Adaptive response to salt involving carbohydrate metabolism in leaves of a salt-sensitive tomato cultivar

Plant Physiology and Biochemistry 45 (2007) 551e559 www.elsevier.com/locate/plaphy

Research article

Adaptive response to salt involving carbohydrate metabolism in leaves of a salt-sensitive tomato cultivar Aminata Khelil a,b, Thierry Menu c, Be´re´nice Ricard c,* a Equipe Osmoadaptation et Me´tabolismes de Stress, UMR CNRS 6026, Universite´ de Rennes I, Rennes, France De´partement de Biologie, Faculte´ des Sciences et Sciences de l’Inge´nieur, Universite´ de Ouargla, Ouargla, Algeria c UMR Physiologie et Biotechnologie Ve´ge´tale, INRA Bordeaux, B.P. 81, F-33883 Villenave d’Ornon Cedex, France

b

Received 30 August 2006; accepted 14 May 2007 Available online 18 May 2007

Abstract A salt-sensitive genotype of Solanum lycopersicum cv. Volgogradskij was submitted to a 6-day treatment with high salt (100, 200 mM NaCl), allowed to recover for 6 days and then submitted to a second period of salt stress in order to study changes in carbohydrate metabolism related to salt adaptation. The ion, soluble sugar and starch contents, as well as sucrose biosynthetic and sugar mobilizing enzyme activities and transcript levels were determined during the salt stress/recovery/stress cycle. Sodium ions were found to accumulate preferentially in old leaves. Young leaves accumulated lower levels of sodium ions but maintained control levels of potassium ions. Hexoses accumulated to higher levels and starch was better maintained in young compared to old leaves during the two salt treatments. Sucrose accumulated dramatically only in old leaves during the initial salt treatment. Sugar accumulation was not related to decreases in the activities of sugar mobilizing enzymes, acid (EC 3.2.1.25) and neutral (EC 3.2.1.26) invertases, sucrose synthase (EC 2.4.1.13) and hexokinase (EC 2.7.1.1). The activity of the biosynthetic enzyme sucrose phosphate synthase (EC 2.3.1.14) was linked to changes in sucrose levels but not with transcript levels. These results point to the importance of post-transcriptional regulation. Transcriptional regulation could nevertheless be seen in the down-regulation of ribulose bisphosphate carboxylase small subunit (EC 4.1.1.39) in old compared to young leaves, but this was not related to sugar levels. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Solanum lycopersicum; Salt stress and recovery; Carbohydrate metabolism

1. Introduction Salinity of soil and water caused by excessive amounts of salt, most commonly NaCl, generates both osmotic and ionic stress. Excess salt reduces water potential, causes ion imbalance or disturbs ion homeostasis and has toxic effects on numerous biochemical processes. The ability of plants to tolerate salt is thus a multigenic trait and is generally acknowledged to be determined by multiple biochemical pathways, Abbreviations: HXK, hexokinase; INV, invertase; RbcS, ribulose bisphosphate carboxylase small subunit; SuSy, sucrose synthase; SPS, sucrose phosphate synthase. * Corresponding author. Tel./fax: þ33 5 57 12 25 41. E-mail address: [email protected] (B. Ricard). 0981-9428/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2007.05.003

among which are those that facilitate water retention or acquisition, maintain ion homeostasis and protect various vital functions (for review, cf. [30]). Yet numerous authors have attempted to enhance salt tolerance through genetic transformation with individual genes [8]. Some degree of success has been obtained. For instance, transgenic tomato plants which overexpressed a vacuolar Naþ/Hþ antiport were able to grow and produce fruit in high salt conditions. High levels of sodium accumulated in leaves but were compartmentalized into the vacuole [39]. In contrast to genetic transformation, the success of marker-assisted selection of salinity-tolerant genotypes has been more limited. Cultural techniques have given more positive results. The early success in adapting cell lines to salinity [3] implied that the genetic potential for salt tolerance was present and could be activated by exposure to salt.

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Thus, pretreatment of seed with salt increased salt tolerance during germination and early growth [33]. Overall plant growth under salinity is significantly reduced due to the inhibition of leaf expansion and a decrease in photosynthesis [25]. The decrease in photosynthesis rate has been attributed mainly to stomatal closure [20] but could also be at least partially a consequence of feedback inhibition due to the increase of sucrose in source leaves [31,34]. Carbon metabolism regulated by sugar could therefore play a significant role in an adaptive response to salinity, via osmotic and/or metabolic adjustments. Changes in carbon metabolism have only rarely been studied in relation to tolerance to salt stress. One of these studies compared carbon metabolism in a salttolerant tomato ecotype (cv. Pera) with that in a salt-sensitive genotype (cv. Volgogradskij). Balibrea et al. [2] suggested that the difference in salt tolerance could be related to differences in the ability to regulate carbon allocation and partitioning and sucrose metabolism. In this study, we asked whether and how carbon metabolism was modified in the salt-sensitive genotype (cv. Volgogradskij) during salt stress and recovery from stress followed by a second salt stress. Plants grown hydroponically for 3 weeks were submitted to two 6-day periods of high salt treatment (100 and 200 mM NaCl) interrupted by return to nonsaline conditions for 6 days. We determined the ion, soluble sugar and starch contents, as well as sucrose biosynthetic and sugar mobilizing enzyme activities and transcript levels in old and young leaves of plants after 3 days of each 6-day salt stress and recovery period, with the objective of detecting early changes that could be related to salt adaptation. 2. Materials and methods 2.1. Plant material and growth conditions Solanum lycopersicum (cv. Volgogradskij) seeds disinfected by treatment with sodium hypochlorite for 3 min were washed extensively before germination for 48 h at 25  C in the dark on filter paper imbibed with 200 mM NaCl. Upon germination, 12 seedlings of uniform size were transferred to each of three plastic containers (20  20  8.5 cm). The containers were filled with Hoagland and Arnon [13] nutrient solution supplemented with 1.5 mM Na silicate [7]. A black plastic film placed over the container supported aerial parts. Roots were kept under constant aeration. Plants were maintained in a growth chamber under controlled conditions (14 h at 75% relative humidity and 25  C/10 h at 90% relative humidity, 21  C light/dark regime; irradiance of 20 W m2). The nutrient solution was changed every 7 days and the levels adjusted every 3 days. 2.2. NaCl treatments and plant harvest Three week old plants grown hydroponically as described above were submitted to a 6-day salt treatment by adding 0, 100 and 200 mM NaCl to three containers. After extensive washing of roots, the plants were returned to fresh nutrient

solution for 6 days. A second saline treatment identical to the first was imposed for 6 days. Plant material was harvested after 3 days of each treatment, 2 h after the onset of the light period, in hopes of detecting early adaptive mechanisms. Samples taken in triplicate consisted of three 6-mm discs taken from the first true leaf (basal or old leaf) and the uppermost fully expanded leaf (apical or young leaf) of three different plants. The basal leaf showed no visible signs of senescence. Plants were discarded after sampling. Samples were lyophilized prior to storage at room temperature for chemical analysis or frozen and reduced to a powder under liquid nitrogen prior to storage at 20  C for RNA extraction and enzyme quantification. 2.3. Chemical analysis Ion contents in 5 mg of lyophilized leaf powder were determined by flame photometry as described by Immamul-Huq and Larher [17]. Soluble sugars were extracted from 10e 15 mg of lyophilized powder using the alcohol extraction method described by Brouquisse et al. [5]. Glucose, fructose and sucrose were enzymatically assayed as described by Kunst et al. [24] and adapted by Rolin et al. [32]. Starch was assayed after extraction and conversion to glucose as described by Moing et al. [27]. 2.4. Enzyme extraction and assays Leaf samples (200 mg) were extracted with 10 volumes of medium containing 50 mM TriseHCl (pH 7.5), 10 mM sodium borate, 5 mM dithiothreitol, 15% (v/v) glycerol, 3% (w/v) insoluble polyvinylpyrrolidone, 1% (v/v) Triton X-100. The extract was centrifuged for 5 min in an Eppendorf centrifuge and the supernatant desalted over a Sephadex G-25 column equilibrated in the reaction buffer as described by Helmerhorst and Stokes [12]. Hexokinase (HXK, EC 2.7.1.1), sucrose synthase (SuSy; EC 2.4.1.13), acid (EC 3.2.1.25) and neutral (EC 3.2.1.26) invertase activities were determined as described in Bouny and Saglio [4]. Sucrose phosphate synthase (SPS; EC.2.4.1.14) activity was assayed according to Hubbard et al. [14]. 2.5. RNA extraction and analysis by Northern blot and RTePCR Total RNA was extracted using the hot phenol method described by Verwoerd et al. [37] from 125 mg of leaf samples. Total RNA (5 mg per lane) was size-fractionated on a 1.2% (w/v) agarose 6.6% (v/v) formaldehyde gel and then transferred to a nylon membrane (Nytran, Schleicher & Schuell). RbcS probes were amplified from tomato cDNA using primers shown in Table 2. Probes were labeled with a-32P by random priming. Prehybridization and hybridization were carried out under stringent conditions at 65  C as described previously [10]. Semi-quantitative RTePCR was carried out as described by Joube`s et al. [18] using 1e2 mg of RNA and oligo dT for

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RT reactions and 100 ng for PCR reactions. Primers used for the PCR reactions are shown in Table 2.

decrease was reduced in young leaves: 1.4- and 1.1-fold at 200 mM NaCl (Fig. 1B, treatments 1 and 2).

3. Results

3.2. Hexose, sucrose and starch contents in leaves during the salt stress/recovery/stress cycle

During the first 6-day cycle of high salt treatment, a 16- and 30-fold increase in Naþ content occurred in the first true (old or basal) leaf of plants exposed to 100 and 200 mM NaCl, respectively, after 3 days of treatment. The increase in Naþ content was lower in the uppermost fully expanded (young) leaf: 3.6- and 6.3-fold at 100 and 200 mM NaCl, respectively (Fig. 1A, treatment 1). By 3 days of return to non-saline conditions, the Naþ content had decreased to control levels in young leaves but remained higher than control levels in old leaves (Fig. 1A, recovery). During the second salt treatment, the increase in Naþ content was reduced compared to the first salt treatment but again accumulation in old leaves was higher than in young leaves. The increase was 17-fold in old leaves but only 4.5-fold in young leaves of plants grown at 200 mM NaCl (Fig. 1A, treatment 2). Fig. 1B shows that Kþ contents in old leaves decreased similarly during the first and second salt treatment: 1.4- and 1.3-fold at 100 mM NaCl; 3.0- and 2.2-fold at 200 mM NaCl, respectively. The

Na+ (meq. g-1 FW)

3

A

2

Control

NaCl 100mM

NaCl 200mM

Young leaves

Basal leaves

In response to the first salt treatment, hexoses accumulated to higher levels in young than in old leaves according to stress intensity (Fig. 2A, treatment 1). Hexoses returned to control levels during the recovery period. During the second salt treatment, hexose accumulation was slightly reduced in both old and young leaves (Fig. 2A, treatment 2). Sucrose contents increased dramatically and to similar levels at 100 and 200 mM NaCl in old leaves. Sucrose also accumulated in young leaves at 200 mM NaCl but only slightly at 100 mM NaCl (Fig. 2B, treatment 1). Sucrose contents returned to control levels during recovery. During the second salt treatment, sucrose again accumulated but to much lower levels than in the first salt treatment (Fig. 2B, treatment 2). The ratio of hexoses to sucrose can be considered to be a measure of sucrose utilization. In young leaves, sucrose levels during the photoperiod are low, due to export out of Hexose (µmol eq. glucose.g-1FW)

3.1. Naþ and Kþ contents in leaves during the salt stress/ recovery/stress cycle

1

60 50

A

Control

NaCl 100mM

NaCl 200mM

Young leaves

Basal leaves

40 30 20 10 0 Treat ment 1

6

Recovery

Treat ment 1

Treat ment 2

B

Control

Recovery

NaCl 100mM

Treat ment 2

NaCl 200mM

0 Treat Recovery Treat ment 1 ment 2

B

Control

NaCl 100mM

NaCl 200mM

Young leaves

Basal leaves 4 3 2 1

2

Basal leaves

0

Young leaves

Treat ment 1

60

1

0 Treat Recovery Treat ment 1 ment 2

Treat Recovery Treat ment 2 ment 1

Fig. 1. Ion contents of old (basal) and young leaves during the salt stress/recovery/stress cycle. Three-week-old plants grown hydroponically were submitted to a first treatment (treatment 1) without NaCl (empty bars) or with 100 mM NaCl (grey bars) or 200 mM NaCl (black bars) for 6 days. After extensive washing of roots, plants were returned to fresh medium without NaCl for 6 days of recovery followed by a second 6-day period of salt treatment identical to the first (treatment 2). Samples were taken as described in Section 2 on the third day of each treatment. (A) Naþ contents; (B) Kþ contents. Values are means  SD of three replicate samples.

Starch(µmol.eq glucose.g-1FW)

K+ (meq.g-1 FW )

3

Treat Recovery Treat ment 1 ment 2

Sucrose (µmol.g-1FW)

5

50

C

Recovery

Treat ment 2

Treat ment 1

Control

Recovery

NaCl 100mM

Treat ment 2

NaCl 200mM

Young leaves

Basal leaves

40 30 20 10 0 Treat ment 1

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treat ment 1

Recovery

Treat ment 2

Fig. 2. Carbohydrate contents of old (basal) and young leaves during the salt stress/recovery/stress cycle. (A) Glucose þ fructose, (B) sucrose and (C) starch were determined in the same samples used in Fig. 1 and as described in Section 2. Values are the mean  SD of three replicate samples.

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Table 1 Effect of salt treatments on hexose/sucrose ratios in tomato leaves Old leaves

Relative decrease

Young leaves

Relative decrease

Treatment 1 0 mM NaCl 100 mM NaCl 200 mM NaCl

17.8  1.1 3.3  2.3 5.4  1.5

1.00 5.36 3.33

23.8  2.1 22.3  2.2 13.2  3.1

1.00 1.07 1.80

Recovery 0 mM NaCl 100 mM NaCl 200 mM NaCl

13.3  2.3 15.3  1.1 14.3  1.1

1.00 0.89 0.93

17.3  1.1 22.5  1.1 16.7  2.1

1.00 0.76 1.03

Treatment 2 0 mM NaCl 100 mM NaCl 200 mM NaCl

13.6  2.1 12.1  0.1 11.6  1.1

1.00 1.14 1.18

18.9  2.1 26.7  2.1 20.0  3.1

1.00 0.72 0.94

source tissues and active glycolysis. Hexose levels are generally higher, probably in reflection of slower rates of hexose phosphorylation and further metabolism. A decrease in the hexose/sucrose ratio could thus indicate a blockage in sucrose utilization, either at the level of sucrose or hexose metabolism. Table 1 shows that the hexose/sucrose ratio in control tissue was relatively stable, 13.3 to 17.8 in old leaves and 17.3 to 23.8 in young leaves. The first salt treatment resulted in a sharp decrease in the hexose/sucrose ratio of 5.4- and 3.3-fold at 100 and 200 mM NaCl in old leaves compared to a 1.1- and a 1.8fold decrease at 100 and 200 mM NaCl in young leaves. This indicates a blockage in sucrose utilization in old leaves at both

3.3. Sucrolytic enzyme activities and transcripts in leaves during the salt stress/recovery/stress cycle In order to determine whether part of the changes in carbohydrate content could be due to a decrease in enzymes mobilizing sucrose or hexose, we measured the optimal activities of acid (EC 3.2.1.25) and neutral (EC 3.2.1.26) invertases (INV), sucrose synthase (SuSy; EC 2.4.1.13) and the first committed enzyme of the central metabolic pathway of glycolysis, hexokinase (HXK, EC 2.7.1.1) after 3 days of the first salt treatment, the recovery period and the second salt treatment, respectively. After 3 days of the first treatment at 100 mM NaCl, SuSy activity increased 12- and 3.4-fold in old and young leaves, respectively. At 200 mM NaCl, SuSy activity increased 6

A

Control

NaCl 100mM

NaCl 200mM

5 4

Young leaves

Basal leaves 3 2 1

Acid invertase (nkat.mg-1 protein)

SuSy (nkat.mg-1 protein)

6

salt treatments and at the higher salt treatment in young leaves. Removal of salt restored the hexose/sucrose ratio to control levels. During the second salt treatment, the hexose/sucrose ratio remained at control levels in both young and old leaves, indicating that the blockage in sucrose utilization was reversed during recovery from the first salt stress and that sucrose utilization was normal during the second salt stress. Starch contents were better maintained in young than in old leaves during the first salt treatment at 200 mM NaCl (Fig. 2C, treatment 1). Starch contents returned to control levels during recovery in young leaves. The second salt treatment resulted in a decrease in starch content at 200 mM for both old and young leaves (Fig. 2C, treatment 2).

Treat ment 1

Recovery

Treat ment 2

Treat ment 1

Recovery

4

NaCl 100mM

NaCl 200mM

Young leaves

Basal leaves

3 2 1

Treat ment 2

Treat ment 1

3

C

Control

Basal leaves

NaCl 100mM

NaCl 200mM

Young leaves

1

Hexokinase (nkat.mg-1 protein)

Neutral invertase (nkat.mg-1protein)

2

Control

0

0

3

B

5

Recovery

Treat ment 2

D

Control

Basal leaves

Treat ment 1

Recovery

NaCl 100mM

Treat ment 2

NaCl 200mM

Young leaves

2

1

0

0 Treat ment 1

Recovery

Treat ment 2

Treat ment 1

Recovery

Treat ment 2

Treat ment 1

Recovery

Treat ment 2

Treat ment 1

Recovery

Treat ment 2

Fig. 3. Sucrolytic and glycolytic enzyme activities in old (basal) and young leaves during the salt stress/recovery/stress cycle. (A) SuSy, (B) acid invertase, (C) neutral invertase and (D) HXK activities were determined in crude extracts in the same samples used in Fig. 1 and as described in Section 2. Values are expressed in nkat mg1 protein and are means  SD of three replicate samples.

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dramatically (46-fold) in old leaves (Fig. 3A, treatment 1). SuSy activity decreased during the return to non-saline conditions but remained higher than control levels in old leaves. The second salt treatment had no effect on SuSy activity (Fig. 3A, treatment 2). Enhancement of SuSy activity was only partially correlated with increased sucrose levels, a reflection of complex transcriptional and translational regulation including sugar responsiveness [38]. In both old and young leaves, acid INV activity showed a tendency to decrease according to stress intensity during the first treatment and an opposing tendency to increase during the second salt treatment (Fig. 3B, treatments 1, 2). During recovery from the 200 mM salt treatment, acid INV activity increased 2-fold in old leaves but remained similar to control levels at 100 mM NaCl and at both salt concentrations in young leaves (Fig. 3B, recovery). Neutral INV had a slightly different pattern of activity. In old leaves, neutral INV activity increased more at 100 mM NaCl than at 200 mM NaCl (Fig. 3C, treatments 1, 2). In young leaves, neutral INV activity was elevated not at 100 mM NaCl but at 200 mM NaCl (Fig. 3C, treatments 1, 2). Similar to the pattern of acid INV activity, neutral INV activity increased about 2-fold during recovery from treatment with 200 mM NaCl not only in old but also in young leaves (Fig. 3C, recovery). Together with SuSy, neutral but not acid INV activity increased in correlation with increased sucrose and hexose levels during the first salt treatment, suggesting that the cytoplasmic activity plays the more important role in sucrose hydrolysis. During the second salt treatment, acid and neutral INV activities were both correlated with increased hexose and sucrose levels. The 2-fold higher acid and neutral INV activities measured in old leaves during recovery from 200 mM salt was inversely related to sodium ion concentration, suggesting that sodium ion accumulation in old leaves was a major determinant of INV activity during the 200 mM

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salt treatment. At 100 mM NaCl and in young leaves, other factors probably intervene. HXK activity increased about 2-fold in old leaves after 3 days of the first salt treatment and remained at or above control levels during recovery and the second salt treatment (Fig. 3D). In young leaves, HXK activity was similar to control activity during the 100 mM salt stress/recovery/stress cycle and during the first treatment with 200 mM salt. Activity was above control levels during recovery from and during the second 200 mM salt stress (Fig. 3D). In general, the second salt treatment thus had a reduced effect on SuSy, neutral and acid INV and HXK activities. During recovery from and during the second salt treatment, the activities of all four enzymes were at control levels or higher. The sucrolytic enzymes and HXK appeared to be in ample supply throughout the stress/recovery/stress cycle and thus, limited activity was not responsible for sucrose or hexose accumulation. Semi-quantitative RTePCR analyses were carried out using the gene-specific primers shown in Table 2 and total RNA from two independent experiments. RTePCR results were obtained for two different genes encoding SuSy (Sus2, Sus3) and HXK (Hxk1, Hxk2) and for single genes encoding SPS, ADP-glucose pyrophosphorylase (ADP-GPPase, EC 2.7.7.27) and a vacuolar acid invertase (TIV). SuSy transcripts were barely detectable and so were probably too low to show the expected increase of transcripts in old leaves in correlation with increased sugar levels. HXK and TIV genes were weakly but clearly expressed while ADP-GPPase and SPS genes were strongly expressed. Their transcripts analyzed by RTePCR showed only small variations in levels during the stress/recovery/stress cycle (results not shown). We tentatively conclude that transcriptional up-regulation was not responsible for the increase in acid INV activity, during recovery from 200 mM salt treatment in old leaves

Table 2 PCR primers used to amplify gene-specific regions Gene

Accession no.

Primer sequence (50 e30 )

Product size (bp)

LeRbcS-5

M152636

387

LeSPS

AF071786

LeINV(TIV)

Z12025

LeSus2

L19762/AJ011535

LeSus3

AJ01139

LeADP-GPPase

L41126

LeHxk1

AJ401153

LeHxk2

AF208543

LeAct1

U60480

(F) TTG CTA GCA ATG GTG GA (R) GGC TTG TAG GCA ATG AA (F) AAT CGT GGT CAT CAG ACA AGG (R) AAT CCT ACC CCC ATT TTG ACA (F) TAT CAA TAC AAT CCA GAT TCA (R) GTA GAT ACC GGG TAA AAG TCC (F) C TTG AAT GGC CAA TTC AGA TGG AT (R) AT CTT ACG GTA CTT GAG AGC GTA A (F) CAA ATT GAT CCA TAC CAT (R) CTA ACA CTA CAT AAT GTC (F) ATG AAG ATT GAC GAA GAA GGA C (R) ACC ACG GAA TGG TGA ATC TTA C (F) CAC TTA TGT GGA TAA TCT ACC C (R) CCA TTC CAT ATT GAT (A/C)AC CAT (F) TAG CTA TGT AGA CAA TCT CCC T (R) CCA TTC CAT ATT GAT (A/C)AC CAT (F) TGG CAT CAT ACT TTC TAC AAT G (R) CT AAT ATC CAC GTC ACA TTT CAT

(F), (R) are forward and reverse primers. PCR conditions: 95  C 5 min, 30 cycles (95  C 30 s/50  C 30 s/72  C 1 min), 72  C 3 min.

600 706 482 460 517 609 609 615

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of the gene family, permitting a comparison of RbcS levels in response to salt treatment. The 32P-labeled probe was hybridized with a Northern blot of total RNA extracted from old (Fig. 5, upper panel lanes 1, 3, 5) and young (lanes 2, 4, 6) leaves of plants submitted to a stress/recovery/stress cycle. RbcS transcript levels quantified by densitometry and normalized to 25S rRNA (Fig. 5, middle panel) are shown in the lower panel of Fig. 5. RbcS transcript steady-state levels were lower in old (lanes 1, 3, 5) than in young (lanes 2, 4, 6) leaves, irrespective whether the plants had been treated or not with salt. The first treatment with 100 mM NaCl had little effect on RbcS transcripts (lanes 1 and 3, 2 and 4) but levels were sharply reduced at 200 mM (lanes 1 and 5, 2 and 6). During recovery, RbcS transcripts returned to control level (lanes 1, 3, 5 and 2, 4, 6). The second salt treatment had little effect on RbcS transcripts in old leaves (lanes 1, 3, 5) but levels were sharply reduced in young leaves at 200 mM NaCl (lanes 2 and 6). The results of Northern hybridization were confirmed by RTePCR analyses (not shown).

(Fig. 3B). Transcript variation did not appear to play a major role in salt adaptation, at least for the genes tested. However, we cannot exclude the possibility that untested members of the corresponding gene families could respond to salt stress and recovery. 3.4. Sucrose biosynthetic enzyme and transcripts The activity of the major sucrose biosynthetic enzyme SPS increased over 5-fold in basal leaves submitted to the first salt treatment of 100 or 200 mM NaCl (Fig. 4A, treatment 1). During recovery, SPS activity returned to control levels. The second salt treatment resulted in an increase of SPS activity at 200 mM NaCl (Fig. 4A, treatment 2). In young leaves, the first treatment with 100 mM NaCl had no effect on SPS activity. Activity increased about 4-fold at 200 mM NaCl. The similarity between SPS activity and sucrose levels (compare Fig. 4A with Fig. 2B) is consistent with the major sucrose biosynthetic role of SPS. Little variation of SPS transcripts analyzed by RTePCR was seen in response to salt stress and recovery (Fig. 4B), an indication that the increase in SPS activity was probably due to known post transcriptional regulation by metabolites and by reversible protein phosphorylation [16].

4. Discussion Salt tolerance is expected to be a complex trait, in accord with the widespread consequences of salt on plants. The water deficit induced by salt results in osmotic stress, while an excess of sodium ions has disastrous effects on numerous key biochemical processes. To tolerate high levels of salts, plants can adopt different strategies. One is the partitioning of sodium ions to basal (old) leaves, a mechanism which is thought to protect the growing (young) leaves from the toxic effects of

3.5. Effect of salt on RbcS transcripts The tomato RbcS gene family consists of five members [35], all of which accumulate to similar high levels in leaves [36]. Primers designed to amplify the coding region were expected to yield a probe that would hybridize with all members

SPS activity (nkat. mg-1 proteins)

A

2

Control

NaCl 100mM

NaCl 200mM

Young leaves

Basal leaves 1

0 Treatment Recovery Treatment 2 1

Treatment Recovery Treatment 1 2

B

1

2

3

4

5

6

1

2

4

6

1

2

4

5

6

SPS

Actin

Treatment 1

Recovery

Treatment 2

Fig. 4. SPS activity and transcript levels in old (basal) and young leaves during the salt stress/recovery/stress cycle. Samples from old and young leaves were taken on the third day of each treatment as described in Section 2. (A) SPS activity was determined in crude extracts in the same samples used in Fig. 1 and as described in Section 2. (B) RTePCR analysis of SPS and constitutively expressed actin transcripts in old (lanes 1, 3, 5) and young (lanes 2, 4, 6) leaves during treatment without NaCl (lanes 1, 2), with 100 mM NaCl (lanes 3, 4) or with 200 mM NaCl (lanes 5, 6).

A. Khelil et al. / Plant Physiology and Biochemistry 45 (2007) 551e559 1

2

3

4

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6

1

2

3

4

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1

557 2

3

4

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6

3

4

5

6

rbcS

rbcS/25S rRNA

25SrRNA 7 6 5 4 3 2 1 0

1

2

3

4

5

6

1

Treatment 1

2

3

4

5

Recovery

6

1

2

Treatment 2

Fig. 5. RbcS transcript levels in old (lanes 1, 3, 5) and young (lanes 2, 4, 6) leaves during treatment without NaCl (lanes 1, 2), with 100 mM NaCl (lanes 3, 4) or with 200 mM NaCl (lanes 5, 6). Total RNA samples were subjected to electrophoresis on 1.2% agarose/formaldehyde gels and then transferred to nylon membranes. Upper panel: autoradiogram of hybridization signals obtained after probing the blot with 32P-labeled RbcS probes hybridizing to the coding region. Middle panel: EtBr staining of formaldehyde-agarose gel. Bottom panel: RbcS transcript levels normalized to 25S rRNA levels quantified by densitometry of the autoradiogram and stained gel shown in upper panels.

salt. The old leaves are eventually sacrificed [6]. As shown in Fig. 1A, tomato cv. Volgogradskij can be defined as a salt ‘‘includer’’ and preferentially accumulated salt in old leaves. The improved maintenance of Kþ contents in young compared to old leaves (Fig. 1B) revealed reduced perturbation of their ionic contents and has been related to salt tolerance [1]. During the second salt treatment, Naþ contents were reduced 36 and 28% in old and 44% and 37% in young leaves at 100 and 200 mM NaCl, respectively. This points to the possible regulation of sodium uptake at the root or phloem level. Increased reduction of Naþ contents was related to improved maintenance of Kþ in young compared to old leaves. Another strategy for salt tolerance is the use of ions or compatible solutes such as proline and sugars for osmotic adjustment or other protective mechanisms. Thus, transgenic tobacco plants with enhanced levels of proline biosynthesis were salt tolerant [29]. In an evaluation of nutritional and biochemical indicators for improving salt tolerance in tomato, the most salt-resistant cultivars had the highest sucrose concentration, almost twice that of the most sensitive variety [19]. A salt-tolerant tomato genotype (cv. Pera) when compared with salt-sensitive cv. Volgogradskij accumulated more soluble carbohydrates, especially hexoses, but also partitioned more sugars to starch [2]. This behavior was suggested to represent a beneficial response by avoiding metabolic inhibitions while contributing at the same time to osmotic adjustment [2]. In our studies of cv. Volgogradskij, proline accumulation during the first and second salt treatment was minor (results not shown). Hexoses and starch were the major carbohydrates present in leaves and either accumulated to higher levels (hexoses) or were better maintained (starch) in young than in basal leaves during the two salt treatments (Fig. 2). This is consistent with a function of carbohydrates in osmotic adjustment. Sucrose accumulated dramatically only in basal leaves during the first salt stress and always remained the minor carbohydrate (Fig. 2A,B). The ratio of insoluble to soluble carbohydrates was relatively constant in control leaves, 2.6 to 2.8 in

old leaves and 2.0 to 2.2 in young leaves (Fig 2). This ratio decreased during the first treatment according to stress intensity more in old than in young leaves treated with 200 mM NaCl. The decrease was similarly reduced during the second treatment in young and old leaves. These results are compatible with the hypothesis that partitioning of more sugar to starch would improve avoidance of metabolic inhibitions and could be a factor in salt adaptation in both young and old leaves. Carbohydrates can function in osmotic adjustment and osmoprotection, but their major function is to fuel metabolism. Hexose and sucrose accumulation during salt stress is generally attributed to decreased utilization, although reduced sugar export would also result in sugar accumulation. The synthesis and accumulation of sucrose in leaves are determined mainly by the opposing actions of the biosynthetic enzyme SPS [11,15] and sucrolytic enzymes that convert sucrose into hexoses but also initiate starch synthesis (INV and SuSy, 15). Greater salt tolerance in tomato has been related to the induction of SuSy and vacuolar acid INV activities in leaves [2]. In the salt-tolerant cv. Pera genotype, acid and neutral INV but especially SuSy activities were enhanced during the first week of treatment with 50 and 100 mM NaCl [2]. In salt-sensitive cv. Volgogradskij, acid INV was the main activity detected and only a small increase was reported in young leaves during the 3 weeks of treatment with100 mM NaCl [2]. In our experiment, activities were quantified after 3 days of treatment with 100 and 200 mM NaCl; at this time acid INV was likewise the main activity in young and old leaves and was enhanced during the second salt treatment. On the other hand, neutral INV and SuSy activities were largely unaffected by the second salt treatment. Sucrose hydrolysis by acid INV localized in the vacuole might contribute to salt adaptation. During the first salt stress, SPS activity rose dramatically especially in old leaves but also in young leaves at the higher salt stress. SPS activity returned to control levels or lower during return to non-saline conditions in near perfect correlation

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with sucrose contents during the first stress and recovery periods (Figs. 4A and 2B), in confirmation of the major sucrose biosynthetic role of SPS. During the second salt stress, sucrose contents were better correlated with starch decrease (Fig. 2B). Hexoses also accumulated (Fig. 2A), in accord with reduced metabolism. The blockage in hexose utilization presumably occurs downstream of HXK since HXK activity was always at least at control levels (Fig. 3D). These results are in accord with a shift in the balance of sucrose-starch metabolism previously documented by Gao et al. [9]. They also indicate that sucrose synthesis via activation of SPS was dominant during an initial salt stress in old leaves and in young leaves at the higher salt stress (200 mM NaCl). The second salt stress activated SPS only at 200 mM NaCl in basal leaves so that sucrose accumulation was then correlated with the decrease in starch content (Fig. 2B,C). A number of reports have described the induction or repression by sugars of various plant genes, including a gene encoding ADP-GPPase [28] and several photosynthetic genes [23], as well as sucrose synthase and invertase genes [21,22]. Yet RTePCR analyses carried out using total RNA extracted from plants in two independent experiments showed no correlation between levels of transcripts encoding SPS, acid INV, SuSy, ADP-GPPase and carbohydrate levels. There was thus no indication for regulation by sugars of these genes. The only gene whose transcript level varied in old and young leaves was RbcS (Fig. 5). However, no correlation was found between reduced transcript levels and high leaf contents of soluble sugars. Fig. 5 (treatment 1) showed similar low levels of RbcS transcripts in old leaves, irrespective whether sucrose content was low (control) or high (100 and 200 mM NaCl) (Fig. 2B, treatment 1). These results are compatible with down-regulation of RbcS being mediated by leaf senescence rather than sugar accumulation [26]. Acknowledgments We are indebted to support from colleagues of the UMR Physiologie et Biotechnologie Vegetale, INRA, Bordeaux, in particular Agnes Destrac-Irvine and Marie-He´le`ne Andrieu for expert technical assistance and Dr. Annick Moing for her critical comments and advice. AK is grateful to her thesis directors, Franc¸ois Larher and Alain Bouchereau, for their contribution to her work. References [1] G.N. Al Karaki, Growth, water use efficiency, and sodium and potassium acquisition by tomato cultivars grown under salt stress, J. Plant Nutri. 23 (2000) 1e8. [2] M.E. Balibrea, J. Dell’Amico, M.C. Bolarin, F. Pe´rez-Alfocea, Carbon partitioning and sucrose metabolism in tomato plants growing under salinity, Physiol. Plant 110 (2000) 503e511. [3] M.L. Binzel, P.M. Hasegawa, A.K. Handa, R.A. Bressan, Adaptation of tobacco cells to NaCl, Plant Physiol. 79 (1985) 118e125. [4] M. Bouny, P. Saglio, Glycolytic flux and hexokinase activities in anoxic maize root tips acclimated by hypoxic pretreatment, Plant Physiol. 111 (1996) 187e194.

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