect was not much pronouncedin this respect (Fig. 3).
A significant effect of salt stress on instantaneous water use effi-ciency (calculated as A/E) was noted in both cultivars (Fig. 3). Seedpriming in GA3 (150 mg L−1) in the salt tolerant and 200 mg L−1 inthe salt intolerant cultivar was effective in increasing WUE undersaline and non-saline conditions when compared with control(Fig. 3).
3.3. GA3-priming alters ionic concentrations
Soil salinity caused a significant (P < 0.05) increase in shootNa+ concentrations in both cultivars. However, GA3 treatmentswere very effective in reducing the shoot Na+ concentration in saltstressed plants of both cultivars (Fig. 4). In comparison to control,priming with GA3 (150 mg L−1) was the most effective treatmentin lowering shoot Na+ concentrations under both saline and non-saline conditions.
A significant (P < 0.001) reduction in shoot K+ concentrationsdue to soil salinity was observed in both cultivars. GA3 priming wasonly effective in increasing shoot K+ concentration in non-stressedplants of the salt tolerant cultivar. In contrast, hydroprimingincreased shoot K+ concentrations in both cultivars when undersaline conditions (Fig. 4).
Salinity significantly (P < 0.001) decreased shoot Ca2+ concentra-tions in both cultivars. However, GA3 priming did not cause increasein shoot Ca2+ concentration in both cultivars when under salt stress.GA3 priming affected both wheat cultivars differently under non-stressed conditions. Plants of the salt tolerant cultivar raised fromGA3 treated seed had higher shoot Ca2+ concentration while thereverse was true for the salt intolerant cultivar (Fig. 4).
Interestingly, GA3-priming increased shoots Cl− concentrationsin both cultivars when compared with control. The increase in shootCl− concentration was more pronounced at low (100 mg L−1) ratherthan at high (200 mg L−1) GA3 concentrations (Fig. 4).
Salinity caused a considerable increase in root Na+ concentra-tion in both wheat cultivars. Priming with low concentration ofGA3 (100 mg L−1) was the most effective priming treatment in bothcultivars because seeds treated with this priming solution hadmarkedly lower root Na+ concentration as compared to the plantsraised from hydroprimed or non-primed seeds when under saltstress (Fig. 5).
Root zone salinity significantly (P < 0.001) altered root K+
concentration in plants of both cultivars. Low concentration(100 mg L−1) of GA3 in the salt intolerant and a higher concentra-tion (200 mg L−1) of GA3 in the salt tolerant cultivar was slightlyeffective in increasing root K+ concentration when compared with
control in the salt stressed plants (Fig. 5).
Root Ca2+ concentration decreased significantly (P < 0.001) inboth cultivars due to salt stress. When compared with control,100 mg L−1 of GA3 was effective in increasing root Ca2+ concen-
80 M. Iqbal, M. Ashraf / Environmental and Experimental Botany 86 (2013) 76– 85
Fig. 3. Gaseous exchange characteristics of two cultivars of spring wheat at 2.84 dS m−1 (non-saline) or 15 dS m−1 NaCl (saline) when the seeds were primed with solutionsof gibberellic acid (GA3). Bars represent the means ± SE (n = 4). GA-1, GA-2, and GA-3 show solutions of 100, 150 and 200 mg L−1 of GA3, respectively; HP, hydropriming; UT,untreated seeds; A, net photosynthetic rate; gs, stomatal conductance; E, transpiration rate; WUE, instantaneous water use efficiency. LSD0.05, least significant difference at5% level of probability.
Fig. 4. Shoot Na+, K+, Ca2+ and Cl− concentration of two cultivars of spring wheat at 2.84 dS m−1 (non-saline) or 15 dS m−1 NaCl (saline) when the seeds were primedwith solutions of gibberellic acid (GA3). Bars represent the means ± SE (n = 4). GA-1, GA-2, and GA-3 show solutions of 100, 150 and 200 mg L−1 of GA3, respectively; HP,Hydropriming; UT, Untreated seeds. LSD0.05, least significant difference at 5% level of probability.
M. Iqbal, M. Ashraf / Environmental and Experimental Botany 86 (2013) 76– 85 81
F t at 2w 1, GAh f prob
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3a
bIlsfn
tte(cw
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ig. 5. Root Na+, K+, Ca2+ and Cl− concentration of two cultivars of spring wheaith solutions of gibberellic acid (GA3). Bars represent the means ± SE (n = 4). GA-ydropriming; UT, untreated seeds. LSD0.05, least significant difference at 5% level o
rations in the two cultivars under both saline and non-salineonditions (Fig. 5).
A significant increase (P < 0.001) in root Cl− concentration wasbserved in both cultivars due to salt stress. However, cultivarsesponded differently to GA3 application (Fig. 5). The most effectiveriming treatment was 100 mg L−1 of GA3 in the salt intolerant and00 mg L−1 of GA3 in the salt intolerant cultivar in reducing rootl− to a maximum extent.
.4. GA3-priming alters hormonal balance between IAA, IBA, ABAnd SA
Salinity significantly reduced leaf free IAA concentrations inoth wheat cultivars (Fig. 6). Priming with GA3 improved leaf free
AA levels only in the salt tolerant; whereas it reduced leaf IAAevels in the salt intolerant cultivar compared with control underaline conditions. Hydropriming showed contrasting effects on leafree IAA concentrations in either cultivar both under saline andon-saline conditions.
GA3 treatments caused a significant change in leaf free indolebu-yric acid (IBA) concentrations in both cultivars (Fig. 6). All primingreatments decreased leaf free IBA concentrations in the salt tol-rant cultivar when under salt stress. Seed treatment with GA3200 mg L−1) increased leaf free IBA concentration in both wheatultivars when under non-saline conditions. However, the reverseas true for both cultivars when under salt stress.
Seed priming treatments altered leaf free ABA concentrationifferently in both cultivars (Fig. 6). Plants of the salt tolerantultivar raised from seed treated with GA3 (200 mg L−1) had maxi-
um leaf free ABA concentration under both saline and non-saline
onditions. However, in the salt intolerant cultivar, all treatmentsowered leaf ABA concentrations compared with plants raised fromntreated seeds when under salt stress.
.84 dS m−1 (non-saline) or 15 dS m−1 NaCl (saline) when the seeds were primed-2, and GA-3 show solutions of 100, 150 and 200 mg L−1 of GA3, respectively; HP,ability.
Salt stress significantly affected leaf free salicylic acid concentra-tion in both cultivars (Fig. 6). Among treatments, GA3 (150 mg L−1)was very effective in increasing leaf SA concentration in both cul-tivars when under salt stress.
Generally, salt stress caused an increase in Put concentrationsin plants of both cultivars. Priming with GA3 (150 mg L−1) wasvery effective in decreasing Put concentrations in stressed plantsof either cultivar when compared with control (Fig. 7). In contrast,GA3 (200 mg L−1) increased leaf free Put concentrations in plants ofboth wheat cultivars under saline conditions.
Generally, all priming agents lowered leaf free Spd concentra-tion in plants of both cultivars compared with plants raised fromuntreated seed. Priming with 200 mg L−1 of GA3 in the salt tolerantcultivar increased whereas 100 mg L−1 of GA3 in the salt intoler-ant cultivar decreased leaf free Spd concentrations in salt stressedplants when compared with control (Fig. 7). Under non-saline con-ditions, 150 mg L−1 of GA3 was the most effective in decreasing leaffree Spd concentrations in plants of both wheat cultivars.
Salinity altered leaf free spermine (Spm) concentrations differ-ently in both cultivars. Pre-treatment with 200 mg L−1 of GA3 in thesalt tolerant cultivar whereas 100 mg L−1 of GA3 in the salt intol-erant cultivar was the most effective in increasing leaf free Spmconcentrations in salt stressed plants when compared with control(Fig. 7).
4. Discussion
Seed priming with optimal concentrations of some plant hor-mones could increase germination, growth, and yield in differentcrops when grown under salt stress (Kumar and Singh, 1996;Aldesuquy, 2000; Kaur et al., 2002; Iqbal and Ashraf, 2005b, 2007b;
82 M. Iqbal, M. Ashraf / Environmental and Experimental Botany 86 (2013) 76– 85
Fig. 6. Leaf free IAA, IBA, ABA (ng g−1 FW) and SA concentrations (�g g−1 FW) of two cultivars of spring wheat at 2.84 dS m−1 (non-saline) or 15 dS m−1 NaCl (saline) whent eans ±r olebua
IiOeggsftGoiI(wiwtarmsibitGvawptw
he seeds were primed with solutions of gibberellic acid (GA3). Bars represent the mespectively; HP, hydropriming; UT, untreated seed; IAA, indoleacetic acid; IBA, indt 5% level of probability.
qbal et al., 2006a). Generally pre-treatment with GA3 alleviated thenhibitory effect of salt stress on growth of both wheat cultivars.verall, seed pre-treatment with 150 mg L−1 GA3 was the mostffective of all treatments that caused a maximum increase in shootrowth (plant height) in either wheat cultivar. It also improvedrain yield in both cultivars when under salt stress. There is nowtrong evidence that different concentrations of GA are requiredor plant development at different stages of plant growth duringhe life cycle (Takahashi, 1992). For example, the total amount ofA decreased following anthesis in rice (Takahashi, 1986). More-ver, it has been shown that normal biosynthesis of active GAsn stems of barley and oats is dependent on the availability ofAA by their inflorescences and therefore, affects shoot elongationWolbang et al., 2004). In the present studies, seed pre-treatmentith GA3-induced better shoot growth and grain yield production
n genetically diverse wheat cultivars suggested that pre-treatmentith GA3 could have affected the hormone homeostasis possibly
hrough the availability of active GAs at later plant growth stages,nd thus exerted long-term effects on treated plants.Tillering iseduced by salinity, with the primary and secondary tillers beingore affected than is the mainstem (Ruana et al., 2008). In our
tudies, priming with GA3 was very effective in improving salinity-nduced decrease in number of grains per ear on mainstem inoth wheat cultivars. However, GA3 treatment (100–150 mg L−1)
ncreased the number of fertile tillers per plant in the salt intoleranthan in the salt tolerant cultivar when under salt stress. Therefore,A3-mediated increase in grain yield in the salt intolerant culti-ar was due to the increased number of fertile tillers per plantnd grain weight rather than number of grains per ear particularly
hen under salt stress. In contrast, the increase in grain numberer ear on mainstem observed with tiller reduction in the saltolerant cultivar may be attributed to the increase in grain yieldhen under salt stress. It has earlier been shown that GA-treated
SE (n = 4). GA-1, GA-2, and GA-3 show solutions of 100, 150 and 200 mg L−1 of GA3,tyric acid; ABA, abscisic acid; SA, salicylic acid. LSD0.05, least significant difference
tissues act as strong sinks to induce carbohydrate translocation(Weaver et al., 1969). GA-treated plants have also been shown todecrease overall starch accumulation (Davis et al., 1988). Therefore,it appears that GA3 applied as pre-treatment acts to some extentlikewise that of its foliar application at later plant growth stages.However, the additional effects of GA3 priming on germination,early seedling growth and development make it more attractivecompared with its foliar application. GA3-mediated increase in thenumber of grains per ear on mainstem can be explained in view ofthe findings of Davies (1995) and Magome et al. (2004). They havedemonstrated the involvement of gibberellins in the induction andpromotion of flowering in many plants. In addition, its applicationcould modify the flower sex expression in some plants. We sug-gest that GA3-priming might have increased seed set efficiency bycontrolling flowering and/or assimilates partitioning within newlyborn sinks, and thus, increased number of seed per ear on main-stem. Interestingly, GA3-priming did not decrease fertile tillers perplant in the either cultivar when compared with the control. Con-trastingly, foliar application of GA3 changed the allocation patternof carbohydrates within shoots, and thus inhibited tiller productionin rice (Jung, 1986; Yim et al., 1997). Similarly, foliar applicationof GA3 caused no significant change in grain yield, but increasedgrain size in both salt intolerant and tolerant wheat cultivars andthe ameliorative effect of the GA3 treatment was more pronouncedin the salt intolerant than in salt tolerant wheat cultivar (Ashrafet al., 2000). Although GA3 priming increased grain yield in bothcultivars, it did not cause consistent effect on gaseous exchangecharacteristics in both cultivars when under salt stress. Amongthe factors responsible for yield compensation in salt stressed
wheat, are a reduction in the assimilation requirements of vege-tative organs and more efficient utilization of assimilated supplies(Grieve et al., 1992). Our results suggested that GA3-pre-treatmentaffect differently to that of foliar application at least in produc-
M. Iqbal, M. Ashraf / Environmental and Ex
Fig. 7. Polyamines concentrations of two cultivars of spring wheat at 2.84 dS m−1
(non-saline) or 15 dS m−1 NaCl (saline) when the seeds were primed with solutionsof gibberellic acid (GA3). Bars represent the means ± SE (n = 4). GA-1, GA-2, and GA-3show solutions of 100, 150 and 200 mg L−1 of GA3, respectively; HP, hydropriming;Us
it
iHemaufiomTIwpci
T, untreated seeds; Put, putrescine; Spd, spermidine; Spm, spermine. LSD0.05, leastignificant difference at 5% level of probability.
ng tillers and mediating the translocation of carbohydrates withinhem.
GA3 altered the pattern of distribution and accumulation ofons in shoots and roots of both cultivars when under salt stress.owever, pre-treatment with GA3 altered ion homeostasis to somextent similarly in the two cultivars. For instance, pre-sowing treat-ent with GA3 decreased Na+ and Cl− concentrations in the roots
nd Na+ concentration in the shoots of both cultivars when grownnder saline conditions. Thus, pre-treatment with GA3 had a bene-cial effect on ion homeostasis under saline conditions irrespectivef cultivars. However, higher concentration of GA3 reduced K+ accu-ulation in the shoots of both cultivars when under salt stress.
he decrease in K+ concentration is not in line with the findings ofbrahim and Khafaga (1986), and Aldesuquy and Ibrahim (2001),
ho had found increased K+ absorption by the plants with GA
3re-treatment. Pre-sowing treatment with 100 mg L−1 of GA3 alsoaused an accumulation of Ca2+ in the roots of both cultivars and K+
n the salt intolerant cultivar when under salt stress. Maintenance
perimental Botany 86 (2013) 76– 85 83
or increase of Ca2+ concentration in the root could induce mainte-nance of K+, since the presence of Ca2+ seems to be necessary forK+–Na+ selectivity, and for the maintenance of an appropriate K+
concentration in plant cells (Subbarao et al., 1990; Iqbal and Ashraf,2010).
Pre-treatment with GA3 affected the leaf free auxins (IAA andIBA) pool differently in both cultivars when under salt stress. Theauxin indole-3-acetic acid (IAA) has been shown to promote GAbiosynthesis in different species (Wolbang et al., 2004). However,GA3 priming did not show consistent effect on leaf free auxins poolin both cultivars in the present studies. This could be due to tworeasons; firstly, IAA conjugation potential of the plants, as well as,their capacity to hydrolyse them varies at different developmentalstages and/or under different stresses (Ludwig-Muller et al., 1996),and secondly, it has generally concluded for all plants studied, thatthe majority of the IAA pool is in the conjugated form (90% of thetotal IAA) (Cohen and Bandurski, 1982). Therefore, GA3 primingcould have affected synthesis and turnover of auxins, but it did notaffect the metabolism in terms of the type of conjugates formed(Eklof et al., 1997).
Salicylic acid is known to play an important role in the plantresponses to adverse environmental conditions including salt stress(Borsani et al., 2001; Shakirova et al., 2003; Iqbal and Ashraf,2010). Pre-sowing treatment with 150 mg L−1 GA3 was the mosteffective in increasing grain yield and leaf SA concentration inboth cultivars when under salt stress. Recently, Alonso-Ramírez etal. (2009) have reported that GA-responsive gene and exogenousaddition of GAs are able to counteract the inhibitory effects of dif-ferent adverse environmental conditions in seed germination andseedling growth of Arabidopsis through modulation of SA biosyn-thesis. Pre-treatment with all GA3 concentrations altered leaf freeABA levels differently in both cultivars under saline conditions. Pre-treatment with GA3 alleviated the stimulatory effect of salinity onthe synthesis of ABA in the salt intolerant cultivar, in line withthe findings of Younis et al. (1991) and Aldesuquy and Ibrahim(2001). However, the increased levels of ABA in the salt tolerantcultivar might be due to their increased synthesis in that culti-var. Such type of increased synthesis of ABA in plants under salineconditions is already reported (Ludewing et al., 1988; Aldesuquyand Ibrahim, 2001; Iqbal et al., 2006a). ABA regulates many agro-nomically important aspects of plant development, including thesynthesis of seed storage proteins and lipids, the promotion ofseed desiccation tolerance and dormancy, and the inhibition ofthe phase transitions from embryonic to germinative growth andfrom vegetative to reproductive growth (Leung and Giraudat, 1998;Rock, 2000; Rohde et al., 2000). The ABA level can also regulatethe pathways of photoassimilate utilization in leaves by partition-ing carbon flows either to the synthesis of high-molecular-weightcompounds (cellulose, hemicellulose, and proteins), used for cellgrowth in leaves, or to the synthesis of transport forms of carbo-hydrates (Kiseleva and Kaminskaya, 2002). Thus, the increase ingrain yield in the salt tolerant cultivar was probably due to theincreased rate of translocation of photosynthates from leaves tograins caused by GA3 pre-treatment through increased concentra-tions of ABA (Dewdeny and McWha, 1978; Aldesuquy and Ibrahim,2001; Iqbal et al., 2006b). Reduction in fertile tillers production inthe salt tolerant cultivar also supports this speculation.
Pre-treatment with 150 mg L−1 of GA3 diminished the salt-induced accumulation of Put in both wheat cultivars. It is knownthat salt stress upregulates the activity of arginine decarboxylase(ADC) and ornithine decarboxylase (ODC), key enzymes requiredfor the biosynthesis of Put via two alternative pathways (Friedmanet al., 1989; Lee and Chen, 1998). In particular, enhancement of
ADC leads to the accumulation of Put in wheat (Basu et al., 1988),rice (Chattopadhyay et al., 1997), and mustard (Mo and Pua, 2002)under saline conditions. Moreover, an increase in polyamine under
8 nd Ex
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4 M. Iqbal, M. Ashraf / Environmental a
tress may be caused by de novo synthesis or reduced degradation,lthough the exact mechanism remains a matter of debate (Iqbalt al., 2006c; Liu et al., 2007). Thus, it is possible that GA3 primingight have down-regulated the conversion of arginine to Put, pos-
ibly by reducing the activity of ADC under conditions of salt stressIqbal and Ashraf, 2006). The other possibility is that GA3 mightave increased the degradation of Put when under salt stress. Sev-ral studies have suggested the capability of the plant to convertut into other polyamines (Spd and Spm) as a function that closelyelated with the ability of the plants to develop under stressful envi-onments (Bouchereau et al., 1999; Iqbal and Ashraf, 2006). In viewf GA3-induced better performance of stressed plants of both wheatultivars, it is likely that GA3 affected plants ability to synthesizeAs.
Pre-sowing seed treatment with GA3 altered leaf free Spd andpm concentrations differently in salt stressed plants of bothheat cultivars. For example, priming with higher concentration
200 mg L−1) of GA3 was very effective in increasing polyaminesSpd and Spm) in the salt tolerant cultivar when under salt stress,hile reverse was true for the salt intolerant cultivar. These results
upport the findings of Kasukabe et al. (2004), who have found thatn comparison to stress-intolerant plants, stress-tolerant plantsenerally had a large capacity to enhance PA biosynthesis inesponses to stress, resulting in a 2–3-fold increase of endogenousA levels over those in unstressed plants (Kasukabe et al., 2004). Inresent studies, the differential response of plants of both cultivarso accumulate PAs in response to salinity and GA3-priming, sug-ested that change in hormonal balance could have affected the PAsontent or their distribution in plants (Rastogi and Davies, 1991;qbal et al., 2006c).
. Conclusions
Considering consistency and effectiveness of the priming treat-ents used in the present studies, 150 mg L−1 GA3 was the most
ffective in alleviating the adverse effect of salt stress on grain yieldf both wheat cultivars. However, GA3-mediated increase in grainield in the salt intolerant cultivar was due to the increased numberf fertile tillers per plant and grain weight rather than number ofrains per ear particularly when under salt stress. In contrast, thencrease in grain number per ear on mainstem observed with fer-ile tiller reduction in the salt tolerant cultivar may be attributedo the increase in grain yield when under salt stress. Moreover,eed priming with GA3 altered the uptake and pattern of accu-ulation of different ions between shoots and roots in the adult
lants of wheat under saline conditions. Even though GA3-primingid not show consistent effect on gaseous exchange characteris-ics and the concentrations of auxins in the salt stressed plants ofoth cultivars, plants of both cultivars raised from GA3-treaed seedad higher SA concentration in the leaves under saline conditions.he decrease in polyamines (Put and Spd) and ABA concentrationsn the salt stressed plants of the salt intolerant cultivar raised fromA3-treated seed suggested that these plants might have faced lesstress compared with control. Therefore, the beneficial effects ofA3-priming can be attributed to its effect on hormonal homeosta-is and ionic uptake and partitioning (within shoots and roots) inhe salt stressed wheat plants.
cknowledgements
The research was partially supported through Research Ori-ntation Scheme (Promotion of Research Program, University ofgriculture, Faisalabad, Pakistan). The authors wish to express theirppreciation to the reviewers of the research article.
perimental Botany 86 (2013) 76– 85
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