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Environmental and Experimental Botany 69 (2010) 1–8

Contents lists available at ScienceDirect

Environmental and Experimental Botany

journa l homepage: www.e lsev ier .com/ locate /envexpbot

arly activation of plasma membrane H+-ATPase and its relation to droughtdaptation in two contrasting oat (Avena sativa L.) genotypes

ong-Shan Gonga,b,1, You-Cai Xionga,c,d,∗,1, Bao-Luo Mac, Tian-Ming Wangd, Jian-Ping Ged,iao-Liang Qina, Pu-Fang Lia, Hai-Yan Konga, Zi-Zhen Lia,b, Feng-Min Lia,∗∗

MOE Key Laboratory of Arid and Grassland Ecology, Lanzhou University, Lanzhou 730000, ChinaSchool of Mathematics and Statistics, Lanzhou University, Lanzhou 730000, ChinaEastern Cereal and Oilseed Research Center (ECORC), Agriculture & Agri-Food, Canada, 960 Carling Avenue, Ottawa, Ontario, Canada K1A 0C6MOE Key Laboratory of Biodiversity and Ecological Engineering, Beijing Normal University, Beijing 100875, China

r t i c l e i n f o

rticle history:eceived 4 February 2009eceived in revised form 27 July 2009ccepted 22 February 2010

eywords:lasma membrane H+-ATPasesmotic regulationrought stressvena sativa L

a b s t r a c t

Major objective of this study is to elucidate the effect of early activation of root hair cell plasma membrane(PM) H+-ATPase on drought adaptation in plants. Pot-culture experiments were carried out to determineoat (Avena sativa L.) genotypic differences in water maintenance, osmotic adjustment and PM H+-ATPaseactivity at the seedling stage. Two oat genotypes with contrasting drought sensitivity, Dingyou6 (A. ver-nasativa, drought-tolerant cultivar) and Bende (A. venanuda, drought-sensitive cultivar) were subjectedto soil drought stress under environment-controlled growth chamber conditions. At 21 days after emer-gence, water supply was withheld to allow soils in pots to dry. Our results showed that drought-tolerant“Dingyou6” maintained significantly greater RWC and osmotic potential (OP) in roots and leaves, and alsohad larger root-to-leaf ratios of RWC and OP than drought-sensitive “Bende” along with 14-day dryingprocess, suggesting that drought-tolerant cv. possesses superior root-to-leaf hydraulic conductivity, andstronger regulatory ability to drought stress. Analysis of the PM H+-ATPase activity and the root and leafosmolyte contents provided further chemical evidence for this result. Biosynthesis of leaf proline andglycine betaine (GB) followed a similar trend as the activities of root hair cell PM H+-ATPase prior tointermediate stress (around 35% FWC). Significant increase in the activity of PM H+-ATPase was observedat the SWC of about 45–50% FWC, without detectable changes in leaf and root RWC simultaneously.

This demonstrated that there existed an early-warning response in roots before the onset of significantdecrease in plant RWC. Moreover, the interspecific difference in the timing of triggering early responsewas obvious. Drought-tolerant “Dingyou6” initiated early response at about 50% FWC, but at about 45%FWC for drought-sensitive cv. This study implies that early activation of root hair cell PM H+-ATPase trig-gers the increased biosynthesis of major osmolytes, which, in turn, leads to the up-regulation of watermaintenance system.

. Introduction

Plants are able to respond to drought stress by altering theirellular metabolism and invoking complex defense mechanisms

Abbreviations: RWC, relative water content; SWC, soil water content; OP,smotic potential; OA, osmotic adjustment; PM, plasma membrane; GB, glycineetaine.∗ Corresponding author at: MOE Key Laboratory of Arid and Grassland Ecology,

anzhou University, Lanzhou 730000, China. Tel.: +86 931 8914500;ax: +86 931 8914500.∗∗ Corresponding author.

E-mail addresses: xiongyc@lzu.edu.cn (Y.-C. Xiong and F.-M. Li).1 The former two authors are the first co-authors.

098-8472/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.envexpbot.2010.02.011

© 2010 Elsevier B.V. All rights reserved.

(Bohnert and Jensen, 1996; Chaves et al., 2002). Their survival andadaptation under drying soil depend on their intrinsic regulatoryability to perceive stimulus, generate and transmit this “drying”signals, and initiate various biochemical changes (Blackman andDavis, 1985; Bohnert and Jensen, 1996; Ober and Sharp, 2003;Fan et al., 2008). During soil drying, early response of plants tostress is closely linked with immediate survival and gradual accli-mation under drought stress (Augé and Duan, 1991; Bohnert andSheveleva, 1998; Chaves et al., 2002; Xiong et al., 2006a; Fan et al.,2008). Due to the complexity of regulatory mechanism, this early

response is extensively considered a coupled root-to-shoot processat the whole plant level. Previous efforts had been paid on eluci-dating how the early response operates, but little is known aboutthe quantitative effect of this early-warning mechanism on cropgrowth and adaptation. Among recent progresses, non-hydraulic

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D.-S. Gong et al. / Environmental a

oot-sourced signal is so far affirmed to be a unique “early-warning”esponse to soil drying in plants (Blackman and Davis, 1985; Chavest al., 2002; Xiong et al., 2006b; Fan et al., 2008).

Since Blackman and Davis (1985) found root-sourced chem-cal signals formed when soil was drying, many subsequentxperiments have elucidated how root–shoot communicationight operate (Blackman and Davis, 1985; Ludlow et al., 1989;

roker et al., 1998; Mingo et al., 2003; Dodd et al., 2003; Normant al., 2004; Xiong et al., 2006a,b). This mechanism enables plantso “sense” drought in the roots and is expressed as an alterationf physiological parameters in the leaves (Davis and Zhang, 1991;owing et al., 1990). This is a typical “early-warning” response oflants to drought (Blum and Johnson, 1993). Continuing drought

nitiates up a hydraulic gradient between the leaf and the dryingoil, which speeds up the development of leaf water deficit by lossf leaf turgor pressure (Blum and Johnson, 1993; Comstock andonathan, 2002). On the basis of knowledge about plant water rela-ion, the triggering mechanism of this “early-warning” response isikely summed up in the regulative role of root hair. Root hair as a

ajor organ of water uptake can respond to reduced soil watervailability, which may act as the signal resource from root-to-hoot. Growing root hair showed a variety of significant responseso osmotic stress including electrical signal, which is likely due tohe presence of osmo-sensor (Lew, 1996). The plasma membranePM) H+-ATPase of root hair cell is an essential protein that may be

ainly responsible for the onset of root-to-shoot signal.The PM H+-ATPase (EC 3.6.1.35) has been called a ‘master

nzyme’ responsible for a broad range of physiological processesSamuels et al., 1992; Gévaudant et al., 2007; Liu et al., 2008), whichnclude water maintenance, osmotic regulation and other adaptive

echanisms under drought stress (Ober and Sharp, 2003; Liu etl., 2005). It is an important proton pump that translocates pro-on out of cells when ATP is hydrolyzed (Liu et al., 2005). However,

ost of the information regarding the physiological effect of theM H+-ATPase has so far come from partial studies focusing onertain organ or tissue. In most cases, overall effect of the PM H+-TPase at the level of whole plant was largely lacked. The regulatoryechanism of root hairy cell PM H+-ATPase to drought adapta-

ion at whole plant level may be closely associated with root–shootydraulic conductance, osmotic adjustment and other unclear pro-esses.

We therefore proposed a hypothesis that the initiation of PM+-ATPase in root hair cells might play a critical role in regulat-

ng the root-to-shoot communication. In higher plants, there isow substantial evidence that glycine betaine (GB) and proline arewo major organic osmolytes that accumulate in a variety of plantpecies in response to drought stress (Hanson and Burnet, 1994;a et al., 2004; Ashraf and Foolad, 2007). These compounds nor-ally accumulate in large quantities in response to dehydration

tress (Mohanty et al., 2002; Yang et al., 2003; Kavi Kishore et al.,005). This process is thought to play adaptive roles in mediatingsmotic adjustment (OA) and protecting subcellular structures intressed plants. Thus, the changes of PM H+-ATPase activity haveeen contemplated to increase the concentrations of these com-ounds in plants grown under stress conditions to increase theirtress tolerance. Increased proline and GB may also function as pro-ein compatible hydrotropes (Srinivas and Balasubramanian, 1995)hat help the generation of ATP for repairing of stress-induced dam-ges and accordingly improve the ability of water maintenance ineaves. Osmotic adjustment is generally considered an importantomponent of drought resistance (Blum et al., 1999; Cattivelli et

l., 2008). Those cultivars with better osmotic adjustment abil-ty had better performance of stress adaptation under droughttress, enabling plants to maintain water absorption and turgorressure (Morgan, 1983, 1995; Moinuddin et al., 2005; Fan et al.,008).

erimental Botany 69 (2010) 1–8

Existing researches showed that the difference among geno-types or species in drought adaptation can be traced to differentcapacities for water acquisition and transportation (Chaves et al.,2002; Ma et al., 2004; Xiong et al., 2006b). The ability of plants towater maintenance and hydraulic conductance may be critical todrought acclimation development. This type of drought acclimationin plants is evolutionarily innate defense ability and can mecha-nistically act as immune responses as in animals (Nürnberger andKemmerling, 2006). Plant species (or plant non-cultivar-specific)and plant cultivar-specific resistance are two distinct but evo-lutionarily interrelated types of resistance that constitute plantinnate immunity. Drought-tolerant type and drought-sensitive onein crops had differentiated stress response to drought (Schwanzand Polle, 2001; Zhu et al., 2005; Fan et al., 2008; Li et al., 2009).Hydraulic properties of roots are regulated by root-sourced signalsuch as ABA (Mahdieh and Mostajeran, 2009).

Our previous work showed that the soil moisture at whichnon-hydraulic root-sourced signal (nHRS) was triggered was posi-tively correlated with that of hydraulic root signal (HRS) in eightold or modern wheat varieties. Earlier onset of nHRS signifi-cantly affected drought tolerance and yield performance (Xionget al., 2006b). However, its regulatory mechanism and the differ-ence between cultivars are so far unclear. In this study, we chosetwo oat genotypes with contrasting drought resistant, Dingyou6(drought-tolerant cultivar) and Bende (drought-sensitive cultivar),as experimental materials to reveal whether the early activation ofPM H+-ATPase in root hair cells is related to osmotic adjustment andwater maintenance. The results will provide a better understandingon the root-to-shoot regulatory mechanism on whole plant level forthe crops grown in drying soil.

2. Materials and methods

2.1. Description of experiments

Seeds of two oat (Avena sativa L.) genotypes with contrast-ing drought sensitivity, Dingyou6 (A. vernasativa, drought-toleranttype) and Bende (A. venanuda, drought-sensitive type) that weresupplied by Dryland Agricultural Research Center of Dingxi, Gansu,PR China, were grown in pot-culture condition as follows. Plantmaterial preparation and water supply control was made accordingto our previous methods (Xiong et al., 2006a).

Oat seeds were surface-sterilized in 0.5% NaOCl for 15 min,rinsed in distilled water for 15 min, and grown in plastic potsof diameter 36 cm and height 30 cm in a growth cabinet (Con-viron PGV36, Asheville, North Carolina, USA) under controlledenvironmental conditions (light/dark regime of 16/8 h at 20–25 ◦C,relative humidity of 60–70%, photosynthetic photon flux densityof 300 �mol m−2 s−1). Twenty seedlings were placed into each pot.After 21 days of growth in cabinet, drought stress treatment wasstarted by withholding water to well-watered seedlings. The soilwater contents (SWCs) were determined gravimetrically everydayby weighing pots throughout the whole drying period (Xiong et al.,2006a,b).

According to soil water characteristic curve calculated by therelationship between soil suction and soil moisture, water avail-ability gradients were categorized into sufficient water supply (CK,65% field water capacity (FWC)), mild stress (MS, 45% FWC), inter-mediate stress (IS, 35% FWC) and serious stress (SS, 20% FWC),respectively (Table 1). To facilitate development of the relationship

between soil moisture and plant physiological parameters (RWC,OP and enzyme activities), a variety of SWCs measured in a con-tinuous drying episode were classified into a series of soil watergradients, in which the soil water content was at the levels of 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% and 65% FWC (with a fluctua-

D.-S. Gong et al. / Environmental and Exp

Table 1The relationship between soil suction and soil water content.

Moisture gradients Soil water content (% FWC) Soil suction (kPa)

Well-watered (CK) 65 187.5 ± 32.6Mild stress (MS) 45 361.1 ± 40.4Intermediate stress (IS) 35 801.4 ± 105.3Severe stress (SS) 20 1517.5 ± 244.2Bulk density (g/m3) 1.31

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he relationship between soil suction and soil water content accords with thequation: Y = 117.5X−1.5039, where Y is soil suction (kPa) and X soil water contentR2 = 0.926**, p < 0.01).

ion range of 2.5% in each group; for example, the soil water contentf 35 ± 2.5% was taken as the FWC35 group). In these treatments,WC65 was considered to be the well-watered check group. Dur-ng the process of gradually drying within 2 weeks since the stressnitiation, soil water status was decreased from CK, MS, IS to SSn the 1st, 4th, 7th and 14th day (Fig. 1). The root and leaf samplesere taken on these four treatment stages and used in the followingrotocols.

There are 120 pots for all treatments (30 pots per treatment × 4reatment groups = 120 pots).

.2. Measurements of leaf proline and glycine betaineoncentrations

The leaf and root RWCs were determined in two whole leaveser genotype using the fresh weight (FW) at excision, the saturatedeight (SW) after 24 h rehydration in distilled water (petioles sub-erged) at 4 ◦C in the dark, and the dry weight (DW) after oven

rying for 48 h at 80 ◦C. The leaf RWC was calculated from theormula: RWC = (FW − DW)/(SW − DW). The determinations of leafnd root osmotic potential were followed by Ball and Oosterhuis2005). Simultaneously, the determination of plasma membranePM) H+-ATPase activity was made according to the method of Shent al. (2005) and the determination of proline and glycine betaineGB) contents was followed by Bates et al. (1973) and Zuniga andorcuera (1987), respectively.

.3. Preparation of PM vesicles

PM vesicles were prepared strictly at 4 ◦C following the methodf Palmgren et al. (1990). Briefly, after drought treatments, thehole-root (6 g fresh weight) and the root segments from primary

oots of approximately 500 plants (constituting approximatelyg fresh weight) were ground in the presence of insoluble

ig. 1. Soil moisture dynamics in pots following water withholding within 16 days.

erimental Botany 69 (2010) 1–8 3

polyvinylpyrrolidone with a homogenizing buffer having 330 mMSuc, 50 mM MOPS-1,3-bis(Tris [hydroxymethyl] methylamino)propane (BTP), pH 7.0, 5 mM EDTA, 5 mM dithiothreitol (DDT),0.5 mM phenylmethylsulfonyl fluoride, 0.2% (w/v) bovine serumalbumin (Sigma–Aldrich, St. Louis, MO, USA, protease free), and0.2% (w/v) casein. The homogenate was filtered through four layersof cheesecloth and centrifuged (10,000 × g, 20 min). The super-natant was ultra-centrifuged (1,000,000 × g, 1 h), and the resultingprecipitate was resuspended with a glass homogenizer suspensionbuffer consisting of 330 mM Suc, 5 mM K-phosphate (pH 7.5), 5 mMKCl, 1 mM DDT, and 0.1 mM EDTA. The homogenate was loaded ona 12-g two-phase system containing 6.5% (w/w) Dextran T500, 6.5%(w/w) polyethylene glycol 3350, 330 mM Suc, 5 mM K-phosphate(pH 7.8), 5 mM KCl, 1 mM DDT, and 0.1 mM EDTA. After the batchprocedure, the resulting upper phase was mixed with a dilutionbuffer that consisted of 330 mM Suc, 5 mM MOPS-BTP (pH 7.5), and5 mM KCl and centrifuged (100,000 × g, 1 h). For the determinationof zeta potential, the PM vesicles were used immediately, or storedotherwise at −80 ◦C until further analysis.

2.4. Determination of H+-ATPase activity in PM vesicles

PM H+-ATPase activity was measured in an assay system con-taining 50 mM MOPS-BTP (pH 6.5), 2.5 mM MgSO4, 50 mM KCl,2.5 mM Tris–ATP, 0.05% (w/w) Brij 58 (polyoxyethylene 20 cetylether, Sigma–Aldrich, St. Louis, MO, USA) to produce inside-outvesicles (Johansson et al., 1995) and an appropriate amount ofH+-ATPase. The reaction was carried out for 30 min at 37 ◦C. Five-hundred microliters of 5% (w/v) cold trichloroacetic acid and2 mL of 0.1 M Na-acetate was added to the mixture and cen-trifuged (2000 × g, 10 min) with a further addition of 0.3 mL of1% (w/v) ammonium molybdate in 0.025 M H2SO4. After a briefincubation (30 ◦C, 10 min), the liberated Pi was measured with aspectrophotometer (model UV-752; Shanghai, China) at 720 nm.The membrane protein was determined with the Bradford (1976)method using bovine serum albumin as standard.

2.5. Statistical analyses

The means of physiological parameters of root and leaf werecalculated from ten and five replications for each combination ofgenotype and SWC, and were compared by least significant dif-ference (LSD) at the 0.05 confidence level. ANOVA residuals wereused for the calculation of the 5% LSD; this was done under theassumption of homogeneity of variances (Levene test). In addition,quadratic regressions were used to describe relationship betweenSWCs and plant osmotic potentials or relative water content inorder to reveal the difference in drought adaptation mechanismbetween two cultivars.

3. Results

3.1. Responses of root and leaf RWC to drying soil

Drying soil led to a continuous decrease in relative water con-tent (RWC) of leaf and root, but the decreasing rate varied withoat genotypes and stress intensions (Fig. 2A and B). At the dryingstages from FWC65 to FWC45, there was only a slight reductionin leaf and root RWC for both oat genotype seedlings, suggestingthat the effect of mild stress (MS, 45% FWC) on plant water sta-

tus was weak. When the stress was aggravated to intermediatestress (IS, 35% FWC) and severe stress (SS, 20% FWC), the RWC ofthese two organs tended to decrease significantly, but evidently,the drought-tolerant “Dingyou6” had a better water maintenancethan drought-sensitive “Bende” (Fig. 2A and B).

4 D.-S. Gong et al. / Environmental and Experimental Botany 69 (2010) 1–8

Fig. 2. Relative water contents (RWCs) of leaf and root (A and B) and the root-to-lg

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ered to −0.274 MPa and −0.188 MPa, respectively. Moreover, the

eaf ratio of RWC (C) in the seedlings of two oat cultivars along with drought stressradients.

When the soil moisture was lowered to 40% FWC, the RWC ofoth leaf and root was decreased significantly, being only 82.5%nd 78.4%, respectively. However, it is interesting that the root-to-eaf ratio of RWC remained at similar level as in early stages. Onlys the SWC further declined to 35%, did both the RWC of root andeaf and the root-to-leaf ratio decrease significantly. With the fur-her development of soil drying, the RWC and the ratio tended toecrease continuously. Regarding the performance of two cultivars,he RWC of two organs was in general higher in drought-tolerant cv.han that of drought-sensitive one. Furthermore, the root-to-leafatio of RWC was higher in drought-tolerant cv. than drought-

ensitive cv. in all three stress treatments (Fig. 2C). The resultshowed that drought-tolerant cv. displayed superior water main-enance and hydraulic conductance abilities to drought-sensitivene.

Fig. 3. Osmotic potentials (OPs) of leaf and root (A and B) and the root-to-leaf ratioof OP (C) in the seedlings of two oat cultivars along with drought stress gradients.

3.2. Osmotic potential variations in root and leaf under drying soil

In order to reveal the regulatory mechanism of water mainte-nance, the variations of osmotic potentials (OPs) in two organs ofoat genotype seedlings were also investigated synchronously. Theresults indicated that the OP of root and leaf remained at the levelsof around −0.23 MPa and −0.13 MPa, respectively at the FWC65,FWC60 and FWC55 treatment groups (Fig. 3A and B). Significantdecrease in the OP values was observed till soil moisture was low-ered to about 50% FWC, which was obviously higher than the criticallevel at which the RWC decreased significantly (i.e. at FWC40, seeFig. 2A and B). At this SWC stage, the OPs of root and leaf were low-

root-to-leaf ratio of OP was synchronously lowered significantly atFWC50, which also varied from the event of RWC.

With the aggravation of soil drought stress, the OP tended tosignificantly further drop in both organs of oat genotypes, and the

D.-S. Gong et al. / Environmental and Exp

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ig. 4. Proline and glycine betaine (GB) contents of leaves (A and B) and plasmaembrane (PM) H+-ATPase activities of root hair cell (C) as a function of drought

tress gradients in two oat cultivars.

Ps were significantly higher in drought-tolerant cv. than that ofrought-sensitive one (Fig. 3A and B), suggesting that the formerad higher level of osmotic regulation than the latter. In addition,he root-to-leaf ratio of OP was wholly higher in the former thanhat of latter one (Fig. 3C).

.3. Plasma membrane H+-ATPase activities of root hair cell andariations of two organic osmolytes

Further evidences of osmotic regulation comparison betweenwo oat genotypes were obtained from the dynamics of osmoticegulatory materials and plasma membrane (PM) H+-ATPase activ-ties of root hair cell (Fig. 4). Time-course data showed that the

iosynthesis of leaf proline and glycine betaine (GB) followed aimilar trend as the activities of root cell PM H+-ATPase prioro intermediate stress (IS, 35% FWC). In the FWC60 and FWC55reatment groups, there were no visible changes in the contents

erimental Botany 69 (2010) 1–8 5

of leaf proline and GB and the activities of root PM H+-ATPase.When the SWC reached around 50% FWC (FWC50), however,all three physiological parameters were found to increase sig-nificantly in drought-tolerant cv., with 11.23 �mol GB/g DW and2.87 �mol proline/g DW in leaves, and 1.51-fold activity of PM H+-ATPase of CK group in roots, respectively. Yet in drought-sensitivecv., there were no significant changes for these parameters at thisstress stage (Fig. 4A–C). It was till the SWC fell down to 45% FWCthat drought-sensitive cv. started to increase significantly for threeparameters (10.98 �mol GB/g DW and 2.86 �mol proline/g DW inleaves; 1.31-fold activity of PM H+-ATPase of CK group in roots).

If considering the dynamics of leaf RWC mentioned above, early-warning responses of both cultivars to stress were observed sincesignificant change in root H+-ATPase activity took place early beforearound 35% FWC at which detectable change in leaf RWC occurred.As for the comparison between cultivars, the SWC of early-warningresponse of drought-tolerant cv. to drought stress was obviouslyhigher than that of drought-sensitive cv. With the developmentof soil drought intension, the contents of both proline and GBtended to increase continuously in leaves of two genotypes, exceptthat the GB content was slightly reduced at the stress stages fromFWC25 to FWC20 in drought-tolerant cv. (Fig. 4A and B). On theother hand, relative PM H+-ATPase activities of root hair cell werefurther increased in both genotype seedlings along with stress gra-dients from FWC40 to FWC35. Yet, the PM H+-ATPase activitieswere rapidly decreased from FWC30 to FWC20, reaching up to asignificantly lower level in FWC20 group compared with CK group(Fig. 4C).

3.4. Effect of early activation of PM H+-ATPase on plant waterrelation and osmotic potential

In order to elucidate the physiological implication of early-warning response at whole plant level, the values of RWC and OP inleaf and root were calculated in each treatment group, respectively.Two-way factorial analyses of variance were used to determine thestatistical significance of changes that occurred in leaf RWC andOP in response to cultivar and SWC. In the meantime, quadraticregression was made to assess the dynamics of leaf RWC and OP.The results indicated that the significant decrease in the OP ofseedlings occurred in FWC50 treatment group, whereas the RWCof plants decreased around 40% FWC (Fig. 5A and B). This differenceprovided further evidence that the plants had an early-warningosmotic response to decreased soil water availability before theplant tissue RWC was reduced significantly.

In comparison with those of drought-sensitive cv., the OP andRWC of plant tissues in drought-tolerant cv. tended to decreasemore slowly. The quadratic regressive equations of RWC andOP for drought-tolerant cv. were y = −0.0191x2 + 2.3595x + 13.548and y = −0.0002x2 + 0.0293x − 1.1682, showing a relatively slowdecreasing trend compared with y = −0.0213x2 + 2.8926x − 9.0682and y = −0.0003x2 + 0.0364x − 1.5001 of drought-sensitive cv. cor-respondingly (Fig. 5A and B).

Based on the above data, a causal relationship between PM H+-ATPase and drought adaptation was developed. Earlier increaseof PM H+-ATPase in root hair cells of drought-tolerant cv. isresponsible for better dehydration tolerance, compared with thatof drought-sensitive cv. This is closely associated with strongerosmotic adjustment on the basis of osmotic regulatory materialsincluding proline and GB (Fig. 5A and B).

4. Discussions

Recent studies have shown that PM H+-ATPase is very sensitiveto abiotic stress, such as extreme temperature (Muramatsu et al.,

6 D.-S. Gong et al. / Environmental and Exp

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ig. 5. Timing effect of activation of PM H+-ATPase on plant water status (A) andsmotic potential (OP) (B) between two oat cultivars.

002; Liu et al., 2008), salinity changes (Gévaudant et al., 2007)nd drought stress (Chen et al., 2005). There are many reports con-erning the activity of PM H+-ATPase in relation to water deficitMichelet and Boutry, 1995; Chen et al., 2005; Liu et al., 2005).he proton-pumping ATPase of plasma membrane can generatehe proton motive force across the plasma membrane and makecritical contribution to the drought adaptation of plants (Serrano,989; Chen et al., 2005). The major objective of this study is to elu-idate root-to-shoot regulatory mechanism of PM H+-ATPase forat seedling grown in drying soil.

Plants have evolved perception systems for drought stress thatrigger non-cultivar-specific (NCS) defense responses (Nürnbergernd Kemmerling, 2006). In present study, the SWC in potsecreased during progressive drying at the seedling stage. Sig-ificant increase in the activities of PM H+-ATPase was observedt about 45% or 50% FWC but there were no significant changesn plant RWC until the SWC was down to about 40% FWCFigs. 2A and B, 4C, and 5A). The threshold range of SWC betweenhe operations of PM H+-ATPase activation and plant RWC was–10% FWC. The data provided critical physiological evidencehat there exists an extensively NCS defense response in plantsrown in drying soil. In addition to the common NCS percep-ion system mentioned above, drought-resistance programs areften also initiated through plant cultivar-specific (CS) recognitionf drying conditions, a recognition specificity that is not known

rom plants (Nürnberger and Kemmerling, 2006). Plant speciesnd plant cultivar-specific resistance represent evolutionarily likedypes of immunity to drought stress (Nürnberger and Kemmerling,006). In this study, drought-tolerant “Dingyou6” responded to

erimental Botany 69 (2010) 1–8

soil drying at about 50% FWC, but the SWC of drought-sensitive“Bende”s responding to soil drying was about 45% FWC (Fig. 4C). Theinterspecific difference in triggering early response was obvious.Accordingly, the threshold range of SWC between the operationsof PM H+-ATPase activation and plant RWC was wider in drought-tolerant cv. than that of drought-sensitive cv.

There exists a signal transduction cascades that mediate acti-vation of innate defense responses including osmotic adjustment(OA). OA is a key mechanism enabling plants under drought tomaintain water absorption and cell turgor pressure (Cattivelli etal., 2008). Tolerance to drought has been associated with higherleaf relative water content of stressed plants (Hsiao, 1973). Ourresults indicate that drought-tolerant “Dingyou6” was relativelytolerant to water stress while “Bende” cv. is sensitive (Fig. 2). Theformer had higher root-to-leaf water content, i.e. better root-to-shoot hydraulic conductance, than the latter. Our previous studiessuggested that the earlier response of plants to drought was closelyrelated to better osmotic adjustment and drought tolerance (Xionget al., 2006a,b; Fan et al., 2008). In this study, better water mainte-nance ability was associated with increased PM H+-ATPase activity,suggesting a possible involvement of PM H+-ATPase along withthe development of drought stress gradients (Figs. 2, 4 and 5). Theplant PM H+-ATPase links ATP hydrolysis to the extrusion of pro-tons from the cytoplasm to the cell exterior (Briskin, 1990; Micheletand Boutry, 1995). This provides a driving force for solute transportat the plasma membrane, consisting of an acid-exterior pH gra-dient and interior-negative electrical potential difference (Briskinand Hanson, 1992; Chen et al., 2005). Chemical root-sourced signalmaterials can be synthesized in root system and transported to theabove ground and exert a series of physiological effect (Xiong et al.,2006a).

Many plant species naturally accumulate GB and proline asmajor organic osmolytes when subjected to different abioticstresses. Previous studies suggest that there exists a positive rela-tionship between accumulation of GB and proline and plant stresstolerance (Ma et al., 2004; Ashraf and Foolad, 2007). In this study,the biosynthesis of leaf proline and glycine betaine (GB) followeda similar trend as the activities of root cell PM H+-ATPase priorto intermediate stress (35% FWC) (Fig. 4). Synchronous change inthe contents of two osmolytes in leaves and the activities of rootcell PM H+-ATPase provided chemical evidence for the process of“early response”. As soil began to dry during the initial stages ofdrought, this kind of rapid and early response of roots is criticalto the survival of the plant (Ober and Sharp, 2003). This responsemay be triggered by turgor sensitive stretch-activated membranechannels or by other osmo-sensing elements (Lew, 1996). Duringthis process, PM H+-ATPase plays an important role in the growthand development of plants, and controls many cellular processes inplants such as secondary active transport, cell pH and turgor (Chenet al., 2005). GB can act as an osmoprotectant in many organisms.The capacity to accumulate GB under drying soil condition prob-ably appeared early in the evolution of angiosperms (Hanson andBurnet, 1994).

In plants, water transport across tissues is a fundamental pro-cess that must be adapted sufficiently to environmental stressesin order to allow plants to survive. Much evidence indicates thatnon-hydraulic root-to-shoot signaling is an important componentof plant response to drought conditions in plants (Davies et al.,1994; Xiong et al., 2006a,b; Fan et al., 2008). Yet, the mechanism ofnon-hydraulic root signaling and its exact effects are still in ques-tion. Davis and Zhang (1991) argued that root-sourced ABA signal

development as a function of soil water availability. This processis largely related to the regulation of ABA-like signal substances onwater transport (Mahdieh and Mostajeran, 2009). Furthermore, thisregulatory behavior varied between plant genotypes of differing

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rought resistance (Li et al., 2009). Our data showed that root-to-hoot ratio of RWC tended to decrease with lowering SWC, androught-tolerant cv. had a higher ratio (Fig. 2C). Logically, higheroot-to-leaf ratio of RWC is parallel with better ability of waterransportation via xylem sap, i.e. relatively advantageous hydrauliconductance. This high RWC ratio is beneficial to the prolongedaintenance of root system vigor. Therefore, drought-tolerant cv.

ad a higher hydraulic conductance than drought-sensitive cv. (Lit al., 2009).

With the aggravation of soil drought, water content gradientrom root-to-shoot implied osmotic adjustment ability at wholelant level. Fan et al. (2008) used spring cultivars differing inrought resistance ability as experimental materials to reveal theooperative relation between root signals and OA. The results sug-ested that the early onset of root signal was positively correlatedith the dynamics of OA (r = 0.93). Elkahoui et al. (2005) found

hat OA is closely involved in the activation of H+-ATPase undersmotic stress. PM H+-ATPase is the most abundant protein in thelant plasma membrane and one of the most active enzymes inelation to drought adaptive functions (Dong et al., 1994). In thistudy, drought stress caused the differential response of the PM+-ATPase between two oat genotypic seedlings and then led toifferent performances of drought adaptation. The critical valuef SWC at which the PM H+-ATPase activity was significantly wasigher in drought-tolerant cv. than that of drought-sensitive oneFig. 4C). Based on the above data, the early adaptive mechanism oflant to drought stress may be explained as the below regulatoryathway:

As a whole, the activation of PM H+-ATPase proves to be criticaln regulating the root-to-shoot communication under water-imited conditions. However, the complexity of biosynthesis andunction of PM H+-ATPase in root hair cells requires more effortsor understanding overall root-to-shoot communication theory.

cknowledgements

This work is financially supported by the Natural Science Foun-ation of China (30670321 and 30970447), the “973” Project2007CB106804) and the Program for New Century Excellent Tal-nts in University (NCET-07-0396).

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