Apoplastic and symplastic solute concentrations contribute to osmotic adjustment in bean genotypes during drought stress

Transcription

1 N. SARUHAN GÜLER, A. SAĞLAM, M. DEMİRALAY, A. KADIOĞLU Turk J Biol 36 (212) TÜBİTAK doi:1.396/iy Apoplstic nd symplstic solute concentrtions contriute to osmotic djustment in en genotypes during drought stress Neslihn SARUHAN GÜLER 1, Aykut SAĞLAM 2, Mehmet DEMİRALAY 2, Asım KADIOĞLU 2 1 Deprtment of Biology, Fculty of Arts nd Sciences, Rize University, 531 Rize - TURKEY 2 Deprtment of Biology, Fculty of Science, Krdeniz Technicl University, 618 Trzon - TURKEY Received: Astrct: The present study investigtes chnges in the inorgnic ions, proline, nd endogenous scisic cid (ABA) contents of the poplstic nd symplstic comprtments of leves from drought-tolernt (Ykutiye) nd droughtsensitive (Zuliye) cultivrs of the common en (Phseolus vulgris L.). Drought stress cused decrese in lef wter potentil nd stomtl conductnce in oth cultivrs. Concentrtions of proline in the drought-tolernt nd droughtsensitive cultivrs incresed in response to drought stress in oth comprtments. The symplstic K + concentrtion decresed in oth cultivrs. However, the opposite trend ws oserved concerning K + concentrtions in the poplstic res. While the symplstic N + concentrtions significntly decresed in the drought-tolernt cultivr, the poplstic N + concentrtions incresed during drought stress. However, N + concentrtions did not significntly chnge in either of the comprtments in the drought-sensitive cultivr. The C 2+ concentrtions in the sensitive cultivr significntly decresed in oth comprtments during drought stress. In the tolernt cultivr, the C 2+ concentrtion significntly incresed in the symplst ut decresed in the poplst. Cl - concentrtions in the tolernt cultivr did not significntly chnge in either comprtment. In the sensitive cultivr, the Cl - concentrtion incresed in the poplstic re ut decresed in the symplstic re. In ddition, while the symplstic sp of the leves exhiited constnt ph vlue, it diminished in the poplst during drought stress. Symplstic nd poplstic ABA concentrtions significntly incresed in oth cultivrs. It might e sid tht inorgnic ions (especilly N +, K +, nd C 2+ ) nd ABA concentrtions chnged etween the poplstic nd symplstic spces to contriute to osmotic djustment under drought stress. In ddition, the drought-tolernt cultivr showed much higher cpcity to mintin osmotic djustment etween the symplst nd the poplst. Key words: Apoplst, symplst, drought stress, tolernce, inorgnic ions, scisic cid Kurklık stresi sırsınd fsülye çeşitlerinde poplstik ve simplstik lnlrdki çözünen mdde konsntrsyonu osmotik regülsyon ktkı sğlr Özet: Kurklığ hsss ve dynıklı 2 fsülye çeşidinin içsel sisik sit (ABA), prolin ve inorgnik iyon içeriklerindeki değişim yprk poplstik ve simplstik lnlrınd rştırıldı. Kurklık stresi her iki çeşitte yprk su potnsiyeli ve stom iletkenliğinde zlış neden oldu. Kurklığ dynıklı ve hsss çeşitlerdeki prolin konsntrsyonu her iki lnd kurklık stresine cevp olrk rttı. Simplstik K + konsntrsyonu her iki çeşitte zldı. Bun krşılık K + konsntrsyonuyl ilişkili ters ir eğilim poplstik lnd görüldü. Simplstik N + konsntrsyonu dynıklı çeşitte önemli ölçüde zlırken poplstik N + konsntrsyonu rttı. Diğer trftn, N + konsntrsyonu hsss çeşitte her iki lnd önemli ölçüde değişmedi. Kurklık stresi sırsınd hsss çeşitte C 2+ konsntrsyonu her iki lnd d zldı. 151

2 Apoplstic nd symplstic solute concentrtions contriute to osmotic djustment in en genotypes during drought stress Dynıklı çeşitte C 2+ konsntrsyonu simplstt rttı poplstt ise zldı. Dynıklı çeşitteki Cl - konsntrsyonu her iki lnd önemli ölçüde değişmedi. Hsss çeşitteki Cl - konsntrsyonu ise poplstik lnd rttı simplstik lnd zldı. Ayrıc kurklık stresi sırsınd simplstik ph değişmezken, poplstic ph zldı. Simplstik ve poplstik ABA konsntrsyonu her iki çeşitte önemli ölçüde rttı. Sonuç olrk, inorgnik iyonlr (özellikle N +, K + ve C 2+ ) ve ABA konsntrsyonu kurklık stresi sırsınd osmotik regülsyon ktkı sğlmk için poplstik ve simplstik lnlr rsınd değişti. Ayrıc kurklığ dynıklı çeşidin poplstik ve simplstik lnlr rsınd osmotik regülsyonu devm ettireilmek için çok dh üyük ir kpsiteye ship olduğu sonucun vrıldı. Anhtr sözcükler: Apoplst, simplst, kurklık stresi, tolerns, inorgnik iyonlr, sisik sit Introduction Drought is one of the most importnt stresses in crop production ecuse it ffects lmost ll plnt functions (1). The decrese in osmotic potentil in response to wter stress is well-known mechnism y which mny plnts djust to drought stress (2). Stressed plnts diminish osmotic potentil y ccumulting low-moleculr-weight, osmoticlly ctive compounds clled osmolytes. Under drought conditions, plnts exhiit physiologicl, iochemicl, nd moleculr responses t oth the cellulr nd whole-plnt levels (3). Generlly, the plnts ccumulte some kind of orgnic nd inorgnic solutes in the cytosol to rise the osmotic pressure, therey mintining oth turgor nd the driving grdient for wter uptke (4). One such solute is proline. Proline ccumultion is n importnt indictor of drought stress tolernce in cteri, lge, nd higher plnts (5,6). In ddition to its role s comptile compound for osmotic djustment, proline contriutes to the stiliztion of sucellulr structures, scvenging of free rdicls, nd uffering of cellulr redox potentil under stress conditions (7). Ascisic cid (ABA) is thought to ply n importnt role in the dpttion of plnts to environmentl stress. In ddition to its well estlished role in closing stomt, there is lso evidence tht ABA increses the influx of ions cross memrnes in the root nd encourges the synthesis nd ccumultion of osmoticlly ctive solutes (8). The cell wll poplst, s the extrprotoplstic mtrix of the plnt cell in the lef, hs n ion nd metolite composition distinct from other cellulr comprtments. Furthermore, the composition of the poplstic solution is influenced y the physicochemicl properties of the cell wll, trnsport chrcteristics of the plsm memrne of neighoring cells, poplstic wter trnsport, solute trnsport, nd environmentl fctors (9). The poplst is the first plnt comprtment to encounter environmentl signls in plnts (1) nd it contriutes to plnt development (11). Apoplstic ph exerts strong influence on turgor nd wll loosening, possily vi the control of hydrolytic rections nd intermoleculr interctions etween structurl crohydrtes nd proteins (12). In ddition, it hs een reported tht the inorgnic minerls K +, N +, nd C 2+ ccount for the osmollity of the poplstic fluid (11). The common en (Phseolus vulgris L.) is n importnt crop from the fmily Fcee tht is cultivted worldwide for humn consumption. A wter deficiency during ny of the growth stges of the en species often results in loss of yield (13). Therefore, it is importnt to identify the drought tolernce mechnisms of these species in order to improve its gronomic performnce nd to otin more resistnt cultivrs (14). In the current study, we investigted the extent to which ABA synthesis nd the ccumultion of poplstic nd symplstic ions my contriute to drought tolernce in 2 en cultivrs differing in their tolernce to drought. We determined the chnges of compounds contriuting to osmotic djustment in the poplstic nd symplstic res of the en cultivrs during drought stress. Mterils nd methods Growth of the plnts nd stress ppliction The seeds of common en (Phseolus vulgris L.) cultivrs Zuliye (drought-sensitive) nd Ykutiye (drought-tolernt), whose tolernce levels re known, were otined from the Antolin Agriculturl Reserch Institute in Eskişehir, Turkey. Plnts were 152

3 N. SARUHAN GÜLER, A. SAĞLAM, M. DEMİRALAY, A. KADIOĞLU grown y dily irrigtion in plstic pots (16 cm high, 18 cm in top dimeter, nd 12 cm in ottom dimeter) contining pet nd snd (5:1) in greenhouse (temperture: 25 ± 2 C, reltive humidity: 6 ± 5%, light intensity: 4 μmol m 2 s 1 ) for 3 dys. Drought stress ws pplied y withholding irrigtion t the flowering stge for 1 dys. The following prmeters were mesured in the poplstic nd symplstic spces. Lef wter potentil Lef wter potentil (Ψ lef ) ws mesured with thermocouple psychrometer t 27 ± 1 C (PSYPRO, Wescor, Inc., Logn, UT, USA). Disks of pproximtely 6 mm in dimeter were cut from the youngest fully expnded leves of the plnts nd seled in the C-52 psychrometer chmer. Smples were equilirted for 6 min efore the redings were recorded y wter potentil dt logger in the psychrometric mode. The vlues of Ψ lef were mesured s MP. Stomtl conductnce Stomtl conductnce (g s ) ws monitored with dynmic diffusion porometer (AP4, Delt-T Devices, Cmridge, UK) fter it ws clirted with stndrd clirtion plte following the mnufcturer s instructions. The vlues of g s were mesured s mmol m 2 s 1. Cell memrne stility (CMS) Mesurements of CMS were tken following the protocol of Blum nd Eercon (15). Smples were wshed 3 times in deionized wter to remove electrolytes dhering to the surfce. The smples were then kept in cpped vil (2 ml) contining 1 ml of deionized wter nd incuted in the drk for 24 h t room temperture. The conductnce ws mesured with conductivity meter (YSI Model 345, Yellow Springs, OH, USA). After the first mesurement, the vils were utoclved for 15 min to kill the lef tissue nd relese the electrolytes. After cooling, the second conductivity reding ws tken. These 2 mesurements were crried out individully for ll of the smples from oth the control nd stress tretments. The control gve mesure of lekge solely due to the cutting nd incution of lef disks. The conductnce of the stress smple ws mesure of electrolyte lekge due to drought stress nd ws ssumed to e proportionl to the degree of injury to the memrnes. CMS ws clculted s the reciprocl of cell-memrne injury sed on the method of Blum nd Eercon (15): CMS% = [(1 (T 1 /T 2 ))/(1 (C 1 /C 2 ))] 1, where T nd C refer to the stressed nd control smples, respectively, nd the suscripts 1 nd 2 refer to the initil nd finl conductnce redings, respectively. Anlysis of poplstic nd symplstic proline Apoplstic wshing fluid (AWF) ws extrcted using the vcuum infiltrtion method descried y Nielsen nd Schjoerring (16). Fresh leves were cut into lengths of 1 cm, wshed with deionized wter, nd infiltrted with 32 mm soritol. The leves were then lotted dry with thin pper tissues nd the poplstic solution ws collected in microcentrifuge vils y centrifuging the lef pieces t 145 g for 15 min t 4 C. For the proline extrction of residul lef, dried ground leves (.25 g) were lso used. Smples were homogenized in 5 ml of 3% sulfoslicylic cid nd extrcts were centrifuged t 8 g for 15 min. Proline determintion in the poplst nd symplst ws crried out ccording to the method descried y Btes et l. (17). The proline concentrtion ws determined using stndrd curve. Apoplstic nd symplstic ions The extrction of the poplstic solution for mesurement of the ions ws performed s descried in the section on proline. Following the collection of AWF, the residul lef (.5 g) ws homogenized with liquid nitrogen in 5 ml of deionized wter. The symplstic homogente ws oiled in wter th for 1 min. The precipitte ws removed y centrifugtion (18). Symplstic nd poplstic ion contents (K +, C 2+, Cl -, nd N + ) were mesured with ph/mv/temperture meter (Jenco 623N, Jenco, Sn Diego, CA, USA). Apoplstic nd symplstic ph Apoplstic ph ws directly mesured with the ph/ mv/temperture meter. Smples (.5 g FW 2 ml) were treted for 12 min in sl solution contining.5 mm CSO 4, 5 mm DCMU, nd 2 mm MES, djusted to the required ph (routinely ph 6, unless otherwise indicted) with H 2 SO 4 depending on the presence of the wek ses. At the end of the tretments, the leves were wshed for 3 min t C 153

4 Apoplstic nd symplstic solute concentrtions contriute to osmotic djustment in en genotypes during drought stress with.5 mm CSO 4 to cler the free spce from the externl medium, lotted on filter pper, trnsferred to plstic syringes, nd frozen t 3 C for t lest 3 h. The ph ws directly mesured with the ph/mv/ temperture meter in the cytoplsmic sp otined y squeezing the leves fter freeze-thwing (19). Determintion of poplstic nd symplstic ABA The extrction of the poplstic solution for the mesurement of ABA ws performed with pressure pump (2). Leves were ground in liquid nitrogen, homogenized in distilled wter t rtio of 1:7 (w/v), nd plced overnight in the drk t 4 C. The extrcts were centrifuged t 1, g for 1 min t 4 C, nd the resulting superntnt ws diluted 4 times in stndrd TBS uffer. The ABA in these extrcts ws quntified using the Phytodetek ABA ELISA kit (Agdi Biofords, Evry, Frnce) ccording to the mnufcturer s instructions. Sttisticl nlysis Ech nlysis ws repeted 3 times on mixture of leves from 3 individul plnts with 3 replictes. Vrince nlysis of the men vlues ws performed using Duncn s multiple comprison test (2-wy ANOVA) with SPSS for Windows (Ver. 1., SPSS Inc., Chicgo, IL, USA), nd the significnce level ws 5% (P <.5). Results nd discussion Lef wter sttus nd stomtl conductnce The wter potentil of leves (Ψ lef ) decresed in oth cultivrs during exposure to drought stress. However, the reduction of the wter potentil in the droughtsensitive cultivr ws stronger thn tht oserved in the tolernt cultivr. For exmple, drought stress cused 1-fold reduction of Ψ lef in the droughtsensitive cultivr, ut the reduction in Ψ lef in the drought-tolernt cultivr ws only 5.6-fold when compred to the control (Figure 1). Stomtl conductnce (g s ) ws lso oserved to decrese in oth cultivrs. It ws reduced y 8.4-fold over the control in the sensitive cultivr, while the decrese ws pproximtely 4.7-fold in the tolernt cultivr (Figure 2). Control Stress -.2 * -.4 Lef wter potentil (MP) * -1.2 Ykutiye Zuliye -1.4 Figure 1. Effects of drought stress on lef wter potentil in the leves of en cultivrs. All vlues re mens of triplictes ± SD. Different letters denote significnt differences t P <.5. The sterisks denote significnt differences etween cultivrs for control nd drought ppliction. 154

5 N. SARUHAN GÜLER, A. SAĞLAM, M. DEMİRALAY, A. KADIOĞLU Stomtl conductnce (mmol m -2 s -1 ) * (A) Ykutiye Zuliye * (B ) * Ykutiye Zuliye Cell memrne stility (%) Control Stress Cultivrs Figure 2. Effects of drought stress on stomtl conductnce (A) nd cell memrne stility (%) (B) in the leves of en cultivrs. All vlues re mens of triplictes ± SD. Different letters denote significnt differences t P <.5. The sterisks denote significnt differences etween cultivrs for control nd drought ppliction. Cell memrne stility CMS ws significntly influenced y drought stress. A significnt difference ws oserved in the CMS in oth en cultivrs. The drought-tolernt cultivr expressed higher CMS thn the sensitive cultivr. The men CMS vlues for the tolernt nd sensitive cultivrs were 83.2% nd 76.7%, respectively (Figure 2). Proline chnges in poplstic nd symplstic res Symplstic proline concentrtions significntly incresed in oth cultivrs during exposure to drought stress. In the sensitive cultivr, the rtio of the increse compred to its control ws 4-fold in the symplstic re of the lef, ut the increse in the corresponding rtio of the tolernt cultivr ws 1.5-fold compred to the control. As for the poplst, proline concentrtions significntly incresed in oth cultivrs during drought stress. When compred to the controls, these increses were 1.4- nd 1.5-fold in the tolernt nd sensitive cultivrs, respectively. The symplstic proline concentrtion of the lef ws further noted to e higher thn tht of the poplst in oth cultivrs (Figure 3). Ion nd ph chnges in poplstic nd symplstic res Symplstic K + concentrtions decresed in the drought-tolernt nd drought-sensitive cultivrs Apoplstic proline (μm) S ymplstic proline (μg g -1 FW) Ykutiye Zuliye Control Stress Figure 3. Chnges in poplstic nd symplstic proline during drought stress. All vlues re mens of triplictes ± SD. Different letters denote significnt differences t P <.5. The sterisk denotes significnt differences etween cultivrs for control nd drought ppliction. under stress conditions. As compred to the controls, the rtes of decrese were 1.6- nd 1.12-fold in the sensitive nd tolernt cultivrs, respectively. * 155

6 Apoplstic nd symplstic solute concentrtions contriute to osmotic djustment in en genotypes during drought stress The opposite trend ws oserved concerning K + concentrtion in the poplstic re. When compred with the controls, the rtes of increse in this re were 1.7 in the sensitive cultivr nd 2. in the tolernt cultivr. While symplstic N + concentrtions significntly decresed in the tolernt cultivr, poplstic N + concentrtions incresed during exposure to drought stress. However, N + concentrtions did not significntly chnge in either of the comprtments in the sensitive cultivr. On the other hnd, C 2+ concentrtions in the sensitive cultivr significntly decresed in oth comprtments during drought stress. In the drought-tolernt cultivr, C 2+ concentrtions significntly incresed in the symplst ut decresed in the poplst. The Cl - concentrtion in the tolernt cultivr did not significntly chnge in either of the comprtments. In the sensitive cultivr, Cl - concentrtions incresed in the poplstic re ut decresed in the symplstic re (Tle). Drought stress resulted in decrese in the ph of the en cultivrs poplstic fluid. The symplstic ph did not significntly chnge during drought stress (Tle). ABA chnges in poplstic nd symplstic res The ccumultion of ABA induced y drought ws significntly higher in the drought-tolernt cultivr Tle. Chnges in inorgnic ions nd ph during drought stress. The sme lower cse letters re not significntly different from ech other (P <.5) in ech line. The sme upper cse letters re not significntly different from ech other (P <.5) in ech column. Dt re mens ± SD. Potssium concentrtion (μm) Apoplst Symplst Zuliye 4.5 ±.9 A 7.5 ±.8 A 21.2 ± 1.8 A 13.1 ±.7 A Ykutiye 1.7 ±.2 B 3.4 ±.2 B 16. ±.8 B 14.2 ±.7 A Sodium concentrtion (μm) Apoplst Symplst Zuliye.44 ±.3 B.5 ±.2 B.27 ±.2 A.26 ±.5 A Ykutiye 1.2 ±.3 A 2. ±.1 A.34 ±.3 A.28 ±.2 A Clcium concentrtion (μm) Apoplst Symplst Zuliye.15 ±.3A.9 ±.1A.9 ±.1 A.3 ±.1 B Ykutiye.15 ±.4 A.9 ±.1A 2.52 ±.4 A 5.1 ±.8 A Chloride concentrtion (μm) Apoplst Symplst Zuliye 59.1 ±.2 B 59.7 ±.2 B 55.2 ±.2 B 54.9 ±.2 B Ykutiye 63.3 ±.3 A 63.6 ±.3 A 56.4 ±.2 A 56.4 ±.2 A ph Apoplst Symplst Zuliye 5.3 ±.4 B 5. ±.3 B 6. ±.2 A 6.1 ±.1 A Ykutiye 5.6 ±.1 A 5.1 ±.3 A 6. ±.2 A 6. ±.2 A 156

7 N. SARUHAN GÜLER, A. SAĞLAM, M. DEMİRALAY, A. KADIOĞLU thn in the drought-sensitive cultivr. Moreover, ABA concentrtions in the poplstic nd symplstic res significntly incresed in oth cultivrs. When compred to the control, the rtes of increse in the symplst were 1.3 nd 1.8 for the sensitive nd the tolernt cultivr, respectively. With regrd to the poplst, ABA concentrtions incresed t rtes of 1.3 nd 1.4 for the sensitive nd the tolernt cultivr, respectively (Figure 4). In this study, chnges in the ABA content nd inorgnic solutes of poplstic nd symplstic spces of leves were determined in en cultivrs differing in their tolernce to drought. As expected, the wter potentil of leves (Ψ lef ) decresed in oth cultivrs fter exposure to drought stress. The cpcity of the tolernt cultivr to mintin higher lef wter potentil compred to the sensitive cultivr my e ttriuted to its ility to postpone dehydrtion (21). It is known tht CMS is n indictor of drought tolernce (22). In our work, the CMS ws significntly influenced y drought stress. The drought-tolernt cultivr expressed higher level of CMS thn the sensitive one. Stomtl conductnce lso decresed in oth cultivrs. The negtive effect of drought stress on stomtl conductnce ws lso oserved in soyen y Bunce (23). It is cler tht the higher stomtl conductnce oserved in the tolernt cultivr compred to the sensitive cultivr could give rise to the differences in sensitivity to drought. The ility of cultivr to keep its stomt open despite internl wter stress hs een considered form of drought resistnce (24). In our experiment, the concentrtion of C 2+ significntly decresed in oth comprtments in the sensitive cultivr. This decrese in C 2+ concentrtion my result from its inding to peptic cids in the cell wll in order to firm the wll, mking the plnt more resistnt to drought. On the other hnd, the symplstic C 2+ concentrtion incresed in the drought-tolernt cultivr, wheres the poplstic C 2+ concentrtion declined. In plnts, trnsient increses in cytosolic C 2+ hve een reported in response to diverse rnge of iotic nd iotic stimuli (25,26), ut the specificity of the physiologicl responses is not understood. Recent studies suggest tht C 2+ inding proteins known s clmodulin or clciumdependent protein kinses serve s the primry regultors of internl C 2+ levels in plnt cells nd function to uffer intrcellulr C 2+ levels or trnslte the intrcellulr oscilltions of free C 2+ levels into.25.2 Ykutiye Zuliye * ABA (pmol ml 1 ).15.1 * *.5 Apoplst Symplst Figure 4. Chnges in poplstic nd symplstic ABA in the leves of en cultivrs during drought stress. All vlues re mens of triplictes ± SD. Different letters denote significnt differences t P <.5. The sterisks denote significnt differences etween cultivrs for control nd drought ppliction. 157

8 Apoplstic nd symplstic solute concentrtions contriute to osmotic djustment in en genotypes during drought stress signl-specific cellulr responses (27,28). On the other hnd, enhnced ABA levels trigger n increse in cytosolic C 2+ in gurd cells, nd it hs een suggested tht this might include C 2+ influx cross the plsm memrne (29). Indeed, we found tht ABA incresed in oth comprtments under drought stress. In ddition, C 2+ ions control the efficiency of wter use y inititing stomtl closure (3). Thus, the incresed symplstic C 2+ concentrtions in the tolernt cultivr my e responsile for the reduction in stomtl conductnce. We lso found tht stomtl conductnce significntly decresed in the droughttolernt cultivr. In the current study, symplstic K + concentrtions decresed in oth cultivrs, y out 62% in the sensitive cultivr nd 13% in the tolernt cultivr. Conversely, the poplstic K + concentrtion incresed in oth cultivrs. A decrese in the K + concentrtion of the symplst my e ssocited with the influx of K + towrd the poplst. On the other hnd, n increse in poplstic K + proly indictes displcement of potssium from fixed nionic exchnge sites in the cell wll. Our dt indicte n increse in the poplstic N + concentrtion of the drought-tolernt cultivr fter drought exposure. This result shows tht the tolernt cultivr hs the ility to protect its cytosol from excess sodium. It is interesting to note tht the poplst ws the primry site of sodium ccumultion (31). In the poplst, the ccumultion of N + resulted in decreses in C 2+ (Tle). Additionlly, high poplstic N + nd K + concentrtions in oth cultivrs my e highly regulted y ion trnsport etween the symplst nd poplst. Our findings lso indicte tht Cl - ws lmost eqully distriuted etween oth spces in the tolernt cultivr. The contriution of this fctor to the chnge in osmotic potentil ws insignificnt. In the sensitive cultivr, Cl - concentrtions incresed in the poplstic re ut decresed in the symplstic re. The increse in poplstic Cl - my show tht the Cl - ws trnsported from the symplst to the poplst to contriute to osmotic djustment. In the current study, the symplstic sp of leves exhiited constnt ph vlue of 6. in oth cultivrs during drought stress. In contrst, the ph of the poplst decresed in oth cultivrs. The poplstic ph hs een reported mny times from different species, nd the mjority of vlues vry etween 5.3 (32) nd 6.7 (33). It hs een reported tht incresed symplstic C 2+ dectivtes the plsmlemm H + / ATPse nd lso ctivtes K + /H + symport. The inflow of K + nd H + depolrizes the memrne, nd thus the poplst ecomes less cidic (34). However, symplstic C 2+ decresed in the sensitive cultivr during drought stress. It cn therefore e sid tht C 2+ does not ffect plsmlemm H + / ATPse ctivity or ctivte the K + /H + symport sufficiently, nd the poplst ecomes more cidic s consequence. However, cidifiction of poplstic ph in the tolernt cultivr my e cused y different mechnisms, such s chnges in phosphte nutrition nd delivery. During the drought period, not only the symplstic ut lso the poplstic ABA incresed. We inferred tht lef poplstic ABA concentrtions incresed nd ph did not ffect the distriution of ABA in the poplstic nd symplstic res. In ddition to the well-estlished role of ABA in closing stomt, there is evidence tht ABA hs role in regulting solute ccumultion nd, thus, osmotic djustment (35). Increses in poplstic ABA nd the lef osmolyte content were higher in the drought-tolernt cultivr s compred to the drought-sensitive cultivr. Therefore, it is resonle to suppose tht osmotic djustment my respond to the ABA of the poplstic frction of the leves. In the present study, generl correltion etween ABA nd stomtl conductnce ws found (P <.1, r =.97). The decrese in g s vlues during drought ws ccompnied y significnt rise in ABA. It is known tht proline provides n importnt contriution to osmotic djustment nd dpttion to stress (36). In our study, proline concentrtions significntly incresed in oth comprtments in en cultivrs, with the highest rte of increse eing oserved in the symplstic re of the drought-sensitive cultivr (293%). These findings re consistent with reports of higher lef proline in sensitive genotypes of other species (37-39). The drmtic increse in lef poplst osmollity in plnts sujected to drought stress my e the result of high ccumultion of N + nd K + in the lef cell. 158

9 N. SARUHAN GÜLER, A. SAĞLAM, M. DEMİRALAY, A. KADIOĞLU In the symplst, however, the reltive contriution of C 2+ to the osmollity ws the highest mong ll of the solutes studied. In the tolernt cultivr, it contriuted to symplstic osmollity. In the sensitive cultivr, this figure ws sustntilly lower, mking it necessry for drought-sensitive plnts to synthesize t lest twice s much symplstic proline s droughttolernt ones. Sensitive cultivrs my need to synthesize high levels of proline to compenste for this difference to lnce the intrcellulr osmotic potentil. Moreover, extrcellulr proline levels cn e incresed y incresing intrcellulr proline, which provides considerle support for the role of proline in stress tolernce. We conclude tht the drought-tolernt cultivr hs much higher cpcity to mintin osmotic djustment etween the symplst nd the poplst. Both cultivrs showed chrcteristic differences regrding inorgnic ions in leves. Additionlly, the N +, K +, nd C 2+ concentrtions in the tolernt cultivr were high, nd their reltive contriutions to the osmotic potentil of the tolernt cultivr under drought stress were higher thn tht of proline. Finlly, it might e sid tht inorgnic ions nd ABA concentrtions chnged etween the poplstic nd symplstic spces, contriuting to osmotic djustment under drought stress. Acknowledgment We thnk the Reserch Fund of Krdeniz Technicl University for its support ( ). Corresponding uthor: Neslihn SARUHAN GÜLER Deprtment of Biology, Fculty of Arts nd Sciences, Rize University, 531 Rize - TURKEY E-mil: neslihnsruhn@hotmil.com References 1. Hernández JA, Ferrer MA, Jimenez A et l. Antioxidnt system nd O 2 - /H 2 O 2 production in the poplst of pe leves. Its reltion with slt-induced necrotic lesions in minor veins. Plnt Physiol 127: , Ptks A, Noitskis B. Mechnisms involved in diurnl chnges of osmotic potentil in grpevines under drought conditions. J Plnt Physiol 154: , Hsegw PM, Bress RA, Zhu JK et l. Plnt cellulr nd moleculr responses to high slinity. Annu Rev Plnt Physiol Plnt Mol Biol 51: , Rhodes D, Smrs Y. Genetic control of osmoregultion in plnt. In: Strnge K. ed. Cellulr nd Moleculr Physiology of Cell Volume Regultion. CRC Press; 1994: pp Kvi Kishor PB, Sngm S, Amruth RN et l. Regultion of proline iosynthesis, degrdtion, uptke nd trnsport in higher plnts: its impliction in plnt growth nd iotic stress tolernce. Current Sci 88: , Tnner JJ. Structurl iology of proline ctolism. Amino Acids 35: , Hoque MA, Okum E, Bnu MNA et l. Exogenous proline mitigtes the detrimentl effects of slt stress more thn exogenous etine y incresing ntioxidnt enzyme ctivities. J Plnt Physiol 164: , LRos PC, Hsegw PM, Rhodes D et l. Ascisic cid stimulted osmotic djustment nd its involvement in dpttion of tocco cells to NCl. Plnt Physiol 85: , Dietz KJ. Functions nd responses of the lef poplst under stress. Prog Bot 58: , Go G, Knight MR, Trewvs AJ et l. Self-reporting Aridopsis expressing ph nd C 2+ indictors unveil ion dynmics in the cytoplsm nd in the poplst under iotic stress. Plnt Physiol 134: , Almeid DPF, Huer DJ. Apoplstic ph nd inorgnic ion levels in tomto fruit: potentil mens for regultion of cell wll metolism during ripening. Physiol Plntrum 15: , Ryle DL, Clelnd RE. The cid growth theory of uxininduced cell elongtion is live nd well. Plnt Physiol 99: , Richrds RA. Vrition etween nd within species of rpeseed (Brssic cmpestris nd B. npus) in response to drought stress. II. Physiologicl nd iochemistry chrcters. Aust J Agric Res 29: , Suro GV, Johnsen C, Slinkrd AE. Strtegies for improving drought resistnce in grin legumes. Crit Rev Plnt Sci 14: , Blum A, Eercon A. Cell memrne stility s mesure of drought nd het tolernce in whet. Crop Sci 21: 43-47, Nielsen KH, Schjoerring JK. Regultion of poplstic NH 4 concentrtion in leves of oilseed rpe. Plnt Physiol 118: , Btes LS, Wldren RP, Tere LD. Rpid determintion of free proline for wter-stress studies. Plnt Soil 39: 25-27,

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