Growth, Gas Exchange, Abscisic Acid, and Calmodulin Response to Salt Stress in Three Poplars

Joumal of Integrative Plant Biology 2006, 48(3): 286-293www.blackwell-synergy.com;www.chineseplantscience.comGrowth Gas Exchange Abscisic Acid and calmodulinResponse to Salt Stress in Three PoplarsYu Chang, Shao-Liang Chen*, Wei-Lun Yin, Rui-Gang Wang, Yan-Feng Liu,Yong Shi, Yuan- Yuan Shen, Yue Li, Jie Jiang and Yue Liu(College of Biological Science and Technology, Beijing Forestry University, Beijing 100083, China)AbstractIn the present study, we investigated the effects of increasing salinity on growth, gas exchange, abscisic acid(ABA), calmodulin(CaM), and the relevance to salt tolerance in seedlings of populus euphratica oliv. andcuttings of P,"pupularis 35-44"(P. popularis)and P. x euramericana cv. I-214(P. cV. Italica). The relative growthrates of shoot height(RGRH)for P. CV. Italica and P. popularis were severely reduced by increasing salt stress,whereas the growth reduction was relatively less in P. euphratica. Similarly, P. euphratica maintained highernet photosynthetic rates(Pn)and unit transpiration rates(TRN) than P cv. Italica and P. popularis under condi-tions of higher salinity. Salinity caused a significant increase in leaf ABa and caM in the three genotypes afterthe onset of stress, but Nacl-induced ABA and CaM accumulation was more pronounced in P euphratica,suggesting that P. euphratica plants are more sensitive in sensing soil salinity than the other two poplarsFurthermore, P euphratica maintained relatively higher ABA and caM concentrations under conditions of highsalinity. The higher capacity to synthesize stress signals, namely ABa and CaM, in P. euphratica and thecontribution of this to the salt resistance of P. euphratica are discussed.Key words: abscisic acid; calmodulin; gas exchange; growth; NaCl; PopulChang Y, Chen SL, Yin WL, Wang RG, Liu YF, Shi Y, Shen YY, Li Y, Jiang J, Liu Y(2006). Growth, gas exchange, abscisic acid,and calmodulin response to salt stress in three poplars. J Integrat Plant Biol 48(3),286-293Salt stress signal transduction is made up of multiple pathways al. 1997; Gomez-Cadenas et al. 1998). ABA limits Na* and Clthat consist of ionic and osmotic homeostasis signaling pathways, accumulation in leaves(Karmoker and van Steveninck 1979detoxification response pathways, and pathways for growth regu- Montero et al. 1997), which appears to be the result of a restric-lation(Zhu 2001, 2002). The SOS pathway for ion homeostasis tion of root-to-shoot ion transport via a transpirational stream be-regulation has been established by Zhu(zhu 2001, 2002). cause ABA is a stomatal regulator. Recently, the role of ABA inGenerally, salinity causes increased biosynthesis and accumula- stress signal transduction has been investigated intensively andtion of abscisic acid(ABA), which can modulate physiological the results indicate that ABA can upregulate many stress-respon-reactions in plant responses to salinity( Karmoker and van sive genes(Hasegawa et al. 2000 Zhu 2002 ). ABA has beenSteveninck 1979: Behl and Jeschke 1981; Wolf et al. 1990; Zhao shown to activate LEA-like class genes through the bZIP tran-et al. 1991; Thomas et al. 1992; He and Cramer 1996; Montero et scription factors ABF/AREB (Shinozaki and Yamaguchi-Shinozaki2000; Xiong et al. 2002a). In addition, ABA has been suggested toinduce the gene expression of anti-oxidant enzymes(zhu andReceived 25 Mar 2005 Accepted 25 May 2005Scandalios 1994 Guan and Scandailos 1998a 1998b Guan et alSupported by the National Natural Science Foundation of China(30430430maior role of ABa in adaptation to abioticthe Foundation for the Author of National Excellent Doctoral Dissertation of envirol中国煤化工 any attempts to correlateChina(200152), and the Teaching and Research Award Program for Out- the preCN MHGoplant salinity tolerancestanding Young Teachers in Higher Education Institution of MOE, China Comparative studies nave snown that salt-tolerant Populuseuphratica plants are sensitive to salt stress and their roots in-Author for correspondence. Tel: +86(0)10 6233 8129; Fax: +86(0)10 6233 crease ABA synthesis under conditions of lower salinity(Chen et7855:E-mail:.al. 2002a, 2003a). Furthermore, P euphratica maintains a higherCaM and ABa Response to Nacl Stress in Poplars 287BA concentration under conditions of long-term increasing sa- week of salinity (Table 1)linty( Chen et al. 2001a). In addition to osmotic stress-inducedABA, ion-specific effects(e.g. Na* salts and cr salts)also con-tribute to the accumulation of ABA in P euphratica(Chen et al. Gas exchange response to salt stress2003a). Abscisic acid signal transduction is a complex signal trans-duction network; however, there is no information on the correla- The Pn, TRN, and Cs of control plants fluctuated considerablytion between salt-induced ABA and secondary signals(e.g. Ca2+ over the 4-week study(Table 2), which resulted from variationsand calmodulin(CaM)in trein environmental factors Natural PAR varied between 100 andThe Ca signal has been regarded as a primary signal trans- 1 100 umol." and air temperature ranged from 20 to 30Cducer in plant cells and CaM is the most important Ca* receptor during the observation period. In general, Ph, TRN, and Cs of thewithin cells. It has been demonstrated that there are many en- three poplar genotypes were all reduced by increasing salt stress,ymes that have important functions in plant metabolism that are but there were genotypic differences in the pattern of the gasregulated by Ca*or the Ca */CaM system( Dieter and Dieter 1983; exchange response to salinity( Table 2). P euphratica and P cvZielinski 1998; Sun et al. 2000 ). The role of Ca/CaM in stress ltalica exhibited a rapid decline in gas exchange upon the initiationphysiology has received considerable attention in recent years, of salinity and the Pn, TRN, and Cs of these two genotypesbut contradictory results have been reported Zong et al. (2000) decreased significantly on Day 1, whereas a marked reduction infound that blockade of Ca */CaM signal transduction with CPZ and Pn, TRN, and Cs was observed in P. popularis leaves on Day 3LaCl decreased water content, relative growth rates, and the (Table 2). With an increasing duration of exposure to salinity, Phsurvival of rice plants under conditions of drought, salt, and low and Cs in P. cv. Italica and P popularis plants dropped to verytemperature. Thus, it can be inferred that Ca*/CaM messenger low levels in the last week of salt stress(Table 2). The Pn of thesecontributed to the rice response to stress(Zong et al. 2000). two genotypes decreased by 88%-92% on Day 28 and Cs de-Similarly, Bidwai et al. (1987) found that CaM indirectly stimulated creased by 72%--79%(Table 2). In comparison, a lower reductionplasma membrane ATPase activity in red beet. Conversely, plasma of Pn(61%)was observed in NaCl-treated P euphratica(Tablemembrane AtPase activity of maize seedlings was accelerated 2). In contrast with severely reduced Pn, the salt stress-inducedby tFP but inhibited by CaM( Huang et al. 1997). In a previous decline in TRN was much lower(4%--57%)in the three poplarsstudy, initial salinity increased the uptake and transport of Ca+ tested (Table 2)and the elevated Ca was considered to contribute to salt toler.ance in salinized P. euphratica( Chen et al. 2001a). However, the Leaf ABA concentrationscorrelation between endogenous CaM and salt resistance hasnot been established in poplar genotypes.Salinity caused a significant increase in leaf ABa in three geno-The aim of the present study was to evaluate changes in en- types after the onset of stress, but NaCl-induced ABAaccumuladogenous ABA, CaM, and their contributions to differences in salt tion was more pronounced in P euphratica( Figure 1). GenotypIctolerance exhibited by P euphratica and the salt-sensitive geno- differences in the pattern of the ABA response to salinity weretypes P "pupularis 35-44"(P popularis)and P x euramericana observed on Day 28. The leaf ABA concentration in P euphraticacV. I-214(P. cV. Italica)Table 1. Effects of Nacl on relative growth rates of shoot height ofsultsPopulus euphratica Oliv., P. popularis and P. cv. ItalicaSpecies TreatmentRGRH(mm.cm--d-)Growth response to salt stress1st week 2nd week 3rd weekP. euphraWithin the first week of salinity, Nacl reduced the RGRH of theControl0570a00524a0.0288athree poplar genotypes, although a higher reduction in RGRH wasNaCl00217b0.0268b0.0272aobserved for P. euphratica and P. cv. Italica(Table 1). In the P. popularisfollowing 2 weeks, there were genotypic differences in the RGR,Control0.0677a0.0330a0.031response to increasing soil NaCl. The RGRH was reduced by 36%中国煤化工0002b09072% in saline-treated plants of P. popularis and P cv. Italica and P. cvgrowth reduction was more pronounced in P popularis. ComCNMHG0015 98 0.020 5apared with P. popularis and P cv. Italica, the inhibitory effects of0.0538a0.0101a0.0086bincreasing Nacl were considerably less in P euphratica. The Data are the mean of three plants and values with the same letter inNaCl-treated P euphratica plants maintained a relatively higher the same column are not significantly different(P>0.05).RGRH.ich was 94% of that in control plants at the third relative growth rate of shoot height据288 Journal of Integrative Plant Biology Vol. 48 No. 3 2006Table 2. Effects of NaCl on the net photosynthetic rate( Pn), unit transpiration rate(TRN, and stomatal conductance(Cs)of Populuseuphratica Oliv., P. popularis and P cv. ItalicaeclesTreatmentDays after treatment (d131628P. euphratica4.97a231b243a2.38a2.81a557a1.54b1.71a1.10bP. popularisControl932a2.92a2.94a5.40Nacl5.40b7.34a3.28a2.29a0.65bControl2,32a196b8.55a7.35b500a0.69bP euphratica194a3.54a148a1.58a3.42a083b0.75b0.95aControl707a3.65a128b1.55a0.98b0.93aNaCl8.51a3.17a2.56a1.72a0.40bP. cv. italica2.26b147a057bCs(mmol- m.s")Control83.88134.03a50.1445.35781b47.03b58.7374.00a25a123.53a49.22aP. cv. ItalicaControl125.40a49.15a26.68b35.00a21,41b125.83aNacl97.15b41.50a6025a3237a42.61a26.43bData are the mean of three plants and values with the same letter in the same column are not significantly different(P>0.05was 7. 5-fold that in controls, although ABA levels in control plants popularis under higher salinity (i. e. Day 28: Table 2). These re-had increased markedly on Day 28 compared with levels on Day sults indicate that P euphratica is more salt tolerant than Pcv1 (Figure 1). Conversely, leaf ABA was markedly reduced in sa. Italica and P popularis in terms of growth and co2 assimilationline-treated P cv. Italica and P popularis plants( Figure 1)This is consistent with results of previous studies, which showeda greater salt tolerance for P euphratica irrespective of the soilLeaf CaM concentrationstype used in the experiments or the Nacl conditions in the media(Ma et al. 1997; Chen et al. 2002b)Saline-induced CaM accumulation was clearly seen in the three Compared with P cv. Italica and P popularis, P euphraticagenotypes tested after 1 day exposure to salinity; of note, P. plants had relatively higher TRN under conditions of high salinityeuphratica maintained a typically higher CaM level than did P. cv. (Day 28; Table 2), indicating that P. euphratica had a greater ca-italica or P popularis, regardless of treatment(Figure 2). Leaf pacity for water uptake and transport(Chen et al. 2002b, 2003b)CaM concentrations in saline-treated plants of the three poplar Higher soil Nacl results in a plant water shortage owing to lownotypes declined markedly by Day 28, but the inhibitory effect osmotic potential in the root medium. P euphratica roots wereof Nacl on P euphratica was considerably less( Figure 2)capable of absorbing water under saline conditions, which isfavorable for maintaining cell turgor and cell division. Moreover, Peuphratica retained higher CO 2 assimilation under conditions ofDiscussionhigher salt stress(Table 2). Collectively, the lesser reduction in中国煤化工 notosynthesis can explainEffects of salinity on growth and gas exchangeInYHCNMHGssed P. euphratica plants(TaThe RGRH of P. cv. Italica and P. popularis were markedly re-It is of note that the stress-induced reduction of Ph was higherduced by increasing salt stress, whereas the growth reduction than that of TRN in the three poplars(Table 2). This implies thatwas relatively less in P. euphratica(Table 1). Similarly, P. non-stomatal factors contributed to the reduced CO, assimilationeuphratica maintained a higher Pn than P cv. Italica and P. in addition to stomatal limitationsCaM and aba Response to Nacl stress in Poplars 289O ControlO Contr3000150500P euphratica P. popularis P. cv.P euphratica P. popularis P. cvItalicaItaliB口 ContrBO Controls504000P euphratica P. popularis P. cvP euphratica P. popularis PItalicaItalicaSpecies/treatment (28 d)Species/treatment( 28 d)Figure 1. Leaf abscisic acid(ABA)in Populus euphratica Oliv., P. Figure 2. Leaf calmodulin in Populus euphratica Oliv., P. popularispopularis and P cv. Italica following control or NaCl treatment for and P. cv. Italica following control or Nacl treatment for()1 or(B(A)1or(B)28d.Data are the mean SEM of three plantsData are the mean* SEM of three plantsABA and caM responses to salt stress in poplarcould limit root-shoot salt transport by reducing water flow thatwas caused by ABA-induced stomatal closure( Chen et al. 1997,Leaf ABa content in three poplars increased significantly at the 2002c)beginning of treatment, but the highest level was observed in P. In addition to a transient increase in ABAat the beginning of salteuphratica(Figure 1). This is consistent with our previous study, stress, P euphratica typically maintained a higher ABAconcen-in which a transient increase in ABA in the xylem of roots andor tration under conditions of high salinity. On Day 28, the leaf ABAshoots was more pronounced in P. euphratica compared with concentration in P. euphratica seedlings was 7.5-fold of that insalt-sensitive genotypes, such as, for example, the hybrid P. controls, whereas a marked reduction in leaf ABA concentrationstalassica Kom x(P euphratica+ Salix alba)(Chen et al. 2001a) was found in the other two salt-sensitive poplar genotypes(Figureand P popularis( Chen et al. 2002a). Collectively, we conclude 1). Similar results were observed in a previous study( chen et althat the rapid increase in ABA in P euphratica may result from 2001a)activation of ABAbiosynthetic genes, which is probably mediated abilityy a Ca*-dependent phosphorelay cascade(Xiong et al. 2001, ablyH中国煤化工 a nas a relatively strongCNMHGlated expression of ABA2002b)because there was a significantly increased Ca con- biosynthetic genes Xiong et al. 2002a)centration in P euphratica corresponding to salt-induced ABA( Chen et al. 2001a). TRN of P euphratica decreased evidently CaMafter the initiation of stress(Table 2), indicating that P euphratica There are considerable experiments showing that CaM modulates290 Journal of Integrative Plant Biology Vol. 48 No. 3 2006multiple processes of cellular responses to stress(Paul et al. plasma membrane H-ATPase activity in P euphratica( Chang Y1994; McAinsh et al. 1995; Sun et aL. 2000; Chen et aL. 2003c), et al. 2004, unpublished data). Moreover, we have found increasedespecially in ABA-induced stomatal closure( Liet al. 2002 ) In the activity of tonoplast H*-ATPase and vacuolar ion compartmental-present study, P euphratica exhibited a typically higher CaM level ization in P euphratica cells(Chen et al. 2000, 2002b, 2003b Maafter the onset of salt stress( Figure 2), which was similar to the et al. 2002 ). The higher capacity to synthesize and transport ABApattern seen for ABAconcentrations(Figure 1). Correspondingly, presumably contributes to ion homeostasis in P euphratica understomatal conductance and leaf water loss were decreased (Table conditions of increasing salinity over the longer term. ABA limits2), suggesting that CaM may participate in ABA-induced stomatal Na* and cr concentrations in leaves( Karmokerandvan Steveninckclosure in P euphratica. Root-to-shoot salt transport is conse- 1979: Montero et al. 1997), which may result from ABA-inducedquently limited owing to a reduction in water flow in the transpira- vacuolar ion compartmentalization and the inhibition of salt transtion streamport to root xylem vessels(Behl and Jeschke 1981). Moreover,Endogenous CaM acts as a Ca-activated target enzyme to Kasai et al. ( 1993)proved that ABA increased proton transportregulate a number of enzymes that have important functions in activity in the tonoplast of barley cells, which is favorable for ionplant metabolism(Dieter and Dieter 1983 Zielinski 1998; Sunet al. compartmentalization into vacuoles( Bennett and Spanswick 1983;2000). Current evidence indicates that CaM regulates Ca* Blumwald and Poole 1985; Garbarino and Dupont 1988). QuantitaATPase activity in a variety of cell compartments(Zielinski 1998). tive X-ray microanalysis has shown that P. euphratica has aThe Ca2*-ATPase plays an important role in Ca2+homeostasis higher ability to sequester ions into vacuoles( Fung et al. 1996following a signal-induced increase in cytosolic Ca*. We have Chen et al. 2000, 2002b, 2003b ) Therefore, a longer period offound that salt stress induces Ca*-ATPase activity in P euphratica sustained higher ABA levels may enhance proton transport activ.(Chang Y et al. 2004, unpublished data). It is likely that CaM regu- ity in tonoplast of P euphratica cells, resulting in ion compartates Ca-ATPase activity during conditions of salt stress, lead. mentalization into vacuoles, which contribute to limitation of rooting to the rapid compartmentalization of Ca"*into stores, such as shoot salt transport( Chen et al. 2002b, 2003b)vacuoles, following increases in cytosolic CaThe CaM level declined in the three poplar genotypes with an Osmotic homeostasisincrease in the duration of exposure to salinity( Figure 2 ) This is In addition to the contribution of vacuolar ion compartmentalizationpossibly the result of down-regulated protein synthesis under to osmotic adjustment, concentrations of glycinebetaine and sugconditions of high salinity. Of note, the decline in CaM in P. ars in P euphratica are significantly increased under conditionseuphratica was lower than in the other two poplars, implying that of salt stress(Chen et al. 2001b). ABA may activate the biosyn-P. euphratica has a higher capacity for CaM synthesis under thesis and/or gene expression of the enzyme of these osmolytesconditions of high salinity. Presumably the higher capacity for The effective osmotic adjustment is favorable for maintaining cellCaM synthesis enables P euphratica plants to modulate salt ad- turgor, cell division, and growth in salt-stressed P. euphraticatation reactions when faced with high salinity.plants(Table 1). In addition to their role in osmotic adjustment,these osmolytes can function in detoxification or damage preven.Salt stress signal transduction and salt tolerance intion or repair(Zhu 2001, 2002)P euphraticaAntioxidative stressPlants suffer from ion-specific effects, osmotic stress, and salt- P. euphratica rapidly activated anti-oxidant enzymes after theinduced oxidative stress in the face of salinity. Compared with onset of salt stress, which may reduce the accumulation of reacsalt-sensitive genotypes, P euphratica plants have a higher ca- tive oxygen species(ROs) and the subsequent acceleration ofpacity for salt uptake and transport regulation, osmotic adjustment, lipid peroxidation( Wang et al. 2005). Salt-induced ABAand Ca2+/and detoxification under conditions of salinity. These responses CaM may contribute to the activation of anti-oxidant enzymes durare presumably associated with salt signal transduction in salt- ing the period of salt stress. Yang and Poovaiah (2002)reportedCa*/CaM downregulated H2O2 levels bytic activity of plant catalase. there is now considerable evilon homeostasisdence that aBa induces the gene expression of anti-oxidant en-The regulation of ion homeostasis by the SOS pathway has been zymes huand Srandaline 1@@4. Guan and Scandailos 1998aelucidated by Zhu(Zhu 2001, 2002). It has been demonstrated 1998中国煤化工 at NaCl rapidly increasedthat Na* initiates a Ca*signal that activates the SOS3-SOS2 pro- theCNMHGer salt stress was initiated,teinkinase complex,which stimulates the Nat'H'exchange activ. whereas there was no corresponding change in the salt-sensi-ity of SOS1 and regulates the activities of other transporters(e.g. tive genotype P popularis(Wang et al. 2005 ).Thus,vacuolar H-ATPase, PPase, and the vacuolar Na H* exchanger, inferred that ABA and Ca */CaM activated anti-oxidant enzymesZhu 2001, 2002). Similarly, NaCl induced a marked increase inr the onset of salt stress, which may reduce the accumulationCaM and ABA Response to Nacl Stress in Poplars 291of ROS and the subsequent acceleration of ipid peroxidation in P. was initiated, plants received I L full-strength Hoagland' s nutrientsolution weekly Control plants were kept well watered and werIn conclusion, P euphratica plants were more salt tolerant than fertilized with no addition of Nacl during the experimental perioP. popularis and P cv. Italica in terms of growth, transpiration, Pots were weighed twice a day to maintain soil moisture at ap-and photosynthesis responses to increasing salinity Compared proximately 70%of field capacitywith the other two poplar genotypes, NaCI-induced ABAand CaMwas more pronounced in P euphratica at the beginning of salt Height growth measurementsstress, indicating that P. euphratica is more sensitive in sensing The height growth of three replicated plants for each treatmentsoil salinity than the other two poplars. Furthermore, P euphratica (control and NaCl) was measured after 1, 6, 16, and 23 d expo-maintained relatively higher ABA and CaM concentrations under sure to saline treatment. The last sample time was on Day 23conditions of high salinity. We conclude that P euphratica has a because salt-injured growing tips appeared in saline-treated P.higher capacity to synthesize stress signals (ABa and caM)un- popularis and P cv. italica plants thereafter. Height growth wasder saline conditions, which contributes to the salt resistance of measured from the growing tip to the base of the stem. RGR,P.were calculated as follows:RGRH=(InHznH,)(T2-T1)where H, and H2 are the heights of the plants at times T, and T2,Materials and MethodsPlant materialsGas exchange measurementsGas exchange measurements were performed after 1, 3, 9, 13Seedlings of P euphratica Oliv. and hardwood cuttings of P.cvand 28 d salinity Ph, TRN, and Cs of upper mature leavesItalica and P popularis were used in this experiment. Cuttings of were measured with a CIRAS-2 portable photosynthesis systemP. popularis and P cv. Italica were obtained in winter from the (PP Systems, Hitchin, Herts, UK). Air temperature was 26-30Cnursery of Beijing Forestry University and seedlings of P. and photosynthetically active radiation(Par)was 800-1 100euphratica were obtained from the Xinjiang Uygur Autonomous umol-m-2-s-'per s supplemented with dysprosium lampsRegion of China. Plants were cultured in a greenhouse as de-scribed by Chen et al. (2001a)and a brief description is given Samplingbelow. In mid-April, plants were planted in individual pots(10 L) Leaves were sampled after 1 and 28 d exposure to salinecontaining loam soil collected from the nursery of Be ]ing Forestry treatment. Three replicated seedlings per treatment were sampledUniversity and placed in a greenhouse Plants in pots were irri- at each sampling time. All sampled leaves were frozen in liquidgated two to three times a week, depending on the evaporative nitrogen and then stored at-70oC for ABA and CaM analysisdemand, and received 1 L full-strength Hoagland's nutrient solution every 2 weeks. Plants were raised 4 months prior to the Abscisic acid and caM analysinitiation of salt treatment(August). Forty to 50 uniform plantswhich were 60-80 cm high and had 50-70(P euphratica)or 30- Extraction and determination of ABA Approximately 0.5 g40(P popularis, P. cv. Italica) leaves were used in the following fresh weight samples were ground to fine powder in liquid nitro-gen and homogenized in 1.5 mL extraction solution containing80%methanol and 1 mmol/L BHT Extracts were kept in a refrig.Methoderator at 4C for 4 h and then centrifuged at 1 000g for 15 min at4C. After centrifugation, the residue was re-extracted with 1 mLSalt treatmentextraction solution and kept at 4 C for 1 h and then centrifuged asThe same two treatments were applied plants from all three described above Then, the supernatants were combined andgenotypes: (control; and()NaCl stress. Plants were subjected loaded onto a C18 column. thereafter the eluate was dried byto 28 d of increasing salinity and saline treatment was imposed by vacuum evaporation. The residue was dissolved in 1 mL sample1 L NaCl solution once a week. The initial and second salt treat- diluent and aBa was assayed using an ELISA as described byments were applied by top watering with 1 L of 100 mmo/L Nacl Wu et al, (1988. The ABA reaaent box was obtained from thesolution. the third and fourth salt treatments were applied by top Biote中国煤化工 ultural University(Beijingwatering with 1 L of either 150 or 200 mmo/L NaCl solution at the ChirCNMHGeginning of the 3rd and 4th weeks, respectively. Soil Na*and crconcentrations were determined according to Chen(2001a) and Extraction and determination of CaM Approximately 0.5 gthe NaCl concentration in the soil solution in weeks 1-4 was 58.5, fresh weight samples were ground to fine powder in liquid nitro-81.3, 137.2, and 2 13.8 mmolL, respectively. When salt treatment gen and homogenized in 1.5 mL extraction buffer containing 150292 Joumal of Integrative Plant Biology Vol 48 No. 3 2006mmoVL Nacl, 2 mmol/L EGTA, 50 mmol/L Tris, 1 mmol/L B- nutrient uptake and transport, and ABA of Populus euphratica. Amercaptoethanol, 0.25 mmo//L phenylmethylsulphonyl fluoridehybrid in response to increasing soil NaCl. Trees 15, 186-194PMSF), and 20 mmoVL NaHCO3, PH7. 4. Extracts were kept at 95 Chen S, Li J, Bi W, Wang S(2001b). Genotypic variation in accu-oc for 3 min in a waterbath. Then, samples weifuged atmulation of salt ions, glycinebetaine and sugars in poplar under10 000g for 45 min at 4'C and the supernatant was used for theconditions of salt stress. Chin Bull Bot 18, 587-596 (in ChineseCaM assay. Calmodulin was assayed with an ELISA according towith an English abstract)the methods ofof Zhao et al. (1988). The CaM reagent box was Chen S, Li J, Wang T, Wang S, Polle A, Huttermann A(2002a)obtained from the Biology Department of Hebei Normal UniversityOsmotic stress and ion-specific effects on xylem abscisic acid(Shijiazhuang, China)and the relevance to salinity tolerance in poplar. J Plant GrowthReu21,224-233Data analysisChen S, Li J, Fritz E, Wang S, Huttermann A(2002b ). Sodium anddata were subjected to ANOVA and significant differences chloride distribution in roots and transport in three poplar geno-between means were determined by Duncan s multiple-range test.ypes under increasing NaCl stress. For Ecol Manage 168, 217-Unless stated otherwise, differences were considered statistically significant when P<0.05Chen S, Wang S, Huttermann A, Altman A(2002c). Xylem abscisicacid accelerates leaf senescence by modulating polyaminesand ethylene synthesis in water-stressed intact cuttings of poplarAcknowledgementsTrees16,16-22Chen S, Li J, wang T, Wang S, Polle A, Huttermann A (2003a).GasThe authors thank Professor Juan Bai(Hebei Normal University.exchange, xylem ions and abscisic acid response to Na-saltsShijiazhuang, China) for valuable suggestions regarding the CaMand Cr-salts in Populus euphratica. Acta Bot Sin 45, 561-566.assay. Professor Bao-Min Wang(China Agricultural University, Chen S, Li J, Wang S, Fritz E, HOttermann A, Altman A(2003bBeijing, China)is acknowledged for his technical assistance withEffects of NaCI on shoot growth, transpiration, ionthe ABAanalysiscompartmentation and transport in regenerated plants of Populuseuphratica and P tomentosa. Can J For Res 33, 967-975Chen YL, Zhang XQ, Chen J, Wang XC(2003c). 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