The control of proline consumption by abscisic ... - Wiley Online Library

PHYSIOLOGIA PLANTARUM 117: 213–221. 2003 Printed in Denmark – all rights reserved

Copyright C Physiologia Plantarum 2003 ISSN 0031-9317

The control of proline consumption by abscisic acid during osmotic stress recovery of canola leaf discs Patricia Trotel-Aziza,1,*, Marie-Franc¸oise Niogreta, Carole Deleua, Alain Bouchereaua, Aziz Aziza,1 and Franc¸ois Robert Larhera a Equipe Osmoadaptation et Me´tabolismes de Stress, UMR CNRS 6026, Universite´ de Rennes I, Campus de Beaulieu, 35042 Rennes cedex, France 1 Present address: Laboratoire d’Ecotoxicologie, UPRES EA 2069 URVVC, Universite´ de Reims Champagne Ardenne, BP 1039, 51687 Reims cedex 2, France *Corresponding author, e-mail: patricia.trotel-aziz/univ-reims.fr

Received 6 March 2002; revised 14 June 2002

Some aspects of the function of abscisic acid (ABA) in the control of proline (PRO) metabolism in canola leaf discs (CLD) subjected in vitro to consecutive hyper-osmotic (stress) and hypo-osmotic (recovery) treatments have been investigated. PRO accumulation in response to stress conditions relies on both stimulation of its synthesis via enhancement of transcription of the gene and activity of D1-pyrroline-5-carboxylate synthetase (P5CS) and inhibition of its degradation via inactivation of PRO dehydrogenase (PDH). These changes were partly reversed under recovery conditions. Thus both PDH mRNA and PDH activity increased while P5CS mRNA decreased. Surprisingly P5CS activity remained high even after a 20-h period of rehydration. Exogenously supplied ABA at recovery inhibited net PRO consumption and this could be associated with downregulation of PDH gene expression and PDH activity. Under these conditions ABA hardly upregulated P5CS gene expression while P5CS activity first

transiently decreased and then reached a value close to that found under stressing conditions. Experiments with CLD supplied with either methionine sulphoximine or gabaculine, brought preliminary evidence for a significant synthesis of PRO from glutamate during recovery that replenished the proline pool(s) and provoked a negative effect on the net rate of PRO consumption. Consequently, it is suggested that the availability of both PRO precursors and ABA could be determinant in the control of the amount of residual PRO present in CLD after the period of recovery. This level also seemed to depend on the amount of P5CS transcripts induced under stress conditions. However, the results obtained with turgid leaf discs treated with ABA indicate that the ABA status of the tissues, necessary for inducing the proline response, is not sufficient to determine their PRO content because it remained relatively low despite the stimulation of P5CS expression and P5CS activity.

Introduction Proline (PRO) accumulation is a very common response in higher plants subjected to water stress (Rhodes 1987). This amino acid has been assumed to exert beneficial effects in plant tissues, especially via its involvement in osmotic adjustment (Handa et al. 1986, Nanjo et al. 1999a). It can also act as a buffer in cellular redox potential (Venekamp 1989, Saradhi and Saradhi 1991, Hare et al. 1998), as a scavenger of free radicals (Smirnoff and Cumbes 1989, Saradhi et al. 1995, Hong et al. 2000) and as a stabilizer of subcellular structures (Schobert and Tschesche 1978). Proline is also involved in the synthesis

of cell wall proteins where it is converted to hydroxyproline (Nanjo et al. 1999). The amount of PRO present in a number of plant species is actually osmoregulated since PRO accumulated under stress conditions is rapidly consumed when stress conditions are alleviated (Jeffries et al. 1979, Trotel et al. 1996, Trotel-Aziz et al. 2000) acting as a transient storage compound for reduced nitrogen and reducing power. Proline has also been found to be very abundant in the flowers of angiosperms (Bathurst 1954, Dashek and Harwood 1974, Leport and Larher 1988) where it could

Abbreviations – ABA, abscisic acid; CLD, canola leaf disc; Glu, glutamate; P5C, D1-pyrroline-5-carboxylic acid; P5CR, P5C reductase; P5CS, P5C synthetase; PDH, proline dehydrogenase; PRO, proline; PVPP, polyvinylpolypyrrolidone. Physiol. Plant. 117, 2003

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be involved in fertilization and in pollen germination. Interestingly the gene encoding P5CS, the first enzyme of the glutamate pathway responsible for PRO synthesis, has been recently characterized as a target for the CONSTANS factor that promotes flowering of Arabidopsis in response to long photoperiods (Samach et al. 2000). In addition, the plant oncogene rolD, which has been found to promote flowering when transferred to tobacco plants (Mauro et al. 1996), is now known to encode a functional ornithine cyclodeaminase, which ensures the direct conversion of ornithine to PRO and enhancement of the PRO content under non-stressing conditions (Trovato et al. 2001). The accumulation of PRO under stress conditions relies on both activation of its biosynthesis and inhibition of its degradation (Delauney and Verma 1993, Yoshiba et al. 1997) and quite similar changes in PRO metabolism could also occur during the morphogenetic events associated with flowering and reproductive development. PRO synthesis from either glutamate or ornithine as well as PRO degradation (Fig. 1) have been widely documented during the past 20 years (see for example Huang and Cavalieri 1979, Zhang et al. 1995, Verbruggen et al. 1996, Forlani et al. 1997, Roosens et al. 1998, Yang and Kao 1999, Deuschle et al. 2001, Mani et al. 2002). A reverse pattern of P5CS and PDH genes expression has been demonstrated by Kiyosue et al. (1996) and Peng et al. (1996), especially in Arabidopsis plants subjected to osmotic stress. These authors showed that at dehydration P5CS and PDH gene expression was upand downregulated, whereas during rehydration it was down- and upregulated, respectively. PRO itself could play a role, not solely in inducing PDH gene expression especially in turgid plant tissues (Kiyosue et al. 1996, Verbruggen et al. 1996), but also in promoting the activity of PDH promoter in flower parts (Nakashima et al. 1998). These features could specify very important functions for the PRO accumulated, this compound being

likely to behave as a potent nutrient for stressed plant cells when growing conditions are restored. We have shown that the apparent rate of PRO consumption induced by rehydration of CLD previously subjected to hyperosmotic stress was dependent on their amount of PRO accumulated (Trotel et al. 1996). At the whole plant level, Singh et al. (1973) have also demonstrated that the growth rate of barley plants during their recovery from water stress was positively correlated with the amount of PRO accumulated during drought. These observations strongly suggest that high amounts of PRO stored under stress conditions could be beneficial at recovery and this could have some interest in breeding for tolerance to water stress since a large variability occurs in the capacity of higher plants to accumulate PRO in response to drought conditions (Hanson et al. 1979, Premachandra et al. 1995, Sanchez et al. 1998). If the explanations for such a variability are not established we have recently demonstrated, via experiments performed with CLD, that glycine betaine and the polyamines behave in vitro as suppressors of the PRO response (Larher et al. 1996, 1998). In contrast, the phytohormone abscisic acid (ABA) has been proposed to promote PRO accumulation in response to a variety of environmental stresses (Bray 1997, Capalans et al. 1999, Hare et al. 1999, Swamy and Smith 1999, Trotel-Aziz et al. 2000). It has also been shown that the accumulation of PRO under water stress and its subsequent consumption during rehydration are preceded by an increase and a decrease in the ABA content, respectively (Aspinall 1980, Stewart and Voetberg 1987, TrotelAziz et al. 2000). Furthermore the aba1 mutant of Arabidopsis thaliana, which is ABA-deficient, accumulated PRO to a lesser extent than its wild relative in response to salt stress. This effect was not relying on a decrease in the accumulation of P5CS mRNA (Savoure´ et al. 1997), which suggests that ABA might be acting at sites located either upstream of P5CS (i.e. those responsible for providing substrates for PRO synthesis), or downstream to this key enzyme, for instance at the level of PDH.

Fig. 1. Simplified scheme of proline (PRO) biosynthesis pathways from glutamate (GLU) and ornithine (ORN), and targets of corresponding inhibitors. GSA, g-glutamyl semialdehyde; P5C, D1-pyrroline-5-carboxylic acid; P5CS, P5C synthetase; P5CR, P5C reductase; d-OAT, dornithine aminotransferase; GS, glutamine synthetase and GOGAT, glutamate synthase; PDH, proline dehydrogenase; PCDH, P5C dehydrogenase; a-KG, a-ketoglutarate; Fd, ferredoxine; red, reduced; ox, oxidized; sp, spontaneous reaction. Methionine sulphoximine (white cross) and gabaculine (black cross) are inhibitors of GS and d-OAT, respectively.

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In the present study we have tried to specify the targets for the suppressing effect exerted by ABA on PRO consumption in CLD subjected to recovery conditions following an osmotic shock, in the light. Assuming reciprocal effects at the P5CS and the PDH levels we have investigated the co-ordinated changes occurring in terms of gene expression and enzyme activity during the sequential hyper- and hypo-osmotic treatments, exogenous ABA being added at the onset of the recovery period. In addition a pharmacological approach was done to assess the control exerted by the availability of precursors for PRO synthesis, in setting the PRO content of CLD supposed to consume this amino acid during recovery from osmotic stress.

Materials and methods Plant material and culture conditions The seeds of canola (Brassica napus L.) var. oleifera, cv. Samouraı¨, were germinated in the dark at 25æC, in Petri dishes on soaked filter paper. Selected seedlings were then transferred on 10 cm vermiculite layer in plastic boxes and grown in a culture chamber [14 h light/10 h dark, 25æC/21æC, light intensity 50 mMol mª2 sª1 (from Philips TLF 110 fluorescent tubes and 40 W incandescent lamps), relative humidity 75/90%]. Plants were fertilized with half-strength Hoagland’s solution (Hoagland and Arnon 1938) twice a week. Media were renewed weekly. Leaf disc preparation CLD (10 mM diameter) were cut with a cork borer from the youngest fully expanded leaves on 6-week-old plants. CLD were immediately incubated on a reference medium (5 mM HEPES, 1.5 mM CaCl2, 10 mM KCl, pH 6.0). Osmotic treatments Hyper-osmotic treatments (or stress) were performed by incubating 15 CLD in 100 ml flasks containing 20 ml of a ª1.69 MPa solution prepared by adding 400 g of PEG 6000 to 1 l of the reference medium. At this concentration, PEG 6000 induced a 30% decrease in the water content of CLD within 5 h, associated with a decrease in their osmotic potential from a value close to ª0.9 MPa to ª1.75 MPa (Trotel et al. 1996, Aziz and Larher 1998). Flasks were then covered with Petri dishes and placed under continuous light (50 mMol mª2 sª1 from Philips TLF 110 fluorescent tubes and 40 W incandescent lamps) on a shaking bath at 29æC. Hypo-osmotic treatments (or recovery) were performed by transferring stressed CLD to the reference medium (ª 0.04 MPa) under continuous light for various periods of time. In response to these successive hyper- and hypoosmotic shocks CLD have been shown to accumulate and then to consume large amounts of PRO, mimicking Physiol. Plant. 117, 2003

in short time intervals the typical PRO responses that are successively expressed in the whole plants of B. napus subjected successively to water deprivation for 20 days and to recovery conditions for the same period of time (Larher et al. 1993). ABA and inhibitor treatments ABA [(∫)-cis-trans, approximately 99%, plant cell culture tested] was added at a 50-mM concentration in the recovery medium, which means a 25-mM concentration for the active form of this hormone. When treated under these conditions for 20 h the ABA accumulated up to 370 nmol gª1 DW, i.e. an internal concentration close to 50 mM (i.e. 25 mM for the active form) which is expected to inhibit PRO consumption at recovery by about 70%. This concentration was actually twice that found in CLD subjected to a 12-h osmotic treatment with ª1.69 MPa PEG 6000 (Trotel-Aziz et al. 2000). Methionine sulphoximine (100 mM) and gabaculine (100 mM), as inhibitors of PRO biosynthesis from glutamate and ornithine, respectively, were added with ABA or separately in the recovery medium. All compounds were purchased from Sigma (Sigma-Aldrich, St. Quentin-Fallavier, France). Proline analysis PRO was determined on the crude extracts by the modified ninhydrin method as described by Trotel et al. (1996). Crude extracts were obtained from the CLD rinsed three times with distilled water, then placed into tubes (5 CLD per tube) containing 2 ml of distilled water. The net amount of PRO consumed results from the subtraction between the amount of PRO accumulated during the stress treatment and the amount of residual PRO at the end of the recovery period. This value refers to PRO degradation, PRO incorporation into proteins as well as PRO synthesis. The apparent mean rate for PRO consumption is calculated by dividing the amount of PRO consumed by the duration of the recovery period. For all treatments, especially those that required long incubation times, it has been checked that PRO leakage remains negligible (Trotel et al. 1996). Isolation of P5CS and PDH partial cDNAs by RT-PCR and cloning Total RNA was isolated as described by Verwoerd et al. (1989) from CLD. Total cDNA was obtained from 2 mg of total RNA extracted from osmotic stressed CLD, incubated for 1 h at 37æC with MMLV-reverse transcriptase (200 U), 20 mM dNTP and 2.5 mM T12VG anchor primer. PCR amplification was performed with 400 ng cDNA as a template, 50 pmol of upstream and downstream primers synthesized based on Arabidopsis thaliana P5CS and PDH cDNA nucleotide sequences. A 456-bp and a 215

inium thiocyanate (Goda and Minton 1995) and subsequently blotted onto a nylon membrane (Gene Screen Plus, New England Nuclear, Boston, MA, USA). Hybridizations were then performed according to the membrane manufacturers recommended protocol. The P5CS and PDH cDNA fragments (50 ng) were labelled by random priming (Feinberg and Vogelstein 1984) with a32PdCTP. Each probe was hybridized to the membranes at 64æC for 20 h. Membranes were washed with 2 ¿ SSPE, 0.1% SDS, twice at room temperature for 10 min and then at 65æC for 20 min, before being exposed to Biomax MS film (Kodak, Eastman Kodak Company, Kodak Pathe´ France, Paris, France) with intensifying screens at ª80æC. Enzyme assays

Fig. 2. Kinetics of gene expression and activity of D1-pyrroline-5carboxylate synthetase (P5CS) and proline dehydrogenase (PDH) in CLD subjected to a sequential hyper- and hypo-osmotic treatments. (A) Northern blot analysis of P5CS and PDH transcripts. The 28 S and 18 S rRNA, visualized by staining with ethidium bromide (below), were used as a loading control. (B) P5CS activity in CLD. Data, expressed as a percentage of the maximal value (28 pKat. mgª1 protein which corresponds to 100%), are means of 3 replicates. Vertical bars indicate standard error (). (C) PDH activity in CLD. The corresponding control values remained at 100% (not shown). Data, expressed as percentage of the maximal value (57 pKat mgª1 protein which corresponds to 100%), are means of 3 replicates. Vertical bars indicate . Arrow indicates the transfer of CLD from hyper- to hypo-osmotic medium.

387-bp RT-PCR product corresponding to internal coding region of the P5CS and PDH, respectively, were obtained, cloned in a pUC vector, and sequenced.

PDH activity was determined on intact leaf tissues according to the method of Mitra et al. (1975). This enzyme labilizes the C-H bond occurring at the C5 position of the PRO molecule, which results by the loss to water of tritium from specifically labelled -[5-3H]-proline. The accumulated tritium oxide in the medium is recovered by direct sublimation of aliquots into scintillation vials, the radioactivity being measured by liquid scintillation counting. The PDH assays were performed in triplicate using five CLD (3 mM diameter) incubated for 30 min at room temperature with constant shaking in 2 ml 0.1 M Tris-HCl buffer, pH 7.4 containing the radiolabelled PRO (3.3 ¿ 104 Bq mlª1). P5CS activity was determined in vitro according to the method of Zhang et al. (1995). P5CS was extracted at 4æC from 500 mg of fresh CLD with PVPP (1:1, w/w) and 50 mM Tris-HCl (pH 7.0) containing 10 mM b-mercaptoethanol, 300 mM sucrose and 5 mM MgCl2. The mixture was then centrifuged at 10 000 g for 20 min (4æC). The supernatant constituted the enzyme source. P5CS assays were performed at 37æC in microtubes containing a Tris-HCl buffer (50 mM, pH 7.0), 20 mM MgCl2, 10 mM ATP, 5 mM NADPH, 5 mM -glutamate, -[U-14C]glutamate 1.8 105 Bq mlª1 (Sigma) and the crude extract. Enzymatic reaction was stopped by transferring the microtubes in liquid nitrogen. Amino acids were separated by thin layer chromatography (silica gel, Merck SA, Fontenay-ss-Bois, France) with a phenol/ water/acetic acid mixture (75:25:5, w/v/v) as eluent. Cochromatography with pure amino acids was performed and TLC were sprayed with ninhydrin (0.3% in methanol). Radiolabelled P5C and PRO were revealed and quantified by Instant Imager.

Results

Northern blot analysis

Changes in gene expression and enzyme activity of P5CS and PDH during successive hyper- and hypo- osmotic treatments

Aliquots of 18 mg of total RNA were fractionated by electrophoresis on a 1.3% agarose gel containing guanid-

RNA extracted from CLD subjected or not to sequential osmotic stress and recovery were examined by Northern

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blot (Fig. 2A). P5CS transcripts were not detected in control leaf discs while their level strongly increased as early as 2 h after application of the hyper-osmotic treatment and remained high thereafter. When osmotically stressed CLD were transferred to the recovery hypo-osmotic medium, the level of P5CS transcripts decreased within 4 h. In contrast, the amount of PDH mRNA, which was apparently weak in the control fresh leaf discs, remained very low during the osmotic shock while its production was upregulated within the first hour of recovery, reaching a relatively high level within 10 h of hypo-osmotic treatment. As shown in Fig. 2B, P5CS activity was low in freshly excised CLD. It increased rapidly in response to application of the osmotic shock, since it was doubled within 6 h, then it remained fairly constant until the transfer to the low osmotically active medium. This transfer provoked a rapid and transient decrease of P5CS activity. Later on P5CS activity was surprisingly found to exceed that measured in dehydrated discs. In control CLD, P5CS activity was also subjected to changes although their PRO level remained fairly constant all along the incubation time. PDH activity was relatively high in freshly excised CLD (Fig. 2C). It was rapidly suppressed in CLD incubated under stressing conditions. After the transfer of CLD to the recovery medium, the whole PDH activity was recovered within the first 2 h of rehydration.

significantly affected by the hormonal treatment. This could indicate that consumption of accumulated PRO through PDH could be decreased while PRO synthesis via P5CS could be maintained, these combined effects being sufficient to maintain the PRO pool(s) at level(s) similar to those reached at the end of the hyper-osmotic treatment. Effects of inhibitors of proline biosynthesis on the rate of proline consumption during recovery of CLD added with or without ABA As expected in CLD recovering from osmotic stress conditions and supplemented with ABA in the light, the

Changes in gene expression and enzyme activity of P5CS and PDH during recovery in the presence of exogenous ABA As previously mentioned the apparent rate of PRO consumption by leaf discs previously treated for 20 h with PEG 6000 (ª 1.69 MPa) and then transferred to a medium of high water potential added with ABA was strongly restricted, provided such recovery treatment was performed in the light. Targets for ABA were suspected to be located at the level of key enzymes of PRO metabolism. This was further assessed during this study. Northern blot analysis indicated that the expected stimulation of PDH gene expression after the transfer of the CLD to the reference medium was restricted when this medium was supplemented with 50 mM ABA (Fig. 3A). Within 10 h of recovery in presence of ABA, the amount of PDH transcripts decreased drastically. In contrast, the level of P5CS mRNA was not affected at least for the first 2 h following the onset of rehydration (Fig. 3A). When the recovery medium was supplemented with ABA, stimulation of PDH activity was restricted by 50% compared to that of the control discs (Fig. 3B). Under the same conditions, P5CS activity (Fig. 3C) of the CLD treated with ABA remained quite similar to that of control discs not supplemented with ABA. Thus it can be inferred that the apparent reduction of the rate of PRO consumption under recovery in the presence of extra ABA could be dependent on both a partial recovery of PDH activity and a P5CS activity which was not Physiol. Plant. 117, 2003

Fig. 3. Kinetic of gene expression and activity of PDH and P5CS in response to ABA during recovery of stressed CLD. (A) Northern blot analysis of PDH and P5CS transcripts. (B) PDH activity. (C) P5CS activity in CLD. Recovery medium was added with 50 mM ABA (π ABA) or not (– ABA). Legend as in Fig. 2.

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Table 1. Changes in the apparent mean rate of proline consumption by canola leaf discs subjected successively to stressful conditions PEG 6000 (ª 1.69 Mpa) for 20 h and recovery conditions on a control medium added with ABA and supplemented as indicated with either methionine sulphoximine or gabaculine. At the end of the hyper-osmotic treatment the proline content of CLD was 445 ∫ 18 mMol gª1 DW. Proline was then quantified after the 18 h incubation time on the control medium (ª 0.04 MPa) added with various effectors. Data are means of 3 independent experiments in triplicate, each being performed with 10 leaf discs. A Student test was performed (two-way ) on means within the first column. Values not sharing the same letter are significantly different (P Ω ⬍ 0.001).

Recovery treatments

Residual PRO level (mmol hª1 gª1 DW)

*Apparent PRO consumption (mmol gª1 DW)

Control 50 mM ABA 100 mM Methionine sulphoximine 100 mM Gabaculine 50 mM ABA π 100 mM Methionine sulphoximine 50 mM ABA π 100 mM Gabaculine

127 352 38 149 216 310

318 93 407 296 229 135

b e a b c d

** Apparent mean rate of PRO consumption (mmol gª1 DW)

Effect on apparent PRO consumption (% of control)

17.7 5.2 22.6 16.4 12.7 7.5

– ª 70.6 π 27.7 ª 7.3 ª 28.2 ª 57.6

*The calculated apparent proline consumption was derived from the subtraction of the amount of PRO accumulated during the stress treatment (445 ∫ 18 mmol gª1 DW) from the amount of PRO at the end of the recovery period. ** The apparent mean rate of PRO consumption was calculated by dividing the amount of PRO consumed by the duration of the recovery period.

mean rate of apparent PRO consumption (Table 1) was significantly lower than in those incubated without ABA. When the recovery medium was added with MSO, an inhibitor of glutamine synthetase (GS), this rate increased significantly (P ⬍ 0.001) which indicated that synthesis of glutamine followed by its conversion to glutamate was actually involved in contributing PRO synthesis during recovery. On the contrary, when CLD were fed with gabaculine, an inhibitor of d-ornithine aminotransferase, the apparent rate of PRO metabolization was not modified in comparison with that of CLD incubated on the recovery medium. This suggests that the conversion of ornithine to PRO was not really involved in PRO synthesis under such experimental conditions and in the absence of ABA. When CLD were supplied with ABA and MSO apparent mean rate of PRO consumption was enhanced in comparison with that of CLD supplied with ABA alone, because the glutamate pathway of PRO synthesis was restricted by MSO acting at the level of glutamine synthetase. When ABA and gabaculine were supplied together, the rate of PRO consumption was found to be weakly increased (P ⬍ 0.001) in comparison with that of CLD supplied with ABA alone. This suggests that in the presence of ABA the ornithine pathway could also be concerned to some extent with PRO synthesis.

10 h (Fig. 4), their PRO content was found to be enhanced compared with that of control discs. When the incubation time in the presence of ABA was doubled, their PRO content significantly increased with the external ABA concentration. However, at the highest external ABA concentration (i.e. 50 mM) the PRO level was about 10 times lower than that of CLD subjected to an osmotic treatment with PEG 6000 (ª 1.69 MPa) for the same incubation time (Trotel-Aziz et al. 2000). Northern blot analysis (Fig. 5A) shows that P5CS transcripts were not detected in turgid CLD. However, in presence of 50 mM ABA, the amount of P5CS mRNA increased within 2 h and stayed at a high level for at least 8 h thereafter. The corresponding P5CS activity (Fig. 5B) increased progressively during the first 20 h incubation period. Exogenous supply of ABA resulted in a transient decrease of P5CS activity, which is followed by a rapid increase, the final value being similar to that obtained in stressed CLD. These results indicate that both P5CS gene expression and P5CS activity could also be upregulated in turgid CLD supplied with exogenous ABA.

Other experimental evidence concerning the effects of exogenously supplied ABA on PRO metabolism The question arises to know whether or not the ABA effects on PRO metabolism could also be observed under either non-stressing conditions or during recovery from osmotic shocks of short duration. PRO metabolism in turgid leaf discs When freshly cut CLD were incubated in the reference medium added with different concentrations of ABA for 218

Fig. 4. Proline level in turgid CLD incubated for 10 h or 20 h in the reference medium supplemented with ABA at concentrations ranging from 0 to 50 mM. Freshly cut CLD contained 3 ∫ 1 mMol gª1 DW. Data are means of 2 replicates ∫. Physiol. Plant. 117, 2003

PRO metabolism of osmotically shocked leaf discs transferred to media of high water potential added or not with ABA When freshly cut CLD were subjected to stress conditions for short periods of time (0–4 h), they accumulated small amounts of PRO (up to 30–40 mmol gª1 DW). After their subsequent transfer to the recovery medium added with ABA, PRO level increased with the duration of the previous osmotic shock, provided it exceeded 2 h (Fig. 6). Hence, after a 4-h osmotic shock and a subsequent 20 h of recovery in the presence of ABA, the PRO content of CLD was twice that of the control discs not supplied with the hormone. Such level could rely on both the amount of PRO accumulated during the stressing period and recovery periods and the decrease of its consumption during recovery in the presence of ABA. As shown in the inset of Fig. 6, the relative amount of P5CS mRNA strongly increased in response to these short periods of stress, the highest one being reached through application of a 4-h osmotic treatment.

Fig. 5. Gene expression and activity of P5CS in turgid CLD incubated on the reference medium added with or without ABA. (A) Northern blot analysis of P5CS transcripts in turgid CLD (Control), transferred to (π) the same medium added with (π 50 mM ABA) or without ABA. The 28 S and 18 S rRNA, visualized by staining with ethidium bromide (below), were used as a loading control. (B) P5CS activity in turgid CLD (control), supplemented (arrow) with 50 mM ABA (control π ABA) or not. Data, expressed as percentage of the maximal value (28 pKat mgª1 protein which corresponds to 100%), are means of 3 replicates and vertical bars indicate .

Physiol. Plant. 117, 2003

Discussion In the present study we have investigated some aspects of the regulatory functions exerted by ABA, exogenously supplied, on PRO metabolism in canola leaf explants experiencing in vitro successive osmotically stressful and recovery conditions. The changes induced are expected to mimic, with respect to the PRO response, the effect of sequential dehydration and rehydration that take place in the whole plants subjected to drought and to further irrigation. We previously showed that the recovery treatment consisting in the transfer of osmotically stressed leaf discs to a medium with a high water potential provoked PRO consumption (Trotel et al. 1996). We also demonstrated that when recovery takes place in the light, PRO consumption was restricted by exogenously supplied ABA. This novel effect of ABA was not observed in the dark nor in the light in the presence of inhibitors of photosynthetic electron transport (Trotel-Aziz et al. 2000). Temporal relationships between the time courses of the changes induced by the successive up- and downshock treatments of CLD at both the endogenous levels of ABA and that of PRO were also characterized. This suggested that enhancement of ABA content could indeed be partly responsible for PRO accumulation, while ABA decrease could contribute to PRO consumption. Such hypothesis is strongly supported by the changes in the activity of key enzymes involved in PRO metabolism as well as in the amount of their related transcripts described in this study. First, PRO accumulation in response to hyper-osmotic shock is tightly connected with co-ordinated and opposite adjustments taking place at the transcriptional level of the P5CS and PDH genes. Similar results had already been reported for Arabidopsis and maize (Rayapati and Stewart 1991, Yoshiba et al. 1995, Kiyosue et al. 1996). These changes were found to be closely related with those occurring at the level of enzyme activity. Secondly, when stress conditions were alleviated, after

Fig. 6. Proline level in CLD subjected to short periods (from 0 to 4 h) of an hyper-osmotic stress, then transferred for 20 h to a reference medium (recovery) added with 50 mM ABA or without ABA. The inset shows the relative content of P5CS mRNA in CLD during hyper-osmotic stress. Data, expressed as percentage of the maximal rate of P5CS expression, are means of 3 replicates and bars indicate .

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the full expression of the PRO response the changes previously observed at the transcriptional level of P5CS and PDH genes were reversed. Thus the PDH mRNAs were produced and accumulated within 1 h while those of P5CS decreased progressively to that of the freshly cut turgid leaf discs. Consequently PDH activity increased up to its typical value in freshly cut discs but P5CS activity stabilized at a level as high as that of the stressed CLD. This suggests that the decrease in PRO content observed during the recovery period did not solely reflect the recovery of catalytic property of the PDH but also the maintenance of P5CS activity that clearly hides the net rate of PRO consumption by PDH. This is also supported by the results obtained with CLD transferred to recovery conditions in the presence of methionine sulphoximine. Under these conditions the mean rate of PRO consumption was apparently increased because the proline pool(s) could not be replenished from glutamate whereas PRO is being oxidized or incorporated into proteins. Accordingly when CLD were fed during recovery with glutamate they exhibited an apparent low rate of PRO consumption (Trotel et al. 1996) because of their capacity to absorb glutamate from the external medium and to convert it into PRO. Our present data may also support the fact that PRO consumption during recovery in the light could rely primarily on the decrease in the endogenous level of ABA (Trotel-Aziz et al. 2000) since the decrease in the apparent mean rate of PRO consumption provoked by exogenous ABA could be attributed to its inhibitory effect on the expression of PDH gene and the activity of PDH. At recovery however, the level of P5CS transcripts and that of P5CS activity being independent of the presence of an extra source of ABA (Fig. 3), PRO biosynthesis could also contribute to set the final level of PRO in CLD and consequently the apparent rate of PRO consumption. This is in good agreement with the fact that the residual PRO in CLD fed with ABA at recovery was found to be lower when leaf explants were supplemented with methionine sulphoximine, assuming this compound blocks the GS/ GOGAT cycle via its inhibitory effect on GS. In turgid CLD also, as primarily described in Trotel et al. (1996) low levels of glutamate are suggested to be responsible for the low amount of PRO they accumulated in response to ABA (see Fig. 6) despite the stimulation of P5CS expression and P5CS activity (see Fig. 5). Furthermore, the PRO level of CLD stressed for short incubation times and subsequently incubated on the recovery medium supplemented with ABA also increased with the duration of the osmotic shock, which might be related to the enhanced availability of PRO precursors. In response to these short osmotic shocks, the relative content of P5CS mRNA increased until 4 h of hyper-osmotic treatment and then stabilized, suggesting that increased PRO level in CLD during their recovery from these shocks in medium added with ABA also relies on the enhanced level of P5CS transcripts. This could corroborate the report of Strizhov et al. (1997) showing that the induction of P5CS2 transcription, under salt stress conditions, was strictly reduced in the abi1-1 and aba1-1 Arabidopsis mutants, 220

which are deficient in ABA signal transduction and ABA biosynthesis, respectively (Meyer et al. 1994, Marin et al. 1996, Merlot et al. 2001). Obviously under our experimental conditions the changes in the endogenous content of ABA could also be involved since its level rapidly increased following onset of the osmotic treatment (TrotelAziz et al. 2000). We conclude that the light-dependent inhibitory effect of ABA on the apparent rate of PRO consumption in CLD experiencing rehydration could depend on its capacity to (1) stimulate P5CS gene expression and P5CS activity; (2) restrict PDH transcription and PDH activity; and (3) possibly exert a positive effect on the availability of precursors for PRO synthesis. Acknowledgements – Authors wish to thank P. Lemesle for her excellent technical assistance. This work was supported by the Brittany Regional Council (‘Re´gion Bretagne’, France).

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