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plant productivity and plant distribution (Parker 1963;. Boyer 1982). Adaptation ... novo synthesis (Boggess and Stewart 1976; Stewart 1980;. Rhodes et al. 1986 ...
 Springer-Verlag 1997

Mol Gen Genet (1997) 254:104 – 109

SHORT COMMUNICATION

A. Savoure´ · X.-J. Hua · N. Bertauche M. Van Montagu · N. Verbruggen

Abscisic acid-independent and abscisic acid-dependent regulation of proline biosynthesis following cold and osmotic stresses in Arabidopsis thaliana Received: 22 July 1996 / Accepted: 7 November 1996

Abstract The role of the phytohormone abscisic acid (ABA) in the regulation of proline synthesis was investigated by following the expression of the At-P5S and At-P5R proline biosynthesis genes in Arabidopsis thaliana wild type, in an ABA-deficient aba1-1 mutant as well as in ABA-insensitive abi1-1 and abi2-1 mutants after ABA, cold and osmotic stress treatments. In wild-type and in ABA mutant seedlings, 50 lM ABA or osmotic stress treatment triggered expression of At-P5S, whereas At-P5R accumulation was scarcely detectable. Expression of either gene was mediated by endogenous ABA since transcript levels were similar in wild-type and in ABA-deficient mutant plants. Proline accumulated to a greater extent after osmotic stress than upon ABA or cold treatment. Thus, ABA-treated abi1-1 mutant plants accumulated less proline than the ABA-treated wild type. Upon salt stress, proline accumulated to a lesser extent in aba1-1 and abi1-1 mutant plants, suggesting an indirect role of ABA on proline accumulation during salt adaptation of the plant. These results indicate that the expression of the genes of the proline biosynthetic pathway is ABA independent upon cold and osmotic treatments, although their expression can be triggered by exogenously applied ABA. However, the endogenous ABA content may affect proline accumulation upon salt

Communicated by A. Kondorosi A. Savoure´1 · X.-J. Hua · M. Van Montagu (&) · N. Verbruggen Laboratorium voor Genetica, Department of Genetics, Flanders Interuniversity Institute for Biotechnology (VIB), Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium N. Bertauche Institut des Sciences Ve´ge´tales, CNRS, F-91198 Gif-sur-Yvette Cedex, France Present address: Laboratoire de Biologie Mole´culaire, Universite´ Picardie Jules Verne, 33 rue Saint Leu, F-80039 Amiens Cedex, France 1

stress, suggesting post-transcriptional control of proline biosynthesis in response to NaCl. Key words Abscisic acid · Arabidopsis thaliana · Cold stress · Osmotic stress · Proline

Introduction Osmotic and cold stresses are major limiting factors for plant productivity and plant distribution (Parker 1963; Boyer 1982). Adaptation of plants to stress involves morphological, physiological as well as biochemical changes including the accumulation of osmolytes such as proline (McCue and Hanson 1990). Proline accumulation occurs mainly as a result of de novo synthesis (Boggess and Stewart 1976; Stewart 1980; Rhodes et al. 1986; Voetberg and Sharp 1991) from glutamate via two successive reductions. In plants, the reduction of glutamate to its semi-aldehyde is performed by a single bifunctional enzyme, ∆1-pyrroline-5-carboxylate synthetase (P5CS), and genomic or cDNA clones encoding P5CS have been isolated from Vigna aconitifolia (Hu et al. 1992) and from Arabidopsis thaliana (Savoure´ et al. 1995; Yoshiba et al. 1995). The glutamate semi-aldehyde cyclises spontaneously to pyrroline-5-carboxylate which is then reduced to proline by pyrroline-5-carboxylate reductase (P5CR); genomic or cDNA clones have been isolated from soybean (Delauney and Verma 1990), pea (Williamson and Slocum 1992) and A. thaliana (Verbruggen et al. 1993). The metabolic processes leading to proline accumulation under osmotic or cold stress involves expression of the proline biosynthetic genes (Delauney and Verma 1990; Hu et al. 1992; Verbruggen et al. 1993; Savoure´ et al. 1995; Yoshiba et al. 1995). Many plants accumulate high levels of proline in response to osmotic stress, and proline is thought to play an adaptive role during osmotic stress (for review, see Delauney and Verma 1993). Furthermore, low temperature without decrease of the water

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potential of the plant tissue triggers proline accumulation which may play a role in establishing cold resistance in plants (Chu et al. 1974). The phytohormone abscisic acid (ABA) is known to exercise an important role in mediating the primary responses of plants to environmental stresses such as low temperature, drought and high salinity (for a review, see Giraudat et al. 1994). Exposure to desiccation, salt stress and low temperature is generally accompanied by an increase in endogenous ABA levels prior to proline accumulation (Zeevaart and Creelman 1988). This increase in ABA levels contributes to the activation of a number of water- and salt stress-induced genes (Chandler and Robertson 1994), the products of which are thought to be involved in the protection of the cell or in recovery from stress. An ABA transduction cascade may possibly be involved in the expression of the proline biosynthesis genes (Yoshiba et al. 1995), although several Arabidopsis genes induced by osmotic stress (Gosti et al. 1995) or cold treatments (Gilmour and Thomashow 1991; Nordin et al. 1991) are not regulated by endogenous ABA. The characterisation of ABA biosynthetic mutants reveals that the induction of a particular gene by exogenously applied ABA does not necessarily imply that the regulation of gene expression is ABA-dependent upon stress (Giraudat et al. 1994). These observations suggest that ABA-independent and ABA-dependent pathways interact to regulate the expression of certain genes in response to osmotic and cold stresses. Interestingly, proline accumulates in a tomato flacca mutant that does not contain elevated levels of ABA even after osmotic stress (Stewart and Voetberg 1987), and addition of the ABA biosynthesis inhibitor, fluridone, to wilted barley leaves does not influence proline accumulation (Stewart and Voetberg 1987). However, increased ABA is required for proline accumulation at low water potential in the maize primary root tip (Ober and Sharp 1994). External application of ABA to non-stressed plants is able to trigger proline accumulation in barley (Stewart 1980; Aspinall and Paleg 1981; Pesci 1987), maize (Ober and Sharp 1994), rice (Chou et al. 1991), pea (Hasson and Poljakoff-Mayber 1983; Fedina et al. 1994) and in Arabidopsis (Finkelstein and Somerville 1990; Yoshiba et al. 1995). Although this response is common, tobacco and sunflower leaves (Aspinall and Paleg 1981) as well as spinach and Pennisetum thyphoides (McDonnell et al. 1983), which have a high proline content upon osmotic stress, do not accumulate proline in response to applied ABA. The relationship between proline and ABA is therefore unclear and it is important to determine the contribution of ABA to the regulation of the genes involved in proline biosynthesis. We have investigated the expression of At-P5S and At-P5R, encoding P5CS and P5CR, respectively, and the level of proline after exogenously applied ABA and during cold and osmotic stresses in A. thaliana wild-type and in ABA-deficient aba1-1 and ABA-insensitive abi1-1 and abi2-1 mutant plants. Our data indicate that upon cold and osmotic

stresses, the expression of the genes involved in the proline biosynthetic pathway is independent of the endogenous level of ABA in Arabidopsis. However, proline accumulation may depend on the endogenous level of ABA, suggesting ABA-dependent and ABA-independent post-transcriptional control of proline accumulation, depending on the stress treatment.

Materials and methods Plant material and growth conditions A. thaliana (L.) Heynh., ecotypes Columbia and Landsberg erecta as well 195 mutants in the Landsberg erecta background were used in this study. ABA mutants were the ABA-deficient aba1-1 mutant (isolation number A26; Koornneef et al. 1982) and the two nonallelic ABA-insensitive abi1-1 and abi2-1 mutants (Koornneef et al. 1984). Seeds were kindly provided by M. Koornneef (Agricultural University, Wageningen, The Netherlands). Seeds were surfacesterilised and grown on nylon filters laid on modified Murashige and Skoog medium (K1 medium) (Murashige and Skoog 1962) under 16-h light conditions at 22° C as described previously (Savoure´ et al. 1995). Ten-day-old seedlings grown on filters were transferred to fresh K1 medium for controls or to fresh K1 medium supplemented with ABA (10 or 50 lM), sorbitol (550 mM), or NaCl (250 mM) as described by Verbruggen et al. (1993). For cold treatment, 10-day-old seedlings were placed at 4° C for 24 h. After different incubation times, seedlings were collected, frozen in liquid nitrogen and stored at )80° C prior analysis. Expression analysis Total RNA from 10-day-old seedlings was isolated according to Logemann et al. (1987). RNA was denaturated in formamide and formaldehyde and was then separated by 1.2% agarose-formaldehyde electrophoresis. After transfer to nylon membrane, RNA was fixed by UV cross-linking. Hybridisation conditions were according to Church and Gilbert (1984). The membrane was hybridised at 65° C with full-length At-P5S (Savoure´ et al. 1995) and At-P5R (previously named At-P5C1; Verbruggen et al. 1993) cDNAs which were labelled with 32P-dCTP by the random primer extension method (Amersham, Aylesbury, UK). Membranes were stained with methylene blue as a control for RNA loading. Proline determination Free proline content was measured according to the method using L-proline as standard (Bates et al. 1973).

Results Exogenously applied ABA triggers At-P5S and At-P5R gene expression prior to proline accumulation The effect of exogenous ABA (10 lM or 50 lM) on the expression of At-P5S and At-P5R was tested first (Fig. 1A). After treatment with 10 lM ABA, At-P5S transcripts started to accumulate within 4 h, reaching maximal levels at 8 h, whereas there is no or only a trace of At-P5R transcript accumulation even after 24-h ABA treatment. The two different experiments performed with 50 lM ABA indicate that transcripts started to accu-

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Fig. 1A, B Exogenously applied abscisic acid (ABA)-induced expression of the At-P5S and At-P5R genes responsible for proline biosynthesis. Ten-day-old Arabidopsis thaliana ecotype Columbia seedlings grown in K1 were treated with either the nutritive medium (control), 10 lM or 50 lM ABA for the time indicated. A Total RNA (15 lg) was fractionated in a formaldehyde-agarose gel, transferred to nylon membranes and hybridised with 32P-labelled At-P5S and At-P5R cDNA probes. The number above each lane indicates the time after the initiation of the treatment prior to RNA isolation. Two independent experiments with 50 lM ABA treatment are presented. B Free proline extracted from ABA-treated seedlings treated with either nutritive medium (Control) or with 10 lM or 50 lM ABA for 4, 8 and 24 h. Each value is a mean (± SD) obtained from three independent determinations

mulate to a higher extent around 8 h and that At-P5S was clearly more responsive to ABA than was At-P5R. To elucidate the putative role of endogenous ABA in the accumulation of proline, we have compared transcript accumulation in wild-type A. thaliana ecotype Landsberg and in three different ABA mutants, the ABAdeficient aba1-1 mutant (Koornneef et al. 1982) and the two ABA-insensitive abi1-1 and abi2-1 mutants (Koornneef et al. 1984). The aba1-1 mutant is deficient in ABA biosynthesis and contains less than 5% of the endogenous ABA of the wild-type plant (Koornneef et al. 1982). abi1-1 and abi2-1 are two mutants which are impaired in their responses to ABA although their endogenous ABA levels are the same as those of wild-type plants (Koornneef et al. 1984). Wild-type plants as well as ABA mutants were exposed to 50 lM ABA for 24 h. This ABA treatment was found to trigger At-P5S and At-P5R transcript accumulation, although At-P5R transcript accumulation was very low (Fig. 2A). During ABA treatment, the At-P5S transcript reached the same level in wild type and in the different ABA mutants. The lower At-P5S

Fig. 2A, B Expression of genes responsible for proline biosynthesis after 24-h low temperature (4° C) and ABA treatments in A. thaliana Landsberg erecta. A Total RNA was isolated from 10-day-old seedlings of Arabidopsis wild type (wt), aba1-1-deficient, and abi1-1and abi2-1-insensitive mutants treated for 24 h with either lowtemperature treatment or application of 50 lM ABA. Total RNA (4 lg) was used per lane and the filter hybridised as described in Fig. 1. B Levels of free proline from low-temperature and 50 lM ABAtreated seedlings

mRNA level in the abi2-1 mutant was not statistically different from the ABA-treated wild-type mRNA level observed in repeated experiments (data not shown). At 24 h, higher At-P5S transcript and proline levels are observed with 50 lM ABA than with 10 lM ABA (Fig. 1). However, this fivefold increase in ABA concentration did not result in a proportional increase in proline levels. After treatment of ABA mutants with 50 lM ABA, accumulation of proline was not affected in the aba1-1 mutant although it was slightly and dramatically reduced in the abi2-1 and abi1-1 mutants, respectively, when compared with wild-type plants (Fig. 2B). Cold treatment enhances At-P5S transcript and proline levels in wild type and in ABA mutants Both wild-type plants and ABA mutants were exposed to low temperature for a period of 24 h. This cold

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treatment slightly induced At-P5S expression whereas At-P5R expression remained unaffected (Fig. 2A). No difference in the steady-state levels of At-P5S transcript was observed in either the ABA-deficient or the ABAinsensitive mutants during cold treatment, when compared to cold-treated wild-type plants. In all plants, cold stress caused a twofold increase of the proline content after 24 h (Fig. 2B). Osmotic stress triggers At-P5S and At-P5R expression and proline accumulation We have previously demonstrated that salt stress induces expression of both At-P5S and At-P5R gene expression prior to proline accumulation (Verbruggen et al. 1993; Savoure´ et al. 1995). We further analysed the expression of the proline biosynthetic genes in response to two different osmotic stresses triggered by either 550 mM sorbitol or 250 mM NaCl in wild-type and in ABAdeficient and ABA-insensitive Arabidopsis seedlings. Expression of both genes was induced by sorbitol and NaCl treatments in wild type as well as in the ABA mutants (Fig. 3A). It is interesting that transcript levels of At-P5S and At-P5R genes in the ABA mutants were not significantly affected compared with the wild type. The steady-state levels of At-P5S and At-P5R transcripts were higher in seedlings treated with 250 mM NaCl than with 550 mM sorbitol. Free proline content was determined in wild-type and ABA mutant seedlings after treatment with either sorbitol or NaCl (Fig. 3B). In wild type, sorbitol treatment triggered proline accumulation to an average level of 2.79 ± 0.09 lmol · g–1 fresh weight and NaCl to a higher level up to 4.36 ± 0.25 lmol · g-1 fresh weight. This NaCl-induced proline level has previously been observed in A. thaliana ecotype Columbia seedlings treated with 170 mM NaCl (Savoure´ et al. 1995). After 24-h treatment, sorbitol-treated ABA mutants accumulated proline to a level similar to that of the sorbitoltreated wild-type; this is in contrast to salt-stressed aba1-1 and abi1-1 mutants that accumulated lower proline levels than wild-type and abi2-1 mutant plants. As with exogenously applied ABA, we again observed a discrepancy in abi1-1 between At-P5S transcript levels and proline accumulation.

Discussion The aim of this work was to study the role of ABA in proline biosynthesis in response to exogenous ABA, cold or osmotic stresses. In wild-type Arabidopsis plants, a direct correlation has been observed between At-P5S transcript levels and proline accumulation during ABA, cold and osmotic stress treatments. Using ABA-deficient aba1-1 mutants, we demonstrate here for the first time at a molecular level that the expression of the genes involved in proline biosynthesis is independent of the en-

Fig. 3A, B Expression of At-P5S and At-P5R genes and levels of free proline in response to osmotic stress in A. thaliana Landsberg erecta. Treatments used were with nutritive medium for control (0), 550 mM sorbitol (1) and 250 mM NaCl (2) for 24 h. A Total RNA (4 lg) from 10-day-old Arabidopsis wild type (wt), aba1-1-deficient, abi1-1 and abi2-1-insensitive mutants. The filter was hybridised as described in Fig. 1. B Levels of free proline in wild type and ABA mutants

dogenous level of ABA in A. thaliana upon cold and osmotic stresses. The expression analysis in ABAinsensitive abi1-1 and abi2-1 mutants upon stress has revealed several interesting features in the regulation of the genes in the proline biosynthetic pathway when compared with the pattern of proline accumulation. In non-osmotically stressed seedlings, application of ABA at concentrations as low as 10 lM triggered expression of At-P5S but not At-P5R. At 50 lM ABA, AtP5S was clearly more responsive to ABA than At-P5R. This phenomenon was also observed by Yoshiba et al. (1995) in A. thaliana, although a 100-fold higher concentration of ABA was used. Furthermore, exogenous ABA induced a lower accumulation of proline compared to that found in salt-stressed plants (Figs. 1B, 3B). Increasing the ABA concentration did not lead to an increase in proline levels up to the level observed with 250 mM NaCl. This result indicates that under nonstressed conditions, exogenous ABA-dependent proline accumulation reaches a different plateau from that observed under osmotic stress conditions, which suggests

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the involvement of different transduction cascades. The lower level of proline accumulation in the abi1-1 mutant compared to that of the wild-type plant corroborates the previous observation of Finkelstein and Somerville (1990), although they also observed a weaker accumulation of proline in the ABA-treated abi2-1 mutant. AtP5S transcript accumulation is not impaired in abi1-1 in response to exogenous ABA in contrast to other Arabidopsis genes tested so far. This result could be explained by a leakage of abi1 mutation after exposure to ABA. Since exogenously applied ABA triggers expression of the proline biosynthesis genes and increases of endogenous ABA were reported after cold and osmotic stresses, endogenous ABA may control expression of these genes upon stress. However, analysis of the ABAdeficient tomato flacca mutant revealed that ABA is not required for wilting-induced proline accumulation (Stewart and Voetberg 1987). The use of ABA biosynthetic mutants and/or ABA biosynthesis inhibitors has demonstrated that endogenous ABA indeed contributes to the regulation of several ABA-responsive genes during desiccation or drought (Giraudat et al. 1994). Until now, ABA biosynthetic and/or ABA-insensitive mutants have rarely been analysed for global stress responses. The ABA-deficient mutant was shown to have lost its capacity to acclimatise to cold (Heino et al. 1990). Most of the cold-regulated genes are also responsive to exogenously applied ABA, although they also show a strict ABA-independent regulation under cold stress (Gilmour and Thomashow 1991; Nordin et al. 1991). The interesting observation in this study is the lack of requirement for an endogenous ABA increase to trigger At-P5S expression and proline accumulation in response to cold stress. The cold-induced proline accumulation in the ABA-deficient mutant is similar to the levels observed in the cold-treated wild type, suggesting that, under cold stress, the proline biosynthetic pathway is not regulated by endogenous ABA and, moreover, that proline does not play an important role in the cold adaptation of the plant cell. Cold- and ABA-induced At-P5S may involve different control mechanisms since higher At-P5S transcript levels were observed in response to ABA than to low-temperature treatments. The cold-induced level of proline accumulation was also lower than that observed in salt- or sorbitol-treated wild type and ABA-insensitive mutants. Although both sorbitol and NaCl lead to osmotic stress, different adaptive mechanisms may be involved in which ABA may play different roles. Besides osmotic stress, salt stress leads to fast ion uptake and may provoke ion toxicity in the plant cell if mechanisms which exclude Na+ and Cl) ions do not function. Continuous accumulation of Cl) and Na+ may produce toxic effects disrupting metabolic process (Greenway and Munns 1980). Amzallag et al. (1990) have shown a protective effect of ABA under salt stress, probably due to a decrease of the transpiration rate. Sorghum plants sprayed with ABA show reduced accumulation of Na+ in the

shoot. ABA may also have a role in induction of genes which encode proteins important in the protection of cell metabolism against the effects of salt accumulation. NaCl provokes a decrease in proline accumulation in the aba1-1 and abi1-1 mutants, although At-P5S transcript levels reach a level similar to that in the wild-type plant (Fig. 3A). This lower proline accumulation may be explained in part by a protective effect of ABA against ion toxicity since the reduced accumulation of proline observed in aba1-1 mutants may be indirectly due to the lower ABA content necessary for this cell protection. The interesting observation is the reduced accumulation of proline in the abi1-1 mutant in response to NaCl stress. This result must be related to the effect of ABA treatment on At-P5S transcript and proline accumulation. In both cases, proline accumulation in the abi1-1 mutant is not correlated with At-P5S transcript levels. These results suggest a post-transcriptional control of proline biosynthesis in response to NaCl in which ABI1 plays a role. The ABI1 locus appears to be an important component of the ABA signalling transduction pathway for proline accumulation. Signal transduction cascades independent of ABA have been suggested to be involved during adaptation of plants to osmotic stress. The use of A. thaliana ABA mutants allowed us to demonstrate that the accumulation of proline biosynthetic gene transcripts upon stress is not dependent on the endogenous level of ABA. This result suggests that the expression of the proline biosynthetic genes are dependent upon at least two signal transduction cascades. One cascade is triggered by exogenously applied ABA in the absence of stress and the other, independently of endogenous ABA levels, is triggered by cold and osmotic stresses. The absence of endogenous ABA control at the transcript level may allow a rapid response of the cell to severe osmotic changes by synthesis of the osmolyte proline. At-P5S responds quickly to osmotic stress since At-P5S transcripts started to accumulate within 1 h (A.S. and X.J.H., unpublished results; Yoshiba et al. 1995). This is in contrast to other dehydration-responsive genes, such as rd29B, which are induced only 5 h after the beginning of treatment. rd29B is probably regulated by the endogenous level of ABA produced under drought stress conditions (Yamaguchi-Shinozaki and Shinozaki 1993, 1994), indicating that ABA may act as a secondary messenger for long-term adaptation of the plant cell to environmental stress conditions. Further studies will be necessary to understand how plant cells perceive the cold and osmotic stress signals which rapidly activate a signal transduction cascades triggering proline accumulation. Acknowledgements The authors thank B. Van de Cotte for skillful technical assistance, D. Inze´, J. Giraudat, F. Gosti, M. May and M. Davey for critical reading of the manuscript, M. De Cock for its final preparation, and R. Verbanck and K. Spruyt for the photographic work. This work was supported by grants from the Belgian programme on Interuniversity Poles of Attraction (Prime Minister’s Office, Science Policy Programming, no. 38) and the Vlaams

109 Actieprogramma Biotechnologie (ETC 002). A.S. was supported by a long-term postdoctoral fellowship from the European Molecular Biology Organization and a Human Capital and Mobility post-doctoral fellowship from the European Union (ERBCHBICT-941824). N.V. was a Research Assistant of the National Fund for Scientific Research (Belgium).

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