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• W1978161788 abstract "Accumulation of proline has been observed in a large number of plant species in response to drought and salt stresses, suggesting a key role of this amino acid in plant stress adaptation. Upstream components of the proline biosynthesis signal transduction pathways are still poorly defined. We provide experimental evidence that phospholipase D (PLD) is involved in the regulation of proline metabolism in Arabidopsis thaliana. The application of primary butyl alcohols, which divert part of PLD-derived phosphatidic acid by transphosphatidylation, stimulated proline biosynthesis even without hyperosmotic constraints. Moreover, application of primary butyl alcohols enhanced the proline responsiveness of seedlings to mild hyperosmotic stress. These data indicate that some PLDs are negative regulators of proline biosynthesis and that plants present a higher proline responsiveness to hyperosmotic stress when this regulator is abolished. We clearly demonstrate that PLD signaling for proline biosynthesis is similar to RD29A gene expression and different from the abscisic acid-dependent RAB18 gene expression. Our data reveal that PLDs play positive and negative roles in hyperosmotic stress signal transduction in plants, contributing to a precise regulation of ion homeostasis and plant salt tolerance. Accumulation of proline has been observed in a large number of plant species in response to drought and salt stresses, suggesting a key role of this amino acid in plant stress adaptation. Upstream components of the proline biosynthesis signal transduction pathways are still poorly defined. We provide experimental evidence that phospholipase D (PLD) is involved in the regulation of proline metabolism in Arabidopsis thaliana. The application of primary butyl alcohols, which divert part of PLD-derived phosphatidic acid by transphosphatidylation, stimulated proline biosynthesis even without hyperosmotic constraints. Moreover, application of primary butyl alcohols enhanced the proline responsiveness of seedlings to mild hyperosmotic stress. These data indicate that some PLDs are negative regulators of proline biosynthesis and that plants present a higher proline responsiveness to hyperosmotic stress when this regulator is abolished. We clearly demonstrate that PLD signaling for proline biosynthesis is similar to RD29A gene expression and different from the abscisic acid-dependent RAB18 gene expression. Our data reveal that PLDs play positive and negative roles in hyperosmotic stress signal transduction in plants, contributing to a precise regulation of ion homeostasis and plant salt tolerance. Accumulating evidence suggests a major role for phospholipids to serve as precursors for the generation of secondary messengers in animal and plant cell transduction pathways. Recent reports have indicated that phospholipase D (PLD) 1The abbreviations used are: PLD, phospholipase D; ABA, abscisic acid; PA, phosphatidic acid; PBut, phosphatidylbutanol; P5C, Δ1-pyrroline-5-carboxylate; P5CS, Δ1-pyrroline-5-carboxylate synthetase; ProDH, proline dehydrogenase; OAT, ornithine-δ-aminotransferase. is also involved in water stress signaling through the hydrolysis of phospholipids to generate phosphatidic acid (PA) and free head groups. Water deficit triggers PLD activity and promotes stomatal closing and more recently it was shown that PLD activation mediates ABA signal transduction cascades (1Jacob T. Ritchie S. Assmann S.M. Gilroy S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12192-12197Crossref PubMed Scopus (247) Google Scholar, 2Ritchie S. Gilroy S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2697-2702Crossref PubMed Scopus (172) Google Scholar, 3Gampala S.S. Hagenbeek D. Rock C.D. J. Biol. Chem. 2001; 276: 9855-9860Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 4Hallouin M. Ghelis T. Brault M. Bardat F. Cornel D. Miginiac E. Rona J.P. Sotta B. Jeannette E. Plant Physiol. 2002; 130: 265-272Crossref PubMed Scopus (39) Google Scholar). Twelve PLD genes were identified in the Arabidopsis genome and tentatively grouped into five classes based on sequence similarity and biochemical properties (5Quin C. Wang X. Plant Physiol. 2002; 128: 1057-1068Crossref PubMed Scopus (233) Google Scholar). Biochemical requirements of these different plant PLDs, particularly for Ca2+ concentration, pH, and substrate lipids, clearly showed distinct features, suggesting different metabolic and physiological functions in plants. Drought and high salinity are common environmental stresses that affect plant growth and crop productivity (6Boyer J.S. Science. 1982; 218: 443-448Crossref PubMed Scopus (2901) Google Scholar). They both produce osmotic stress by decreasing the chemical activity of water and leading to a loss of cell turgor. The presence of salt has an additional effect causing ion toxicity because high intracellular concentration of sodium is deleterious to cellular metabolism (7Serrano R. Gaxiola R. Crit. Rev. Plant Sci. 1994; 13: 121-138Crossref Scopus (198) Google Scholar). Plants are sessile organisms, and their survival in a changing environment requires rapid responses to adverse conditions. Water deficit begins with stress perception, which initiates signal transduction pathway(s) to trigger a complex set of adaptive responses. Both drought and salt stresses stimulate the accumulation of compatible solutes that include ions such as K+ or organic compounds such as sucrose, betaines, and proline in the cytosol as a protective mechanism. Accumulation of proline is a wide-spread plant response to environmental stresses, and proline is thought to play a role in the adaptive response (8Delauney J.A. Verma D.P.D. Plant J. 1993; 4: 215-223Crossref Scopus (1406) Google Scholar). The significance of proline accumulation in osmotic adjustment and tolerance is still a matter of debate, and recently, the proline metabolism intermediate pyrroline-5-carboxylate (P5C) was suggested to be highly toxic to the cell by either directly or indirectly triggering apoptosis (9Deuschle K. Funck D. Hellmann H. Daschner K. Binder S. Frommer W.B. Plant J. 2001; 27: 345-356Crossref PubMed Scopus (169) Google Scholar). Proline has also been proposed to serve as a scavenger of radicals, a transient storage of nitrogen, and a source of redox equivalent (10Hare P.D. Cress W.A. Van Staden J. J. Exp. Bot. 1999; 50: 413-434Google Scholar). Higher plants synthesize proline via two different pathways. The first pathway is from glutamate that is converted to P5C by Δ 1The abbreviations used are: PLD, phospholipase D; ABA, abscisic acid; PA, phosphatidic acid; PBut, phosphatidylbutanol; P5C, Δ1-pyrroline-5-carboxylate; P5CS, Δ1-pyrroline-5-carboxylate synthetase; ProDH, proline dehydrogenase; OAT, ornithine-δ-aminotransferase.-pyrroline-5-carboxylate synthetase (P5CS) catalyzing the rate-limiting step in proline biosynthesis upon osmotic stress conditions (11Szoke A. Miao G.H. Hong Z. Verma D.P. Plant Physiol. 1992; 99: 1642-1649Crossref PubMed Scopus (117) Google Scholar, 12Kavi Kishor P.B. Hong Z. Miao G.H. Hu C.A.A. Verma D.P.S. Plant Physiol. 1995; 108: 1387-1394Crossref PubMed Scopus (1081) Google Scholar). P5C is then reduced to proline by Δ 1The abbreviations used are: PLD, phospholipase D; ABA, abscisic acid; PA, phosphatidic acid; PBut, phosphatidylbutanol; P5C, Δ1-pyrroline-5-carboxylate; P5CS, Δ1-pyrroline-5-carboxylate synthetase; ProDH, proline dehydrogenase; OAT, ornithine-δ-aminotransferase.-pyrroline-5-carboxylate reductase. The second pathway is from ornithine that is transformed to P5C through δ-transamination by ornithine-δ-aminotransferase (OAT), and P5C is subsequently reduced to proline. Upon relief from hyperosmotic stress, proline is rapidly degraded by the sequential action of the mitochondrial enzymes proline dehydrogenase (ProDH) and P5C dehydrogenase. Genes encoding enzymes catalyzing proline biosynthesis and degradation have been cloned from various plant species. Strong positive correlation between P5CS transcript and proline levels have been abundantly reported (10Hare P.D. Cress W.A. Van Staden J. J. Exp. Bot. 1999; 50: 413-434Google Scholar) with a reciprocal transcriptional regulation between P5CS and ProDH genes upon and after hyperosmotic stress (13Verbruggen N. Hua X.J. May M. Van Montagu M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8787-8791Crossref PubMed Scopus (160) Google Scholar, 14Kiyosue T. Yoshiba Y. Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1996; 8: 1323-1335Crossref PubMed Scopus (309) Google Scholar, 15Peng Z. Lu Q. Verma D.P. Mol. Gen. Genet. 1996; 253: 334-341PubMed Google Scholar). Among factors involved in the regulation of proline metabolism, ABA and calcium were previously shown to play an important role (10Hare P.D. Cress W.A. Van Staden J. J. Exp. Bot. 1999; 50: 413-434Google Scholar). ABA-dependent and independent signaling cascades have been identified between the initial water stress signal and the expression of drought-responsive genes like genes involved in proline biosynthesis, RAB18 and RD29A (16Savouré A. Hua X.J. Bertauche N. Van Montagu M. Verbruggen N. Mol. Gen. Genet. 1997; 254: 104-109Crossref PubMed Scopus (148) Google Scholar, 17Yamaguchi-Shinozaki K. Shinozaki K. Mol. Gen. Genet. 1993; 236: 331-340Crossref PubMed Scopus (431) Google Scholar, 18Lang V. Palva E.T. Plant Mol. Biol. 1992; 20: 951-962Crossref PubMed Scopus (362) Google Scholar). The two pathways may interact and converge because genes like RD29A possess an ABRE-responsive complex mediating ABA regulation and a dehydration-responsive element that can be activated by osmotic stress but not by ABA (19Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1994; 6: 251-264Crossref PubMed Scopus (1571) Google Scholar). Calcium also plays a role as a signaling molecule in a number of responses to drought and salinity. Water and salt stresses have been shown to induce transient free Ca2+ cytosolic waves derived from either influx from the apoplastic space or release from internal stores. Using Arabidopsis plants expressing cytosolic aequorin, calcium was shown to be necessary but not sufficient for drought and salt induction of P5CS, indicating that an additional signaling factor is involved (20Knight H. Trewavas A.J. Knight M.R. Plant J. 1997; 12: 1067-1078Crossref PubMed Scopus (727) Google Scholar). Despite the importance of the proline accumulation in the adaptive response of plants to osmotic stress and in contrast to metabolic events involved in proline accumulation, the signaling cascades regulating proline metabolism are still poorly characterized. In this report, we describe how PLD negatively regulates the proline biosynthesis pathway in Arabidopsis. We also show that calcium is involved in proline biosynthesis during osmotic stress and provide molecular evidence that this signaling cascade is different from the ABA-dependent pathway. Plant Material—Arabidopsis thaliana (L.) Heynh. ecotype Columbia seeds were surface-sterilized and grown on 0.5× Murashige-Skoog (MS) agar medium (21Murashige T. Skoog F. Physiol. Plant. 1962; 15: 473-497Crossref Scopus (54339) Google Scholar) in 14-cm-diameter Petri dishes as previously described (22Verbruggen N. Villarroel R. Van Montagu M. Plant Physiol. 1993; 103: 771-781Crossref PubMed Scopus (167) Google Scholar). After an overnight period at 4 °C to raise dormancy, the seedlings were placed under continuous illumination with 45 μmol of photons·m–2·s–1 at 22 °C for 12 days. Hyperosmotic Stress and Inhibitory Treatments—Twelve-day-old seedlings were removed from 0.5× MS agar plates and put onto a liquid 0.5× MS medium supplemented with different butanol isomers, verapamil, or EGTA or with water as control for 1 h. Then an equal volume of water, NaCl (200 mm), or mannitol (400 mm) was added to the medium. When plants were treated with 1-butanol (0.5% v/v) and calcium (10 or 50 mm) or with both 1-butanol (0.5% v/v) and potassium (10 or 50 mm), no pretreatment was made. After different incubation times, the seedlings were collected and immediately frozen in liquid nitrogen and stored at –80 °C prior to analysis. In Vivo Phospholipase D Activity—Phospholipids were metabolically labeled by incubating 12-day-old seedlings for 24 h in [33P]orthophosphate (53 MBq·liter–1). Primary butyl alcohols were then added to the medium supplemented with NaCl or mannitol to measure PLD activity by transphosphatidylation through phosphatidylbutanol (PBut) formation. The incubations were stopped by immersing seedlings in boiling water for 5 min. The lipids were then extracted and separated as described by Refs. 4Hallouin M. Ghelis T. Brault M. Bardat F. Cornel D. Miginiac E. Rona J.P. Sotta B. Jeannette E. Plant Physiol. 2002; 130: 265-272Crossref PubMed Scopus (39) Google Scholar and 23Munnik T. Arisz S.A. de Vrije T. Musgrave A. Plant Cell. 1995; 7: 2197-2210Crossref PubMed Scopus (230) Google Scholar, respectively. Radiolabeled PA and PBut were quantified using a PhosphorImager (Amersham Biosciences). PLD activity is presented as the formation of [33P]PBut counts, which is expressed as the percentage compared with nonstressed control plants. The means were compared using analyses of variance followed by protected t tests with the LSMEANS instruction of the SAS GLM procedure. Northern Blot Analysis—Total RNAs were isolated from seedlings harvested in liquid nitrogen by the guanidinium thiocyanate-CsCl purification method (24Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: 4.2.1-4.2.6Google Scholar). RNA samples were denatured and separated by electrophoresis in a 1.2% agarose-formaldehyde gel. After transfer to nylon membrane, the RNAs were fixed by UV cross-linking. The membranes were hybridized at 65 °C with either specific 3′-untranslated regions of AtP5CS1, AtP5CS2, or RD29A or with full length of At-δOAT, ProDH, or RAB18 according to Ref. 25Church G. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1991-1995Crossref PubMed Scopus (7266) Google Scholar. The fragments were labeled with [32P]dCTP using Ready-To-Go™ DNA labeling beads (Amersham Biosciences). Before hybridization, the membranes were stained with methylene blue as a control for RNA loading. The hybridization signals were quantified using a PhosphorImager (Amersham Biosciences). Gel Electrophoresis, Electroblotting, and Immunological Detection— The proteins were extracted as described (26Martinez-Garcia J.F. Monte E. Quail P.H. Plant J. 1999; 20: 251-257Crossref PubMed Google Scholar), separated by SDS-PAGE (27Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar), and transferred electrophoretically to a nitrocellulose filter in a solution of 48 mm Tris, 39 mm glycine, 0.04% (w/v) SDS, and 20% (v/v) methanol at 50 mA for 1 h. For immunodetection, the nitrocellulose filter was incubated in TBS (TBS-T) with 5% nonfat dry milk and 0.05% (v/v) Tween 20 for 1 h at room temperature and then in TBS-T with 0.1% (v/v) rabbit antiserum for 16 h at room temperature. The antisera were obtained by immunization of rabbits with the following proteins: AtP5CS1 (amino acids 5–717), AtProDH (amino acids 1–522), and AtδOAT (amino acids 52–474). The blots were washed with TBS-T. Detection was performed with an ECL assay using horseradish peroxidase-conjugated antibodies (Amersham Biosciences). Equal protein loading and integrity of protein samples were verified by Ponceau S red staining of the blot membrane. Proline Determination—Free proline content was measured using l-proline as standard according to Ref. 28Bates L.S. Waldren R.P. Teare I.D. Plant Soil. 1973; 39: 205-207Crossref Scopus (13285) Google Scholar. Primary Butanol Triggers Proline Accumulation—Recently, PLDs were shown to be involved in drought signaling (29Munnik T. Meijer H.J. Ter Riet B. Hirt H. Frank W. Bartels D. Musgrave A. Plant J. 2000; 22: 147-154Crossref PubMed Google Scholar, 30Katagiri T. Takahashi S. Shinozaki K. Plant J. 2001; 26: 595-605Crossref PubMed Google Scholar), reinforcing the need to investigate their putative role in the regulation of proline metabolism. PLD readily transfers the phosphatidyl moiety of a phospholipid to a primary alcohol rather than water producing PBut at the expense of PA, and because PBut is an inactive lipid formed, butanol treatment will inhibit PA signaling from PLD. Using this tool, we investigated the implication of PLDs in water stress-induced proline accumulation in A. thaliana. In response to 24-h treatments with either 200 mm NaCl or 400 mm mannitol, proline was accumulated to 2.5-fold compared with control in A. thaliana seedlings (Fig. 1A). Interestingly, the proline level was 2-fold increased in 1-butanol-treated plants without osmoticum (Fig. 1A). sec-Butanol, an activator of G protein activity (23Munnik T. Arisz S.A. de Vrije T. Musgrave A. Plant Cell. 1995; 7: 2197-2210Crossref PubMed Scopus (230) Google Scholar), and ter-butanol, an inactive analogue of 1- and 2-butanol (23Munnik T. Arisz S.A. de Vrije T. Musgrave A. Plant Cell. 1995; 7: 2197-2210Crossref PubMed Scopus (230) Google Scholar), did not have any effect on proline accumulation (Fig. 1A). To analyze the biological function of PLD in Arabidopsis, PLD activity was measured by transphosphatidylation reactions in the presence of primary butyl alcohols. The seedlings were incubated with 33P for 24 h to label structural phospholipids and subsequently treated with NaCl or mannitol. Lipids were then extracted and separated by TLC. Because PBut was formed after the addition of 1-butanol, the amount of PBut reflects a cumulative PLD activity under each treatment. The PA level was higher in seedlings upon hyperosmotic stresses compared with control plants, suggesting a higher PLD activity in stress conditions (Fig. 2). When 1-butanol was added, PBut was formed at the expense of PA upon normal growth as well as in hyperosmotic stress conditions. A lower PLD activity was present in seedlings grown in control medium than with NaCl and mannitol because PBut levels were increased by 2.5- and 1.5-fold, respectively, compared with control. In contrast, the increase in PBut formation was not accompanied by a significant decrease in PA formation, suggesting a low impact of the activated PLDs to this PA pool. The fact that 1-butanol stimulated proline accumulation could result from an activation of PLD by 1-butanol or through a diminution of PA signaling from PLDs. Therefore, the effect of different concentrations of 1-butanol was analyzed on proline and PBut levels (Fig. 3). Accumulation of proline increased in a dose-dependent manner in the presence of low 1-butanol concentrations with a maximum at 0.5%. Interestingly 1-butanol concentration above 0.75% diminished proline to lower levels than control. This result contrasted with PBut levels that strongly increased with 1-butanol concentrations above 0.5%, suggesting a stimulation of PLDs by primary alcohol as already reported by 23. We concluded, therefore, that stimulation of proline by 1-butanol was not due to an increase in PLD activity but rather to inhibition of a negative regulation by specific PLDs.Fig. 2In vivo PLD activity in Arabidopsis seedlings in response to various treatments. The seedlings were labeled with 33P for 24 h and then treated with or without (control) 200 mm NaCl or 400 mm mannitol in the presence or absence of 0.5% 1-butanol. The lipids were extracted, separated by TLC, and quantified by phosphorimaging as described under “Experimental Procedures.” A, representative autoradiography from a TLC plate after separation of phospholipids from seedlings treated with or without NaCl or mannitol. The position of PBut, PA, and structural phospholipids are indicated. B, quantification of PA and PBut levels by PhosphorImager. The values are the means of four independent treatments. The results for the PA and PBut are expressed as percentages with respect to nonstressed control seedlings. Based on variance analyses, NaCl and mannitol stresses had a significant effect on PA and PBut levels compared with control (p < 0.05), although 1-butanol did not affect PA level compared with the corresponding control, whatever the treatment (p < 0.05). 1-But, 1-butanol; SPL, structural phospholipids.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Proline and PBut formation are stimulated by 1-butanol in a different dose-dependent manner. The seedlings were prepared as described in the legend to Fig. 2. The proline (A) and PBut (B) levels were measured in seedlings treated with different concentrations of 1-butanol for 24 h. The results are shown as the means ± S.E. of three independent experiments. FW, fresh weight; 1-But, 1-butanol; AU, arbitrary units.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Molecular Analysis of Hyperosmotic Stress-responsive Genes—Next we investigated the effect of 1-butanol on three markers of proline biosynthesis, namely AtP5CS1, AtP5CS2, and OAT, and one marker of proline catabolism, ProDH. AtP5CS1, OAT, and ProDH transcript levels were increased by hyperosmotic treatments with different kinetics (Fig. 1B). AtP5CS1 transcript level was higher 3 h after treatment with either 200 mm NaCl or 400 mm mannitol than it was after 24 h. In contrast, OAT transcript level peaked at 24 h, whatever the treatment. Interestingly, we also observed a significant increased of ProDH RNA level after a 24-h NaCl treatment and to a lesser extent after a 24-h mannitol treatment. Under the same experimental conditions, the AtP5CS2 transcript level did not show any variation, whatever the treatment (data not shown). Application of 1-butanol without osmoticum increased AtP5CS1transcript levels by 1.5-fold after 3 h and over 3-fold after 24 h (Fig. 1B). In NaCl- or mannitol-treated plants, 1-butanol increased AtP5CS1 and OAT transcript levels at 24 h. A slight decrease in P5CS1 transcript level was always observed at 3 h in the presence of 1-butanol, indicating that other PLDs may be partially involved in short time responses. In contrast to NaCl, 1-butanol alone or together with mannitol triggered ProDH transcript accumulation at 24 h. We also investigated protein levels of P5CS, OAT, and ProDH using specific antibodies. P5CS accumulated to the same extent at 24 h of treatment with NaCl or mannitol. 1-Butanol alone triggered P5CS accumulation, although it had no clear effect on P5CS level in NaCl- or mannitol-treated plants. No significant accumulation of ProDH proteins was observed after a 24-h NaCl or mannitol treatment. Two proteins detected by the ProDH antibody were probably reflecting mature and immature forms of the enzyme that localized in mitochondria (14Kiyosue T. Yoshiba Y. Yamaguchi-Shinozaki K. Shinozaki K. Plant Cell. 1996; 8: 1323-1335Crossref PubMed Scopus (309) Google Scholar). However, accumulation of ProDH was observed at 24 h with 1-butanol alone or with mannitol. OAT proteins remained unchanged to a low level, whatever the treatment (data not shown). In contrast to ProDH and OAT, a positive correlation was observed between P5CS transcript and protein levels. sec- and ter-butanol did not show any effect on protein and transcript accumulation, as compared with control (data not shown), suggesting the implication of a PLD in negative regulation of proline biosynthesis in normal conditions. However, the inhibition of PA biosynthesis from PLD is apparently not sufficient to activate proline biosynthesis to the level observed during water stress. The effect of PLD on proline metabolism prompted us to investigate its role on the ABA-dependent RAB18 and ABA-independent RD29A-regulated genes. The transcript level of RAB18 was increased by NaCl and mannitol treatments at a higher level after 3 h and diminished when 1-butanol was added (Fig. 1B). Interestingly, PLD also negatively regulated RD29A, because its transcript level closely matched that of P5CS1, whatever the treatment. Proline Biosynthesis Is Mediated by Calcium—Several reports provided evidence pointing to calcium as an important secondary messenger leading to proline accumulation (for review see Ref. 10Hare P.D. Cress W.A. Van Staden J. J. Exp. Bot. 1999; 50: 413-434Google Scholar). We tested here the effect of verapamil, a phenylalkylamine-type calcium channel inhibitor that predominantly blocks L-type calcium channels (31White P.J. J. Exp. Bot. 1996; 47: 713-716Crossref Scopus (25) Google Scholar), and EGTA, an impermeant calcium chelator, on proline accumulation upon osmotic stress. A strong inhibition of proline accumulation was observed upon treatment of seedlings with 1 mm verapamil in the presence of 200 mm NaCl and to a lesser extent in the presence of 400 mm mannitol (Fig. 4). In NaCl-treated plants, EGTA completely inhibited proline accumulation, whereas the proline level was only reduced by 50% after mannitol treatment. These results show that calcium is a key component for the biosynthesis of proline in plants after a NaCl treatment and to a lesser extent after a mannitol treatment. Primary Butanol Enhanced Proline Responsiveness of Seedlings to Low Hyperosmotic Stress—Because 1-butanol stimulated proline accumulation, we tested whether calcium was another factor involved in proline biosynthesis (20Knight H. Trewavas A.J. Knight M.R. Plant J. 1997; 12: 1067-1078Crossref PubMed Scopus (727) Google Scholar). Calcium alone did not have any effect on proline level at 50 mm (Fig. 5A). Interestingly, the addition of both CaCl2 50 mm with 1-butanol increased the proline content to 2.5 μmol·g–1 fresh weight, which corresponds to the levels observed in NaCl- or mannitol-stressed plants. Like CaCl2, 50 mm KCl alone did not have any impact on proline levels (Fig. 5A). Interestingly, the application of both 10 mm KCl and 1-butanol increased proline levels 2-fold, as was observed with 1-butanol alone. Treatment of seedlings with both 50 mm KCl and 1-butanol caused the same induction of proline biosynthesis as did NaCl or mannitol treatments (2.5 μmol·g–1 fresh weight). These results indicate that the observed increase of proline level is probably due to an osmotic effect and that calcium alone is not a limiting factor for the induction of proline synthesis upon the inhibition of PLD by 1-butanol. 50 mm CaCl2 or KCl did not have any measurable effect on P5CS level (Fig. 5B). However, the addition of 1-butanol together with CaCl2 dramatically increased the P5CS level, this effect being specific and dependent on the dose of calcium, because the application of KCl, even at 50 mm, with 1-butanol did not have any effect on P5CS. However, this increase of P5CS was not correlated with the corresponding accumulation of proline. Interestingly, the ProDH levels decreased when concentrations of CaCl2 or KCl were raised to 50 mm, even with butanol-1. The application of calcium stimulated the increase of AtP5CS1 transcript levels (Fig. 5C) as was already shown in Ref. 20Knight H. Trewavas A.J. Knight M.R. Plant J. 1997; 12: 1067-1078Crossref PubMed Scopus (727) Google Scholar. AtP5CS1 and ProDH accumulation is correlated with their corresponding protein levels in plants treated with both 1-butanol and calcium. When seedlings were treated with both 1-butanol and 50 mm CaCl2, a strong increase of OAT and RD29A transcript levels were detected at 24 h (Fig. 5C), whereas these treatments did not have any effect on RAB18 transcript levels. However, OAT levels were not affected by any treatment with butanol isomers (data not shown). Taken together, these data illustrate fine regulation of proline metabolism by independent negative and positive regulators involving calcium and PLD. Recently, several reports suggested the implication of phospholipids in the mediation of plant responses to environmental stresses, especially drought and salinity. Several PLD genes have been isolated from various plant species, and their molecular characterization has shown their role in early signaling events. In this paper, we have established that PLDs during osmotic stress play a role as negative regulators of proline biosynthesis in A. thaliana. To our knowledge, this is the first case of the implication of PLDs as negative regulators of gene expression in plants. In response to hyperosmotic stress triggered by NaCl, mannitol, or sucrose, an increase of PA level and its conversion to diacylglycerol pyrophosphate has been reported in plants (29Munnik T. Meijer H.J. Ter Riet B. Hirt H. Frank W. Bartels D. Musgrave A. Plant J. 2000; 22: 147-154Crossref PubMed Google Scholar). This PA may come from phospholipase C and/or PLD activities. PLC generates diacylglycerol and indirectly PA because of diacylglycerol kinase activity. However, a recent work demonstrated that PA mainly derives from AtPLDδ activity during dehydration because AtPLDδ antisense transgenic plants are affected in this PA accumulation in response to water stress (30Katagiri T. Takahashi S. Shinozaki K. Plant J. 2001; 26: 595-605Crossref PubMed Google Scholar). PLDα was also shown to play a crucial role in regulating transpiration water loss. Arabidopsis depleted in PLDα are impaired in stomatal closure and show reduced response to ABA (32Sang Y. Zheng S. Li W. Huang B. Wang X. Plant J. 2001; 28: 135-144Crossref PubMed Scopus (142) Google Scholar). Hyperosmotic stress and dehydration stimulated PLD activity in tomato, alfalfa, and resurrection plant Craterostigma plantagineum (29Munnik T. Meijer H.J. Ter Riet B. Hirt H. Frank W. Bartels D. Musgrave A. Plant" @default.
• W1978161788 created "2016-06-24" @default.
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• W1978161788 date "2004-04-01" @default.
• W1978161788 modified "2023-10-18" @default.
• W1978161788 title "Phospholipase D Is a Negative Regulator of Proline Biosynthesis in Arabidopsis thaliana" @default.
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