ISSN 2-4437, Russin Journl of Plnt Physiology, 28, Vol. 55, No., pp. 48 53. Pleides Pulishing, Ltd., 28. RESEARCH PAPERS Physiologicl Chnges in Them in Post-Stress Emerging Ctennthe setos Plnts under Drought Conditions A. Sglm, A. Kdioglu, R. Terzi, nd N. Sruhn Deprtment of Biology, Fculty of Science nd Arts, Krdeniz Technicl University, 68 Trzon, Turkey; fx: 9-462-325-395; e-mil: kdioglu@ktu.edu.tr Deprtment of Biology, Fculty of Science nd Arts, Rize University, 53 Rize, Turkey Received Decemer, 26 Astrct The cpility of Ctennthe setos (Rosc.) Eichler (Mrntcee), pre-exposed to severe drought stress, of dpttion to new drought conditions ws investigted. C. setos unstressed plnts were exposed to drought period of 7 dys. These plnts were nmed s primry-stress plnts (PS). They were trimmed, nd new plnts were produced from their rhizomes. New growing plnts were nmed s post-stress emerging (PSE) plnts. It ws oserved tht lef re nd petiole length of PSE plnts were reduced, s compred to unstressed plnts. When PSE plnts were exposed to second drought-stress cycle, it ws determined tht the degree of their lef rolling ws higher ut their reltive wter content (RWC) ws less thn these prmeters of PS plnts during drought period. However, t the 8% rolling degree, RWC in PSE plnts ws higher thn in PS plnts. Consequently, it cn e sid tht the PSE plnt were more resistnt to new drought stress thn unstressed nd PS plnts. In the leves of PSE plnts, which were exposed to second drought-stress period, the content of solule protein decresed in the erly period of drought, ut incresed in the lter periods. Also, the content of reducing sugr in PSE plnts grdully decresed during the stress period. Proline content incresed mrkedly during drought period, wheres peroxidse ctivity incresed up to the 6th dy nd then decresed. Key words: Ctennthe setos - wter deficit - lef rolling - post-stress emerging plnts - sugr - proline - peroxidse DOI:.34/S24437856 INTRODUCTION Long-term drought cuses destructive effects in plnts []. To overcome these effects, plnts exhiit some chnges in morphologicl, developmentl ptterns s well s physiologicl nd iochemicl processes [2]. One of the morphologicl responses to drought is lef movement [3]. Some plnts, especilly crops, show lef rolling to e le to del with unfvourle conditions, such s trnspirtionl wter losses from dxil surfces nd energy lod on leves resulted from desicction [4]. A decrese in the lef wter potentil retrds lef growth in response to wter loss [5]. Furthermore, reduction in the lef re results in decrese in trnspirtion, cting similrly s stomtl closure, nd reduces wter loss [6]. Stress effect on protein content depends on plnt species []. For instnce, dpttion of tomto plnt cells to wter stress ws ccompnied y decrese in the solule protein level [7]. On the other hnd, exposure to drought increses sugr content in mny spe- POD peroxidse; PS primry stress; PSE post-stress emerging; PVPP polyvinylpolypyrrolidone; RWC reltive wter content; trg wt turgid weight. This text ws sumitted y the uthors in English. cies. For instnce, in clover, reducing sugrs ccumulted s drought stress progressed [8]. Proline lso ccumulted in response to wter stress in lef tissues of plnts [9]. Resistnt lines of some plnts ccumulted high levels of proline. Proline ccumultion contriutes to the immedite recovery of plnts from drought []. Moreover, some enzymes, such s peroxidses, hve een ffected y wter deficit []. Severl physiologicl functions of plnt peroxidses re necessry for plnt stress resistnce. These re removing of ç 2 é 2, oxidtion of toxic reductnts, iosynthesis nd degrdtion of lignin [2]. Ctennthe setos, plnt exhiiting lef rolling to dpt to severe wter deficit conditions, is memer of rhizome-forming herceous plnts (Mrntcee) nd is grown s houseplnt ecuse of their ttrctive ornmentl leves. A notle increse in the degree of lef rolling, proline nd reducing sugr contents ws determined fter exposing C. setos plnts to wter deficit [3, 4]. However, there is no report out lef rolling in post-stress emerging C. setos exposed to second drought-stress cycle. The im of the present study ws to investigte morphologicl dpttion of post-stress emerging C. setos 48
PHYSIOLOGICAL CHANGES IN THEM IN POST-STRESS EMERGING 49 plnts to new drought-stress conditions. We lso studied chnges in solule protein, reducing sugr, nd proline contents, nd peroxidse ctivity of post-stress emerging plnts under incresing drought periods. MATERIALS AND METHODS Plnt growth nd stress pplictions. Ctennthe setos (Rosc.) Eichler (Mrntcee) plnts re perennil hers with sympodil rhizome system. The leves re rdicl nd their sheths together form n pprent stem. The plnts were grown in plstic pots (6.3 cm high, 7.9 cm top nd.2 cm ottom dimeters) contining pet nd snd mix (5 : ), in growth chmer under the following tretment conditions: 6-h photoperiod, 25/2 ë dy/night temperture, 7% reltive humidity, nd 25 µmol/(m 2 s) photon flux density. These plnts were nmed s unstressed plnts. All unstressed plnts were exposed to severe drought period for 7 dys. These plnts sujected to primry stress (PS) were trimmed nd then irrigted. New plnts were produced from the rhizomes of these plnts. These new plnts were clled post-stress emerging (PSE) plnts. The PSE plnts were grown for 9 dys. All plnts hd pproximtely 2 leves. The PSE plnts were not wtered through 7 dys under the lortory conditions. The leves of PSE plnts were hrvested on the (control), 2, 35, 45, 5, 6, nd 7th dys of drought. The hrvested leves were used for nlysis. Mesuring of lef rolling degree nd lef reltive wter content. A degree of lef rolling ws detected ccording to Premchndr et l [5] y mesuring the width of the rolled lef nd tht of the fully unrolled lef t its hlf-length; the degree of lef rolling ws expressed s percentge of the rolled lef width of tht of the unrolled lef. Lef reltive wter content (RWC) ws estimted ccording to the method of Cstillo [6] nd clculted for ech drought period. Lef smples (.5 g) were sturted in ml of distilled wter for 24 h t 4 ë in drkness, nd their turgid weights (trg wts) were recorded. Susequently, they were dried t 65 ë in oven for 48 h, nd their dry weights were determined. RWC ws clculted s given elow: RWC (%) = [(fr wt - dry wt)/(trg wt - dry wt)]. Determintion of solule protein content. For extrction of solule proteins, the leves of PSE plnts were freshly hrvested nd fixed in liquid nitrogen. Thwed leves were homogenized in.5 M sodium phosphte uffer (Merck, Germny) (ph 6.5) with.% polyvinylpolypyrrolidone (PVPP) (Sigm, United Sttes). The homogente ws filtered through cheesecloth. The filtered homogente ws centrifuged t 2 g (3K 8 Sigm, Germny) for 2 min t 4 ë. After centrifugtion, the superntnt ws tken nd used for protein mesurements. Solule protein content ws ssyed ccording to Brdford [7]. Asornce ws red on UV-VIS spectrophotometer (Techcomp 85 II, South Kore) t 595 nm. The solule protein concentrtion ws determined y using stndrd curve nd expressed s mg/g fr wt. Determintion of reducing sugr content. Dry smples of PSE plnt leves were ground in 5 ml of 8% queous ethnol (Merck) with mortr nd pestle. The extrct ws filtered through two lyers of cheesecloth, the filtrte ws centrifuged t 5 rpm (NF 65, Nuve, Turkey) for 5 min, nd the superntnt ws stored t 4 ë for further nlysis. Reducing sugr content ws determined ccording to the method of Ross [8]. The superntnt ( ml) ws dded to 3 ml of dinitrophenol solution (.38 M). Tues with this mixture were incuted t 65 7 ë for 6 min in wter th nd then llowed to cooling in cold wter. Color chnge ws estimted using UV- VIS spectrophotometer t 6 nm. Determintion of proline content. The method of Btes et l [9] ws used for proline determintion. Free proline ws extrcted from the leves of PSE plnts using queous sulfoslicylic cid (Acros Orgnics, United Sttes). The filtrte ( ml) ws mixed with equl volumes of glcil cetic cid (Riedel-de Hén, Germny) nd ninhydrin regent (.25 g ninhydrin (Merck), 3 ml of glcil cetic cid, 2 ml 6 M ç 3 êé 4 (Merck)) nd incuted for h t ë. The rection ws stopped y plcing the test tues in cold wter. The smples were rigorously mixed with 3 ml toluene (Merck). The light sorsion of toluene phse ws estimted t 52 nm on UV-VIS spectrophotometer. The proline concentrtion ws determined using stndrd curve. Free proline content ws expressed s mg/g dry wt. Mesurement of peroxidse ctivity. Totl peroxidse ctivity ws determined spectrophotometriclly using the method of Rodriguez nd Snches [2]. Freshly hrvested leves were frozen in liquid nitrogen. The leves were then ground in liquid medium, which contined 5 mm sodium phosphte uffer, ph 6.5 (Merck) with.% PVPP (Sigm). The homogente ws filtered through cheesecloth. The filtered homogente ws centrifuged t 2 g (3K 8 Sigm, Germny) for 2 min t 4 C. The peroxidse ctivity ws nlyzed in 5 mm phosphte uffer (ph 6.5) contining 4 mm guicol (Sigm) nd 26 mm ç 2 é 2 (Merck). The increse in sornce t 42 nm ws recorded within 8 s fter dding 26 mm ç 2 é 2. Peroxidse ctivity ws expressed s A 42 /(min g fr wt). Sttisticl nlysis. Mens of 23 replictes of lef re nd petiole length were compred y one-smple t test t p.5. Mens of six replictes of lef rolling, solule protein, reducing sugr, free proline, nd POD ctivity, nd lef RWC were compred using the ANOVA test t p.5.
5 SAGLAM et l. Tle. Comprison of lef re nd petiole length in unstressed nd PSE plnts Plnts Lef re, cm 2 Petiole length, cm Unstressed 89.7 ± 25. 44.9 ± 8. PSE 27.5 ± 7.2.6 ± 3. Note: Different letters denote significnt differences mong mens (p.5; one smple t test). Vlues re men nd stndrd devitions (n = 23). RESULTS Effect of Wter Deficit on Lef Morphology nd Reltive Wter Content A significnt (p.5) reduction ws found in the lef re (27.5 ± 7.2 cm 2, n = 23) nd petiole length (.6 ± 3. cm, n =23) in PSE plnts, wheres these prmeters were equl to 89.7 ± 25 cm 2 (n = 23) nd 44.9 ± 8 cm (n = 23) in unstressed plnts, respectively (Tle ). A remrkle increse in the rolling degree nd decrese in RWC of the leves in oth PS nd PSE plnts were determined with n enhnced drought period (Tle 2). Leves of PSE plnts were rolled erlier thn those of PS plnts. For exmple, on the 2th dy of drought period, when no rolling ws oserved in PS plnts, the degree of lef rolling in the PSE plnts ws 67%. However, t the similr degrees of lef rolling, RWC in the PSE plnts ws higher thn tht in the PS plnts. For instnce, t 8% rolling degree, RWC in PS nd PSE plnts ws 74 nd 79%, respectively. Effect of Drought Stress on Solule Protein nd Reducing Sugr Contents in PSE Plnts In PSE plnts, sudden decrese ws seen in the content of solule protein on the 2th dy of drought stress. Then, the level incresed significntly on the 35th dy. A susequent grdul increse etween the 45th nd 7th dys of drought period ws insignificnt (Fig. ). An ovious decrese ws lso determined in the content of reducing sugr during drought period, prllel to the increse in lef rolling of PSE plnts under the stress. The lowest content of the sugr in PSE plnts (.69 ±.7 mg/g dry wt) ws oserved on the 7th dy of drought stress (Fig. ). Effect of Drought Stress on Proline Content in PSE Plnts The free proline content in PSE plnt ws found s 4.7 ± 2.5 mg/g dry wt on the first dy of drought stress. Then it decresed on the 2th dy of drought period. However, it grdully incresed from the 2th dy up to the 7th dy. The lowest nd highest proline contents were determined s 29. ± 2.5 mg/g dry wt on the 2th nd 23. ± 5.9 mg/g dry wt on the 7th dy of the drought period, respectively (Fig. 2). Effect of Wter Deficit on Peroxidse Activity in PSE Plnts The peroxidse (POD) ctivity rise in stressed PSE plnts ws oserved through drought stress period (Fig. 2). Until to 6th dy of drought, shrp increse in peroxidse ctivity in PSE plnts ws found. Then the ctivity decresed on the 7th dy of drought period. Although peroxidse ctivity on the 7th dy of drought period ws lower thn tht of the 6th dy of drought, it remined 6.3-fold higher thn the initil one (Fig. 2). DISCUSSION There were ovious reductions in the lef re nd petiole length of PSE plnts in comprison to the unstressed plnts. The lef re nd petiole length of PSE plnts decresed y 69.3 nd 76.4%, respectively, s compred with those of unstressed plnts. Similrly, Fernndez nd Reynolds [2] reported out chnges in specific lef re in response to drought in eight peren- Tle 2. Effects of drought stress on the degree of rolling nd reltive wter content in leves of PS nd PSE Ctennthe setos plnts Drought period, dys Degree of rolling *, % Reltive wter content **, % PS plnts PSE plnt PS plnts PSE plnt 94 ± e 92 ± 2 d 2 67 ± 3 9 ± 2 e 86 ± 2 c 35 2 ± 7 8 ± 2 87 ± d 79 ± 45 47 ± 5 82 ± 4 84 ± c 78 ± 5 62 ± 7 c 82 ± 5 8 ± 2 77 ± 3 6 78 ± 4 d 86 ± 2 75 ± 76 ± 2 7 8 ± 3 d 87 ± 74 ± 2 76 ± 2 Note: Different letters denote significnt differences mong mens (p.5; ANOVA). Vlues re men nd stndrd devitions of eight (*) nd three (**) replictes.
PHYSIOLOGICAL CHANGES IN THEM IN POST-STRESS EMERGING 5 Solule protein, mg/g fr wt 3.5 3. 2.5 2..5..5 f c c d de ef 2 2 3 4 5 6 7 Drought durtion, dys.4.2..8.6.4.2 Reducing sugrs, mg/g dry wt POD ctivity, A 42 /(g fr wt min) 3 25 2 5 5 2 2 3 4 5 6 7 Drought durtion, dys c e c e d 2 e d 3 25 2 5 5 Free proline, mg/g dry wt Fig.. Effects of drought stress on () solule protein nd (2) reducing sugr of PSE plnts. Different letters denote significnt differences mong mens. Men comprison ws performed using the ANOVA test t p.5. Verticl rs indicte stndrd devitions of six replictes of ech tretment. Fig. 2. Effects of drought stress on POD () ctivity nd free proline (2) content of PSE plnt. Different letters denote significnt differences mong mens. Men comprison ws performed using the ANOVA test t p.5. Verticl rs indicte stndrd devitions of six replictes of ech tretment. nil grsses. They lso indicted tht drought reduced growth y 5 nd 68% (moderte nd severe drought, respectively) compred to the well-wtered controls. Also, Mroco et l [22] found tht drought-stressed C 4 grsses displyed lower specific lef re thn tht of the well-wtered plnts. Mny reserchers hve reported tht lef rolling is n dptive response reducing wter loss vi trnspirtion [23, 24]. Turgut nd Kdioglu [3] lso found the lef rolling response during drought stress in C. setos. The prolem ws which ws the response of PSE plnts mnifesting in lef rolling? To find n nswer the question, the degree of lef rolling during drought period ws mesured in PSE plnts. In the present study, first lef rolling of PSE plnts ws determined on the 2th dy fter withholding wter. However, first lef rolling for PS plnts ws noted only on the 35th dy of drought. The highest lef rolling degree (87%) of PSE plnts ws oserved on the 7th dy fter withholding wter. In other words, we determined tht the lef rolling response in PSE plnts ws quicker thn this of PS plnts, which were exposed to the stress. In ddition, t 8% rolling degree, RWC in PSE plnt ws higher thn tht of PS plnts. By rolling their leves erlier with higher rolling degree, the PSE plnts might keep trnspirtionl wter loss t lower level nd provide itself chnge to sve more wter. In ddition, it is well known tht the reduction in the lef re results in decrese in trnspirtion, which reduces wter loss [6]. A decline in the lef RWC ws oserved in numerous plnts s response to wter deficit [25]. In the content of solule protein, significnt decrese ws oserved on the 2th dy of drought, ut in the lter periods, the increses were found in the leves of PSE plnts. The oserved decrese in the solule protein content on the 2th dy of drought my e due to stress hitution. On the other hnd, the increse in the solule protein content fter the 2th dy of drought suggested tht new proteins might e synthesized, such s key enzymes for osmolytes iosynthesis, sugr nd proline trnsporters, detoxifiction enzymes, nd vrious proteses [26]. In previous studies, drought-induced decrese in the solule protein content ws reported for PS plnts of C. setos [4]. For exmple, solule protein contents of PS plnts were detected on the 32, 48, nd 64th dys of drought stress s 2.,.6, nd.5 mg/g fr wt, respectively. These decreses for PS plnts comprised 6, 36, nd 4% of their initil vlues for the 32, 48, nd 64th dys of drought, respectively (unpulished dt). On the other hnd, the declines in the protein content of PSE plnts in the present study comprised 9.9,., nd 7.8% of 35, 5, nd 6th dys of drought, respectively. Our results showed tht reducing sugr levels of PSE plnts grdully decresed with incresed drought periods. In contrst, grdul increse in reducing sugr content of PS plnt leves ws reported during drought stress of Ctennthe [27]. Sruhn et l. [27] found tht the reducing sugr content of PS plnts scended from 44.8 to 65.3 mg/g dry wt y strting from the 6th to 28th dys of drought. However, the incresed concentrtion of reducing sugr provides for plnt dpttion to stress [7]. On the other hnd, the overll low contents of reducing sugrs of PSE plnts under stress conditions my e due to generl decrese in totl metolic ctivity cused y stress. It is lso possile tht more rpid hexose metolism, supplying compounds for wter lnce, is involved in decresing of reducing sugrs [28].
52 SAGLAM et l. The increses in the concentrtion of proline in PSE plnt leves were found to e remrkle during drought stress. These results in PSE plnts were in ccordnce with those for PS plnts during drought stress s reported previously [27]. In this previous study the vlues of proline content in PS plnts were 38.8 nd 28.3 mg/g dry wt on the 6 nd 28th dys of drought stress, respectively. It hs een found tht proline hs mjor role in mintining osmotic djustment nd dpttion to stress nd protects memrnes nd proteins from the dverse effects of environmentl stresses [29, 3]. In the present study, grdul increse in peroxidse ctivity with enhnced drought period in PSE plnts cn e explined s form of defense, y which the plnt cn reduce oxidtive injury rising from ROS, especilly ç 2 é 2, forming during drought stress. Similrly, the increses in POD ctivity were otined for rice cultivrs nd pe plnts under wter stress conditions [, 3]. Similr increses in the ctivity of POD were lso determined during drought stress in the PS plnts of C. setos [32]. In the previous study, POD ctivity in leves of PS plnts on the 32, 48, nd 64th dys of drought were 3.7-, 7.3-, nd -fold higher thn tht of initil plnts [32]. However POD ctivity in leves of PSE plnts on the 35, 5, nd 6th dys of drought were 4.5-, 7.6-, nd 7.8-fold higher thn PSE control in the present study. Consequently, the present results show tht PSE plnts displyed different metolic responses in comprison with PS plnts. It cn e climed tht the lef rolling mechnism ws not drsticlly ffected in PSE plnts fter creting secondry severe wter deficit conditions. However, n erlier lef rolling response ws determined in PSE plnts s compred to the PS plnts. Our dt indictes tht the lef rolling is necessry voidnce mechnism for the plnt survivl under extreme drought conditions, despite of the whole plnt reduction. 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