Plant Molecular Biology

, Volume 38, Issue 5, pp 755–764 | Cite as

Isolation and characterization of two different cDNAs of Δ1-pyrroline-5-carboxylate synthase in alfalfa, transcriptionally induced upon salt stress

  • Idit Ginzberg
  • Hanan Stein
  • Yoram Kapulnik
  • Laszlo Szabados
  • Nicolai Strizhov
  • Jeff Schell
  • Csaba Koncz
  • Aviah Zilberstein


Two different cDNA clones, MsP5CS-1 and MsP5CS-2, encoding Δ1-pyrroline-5-carboxylate synthase (P5CS), the first enzyme of the proline biosynthetic pathway, were isolated from a λZap-cDNA library constructed from salt stressed Medicago sativa roots. MsP5CS-1 (2.6 kb) has an open reading frame of 717 amino acids, as well as a non-spliced intron at a position corresponding to the evolutionary fusion point of the bacterial proA and proB genes. MsP5CS-2 (1.25 kb) is a partial clone. The clones share 65% identity in nucleotide sequences, 74% homology in deduced amino acid sequences, and both show a high similarity to Vigna aconitifolia and Arabidopsis thaliana P5CS cDNA clones. Southern blot analysis confirmed the presence of two different P5CS genes. The effect of salinity on the transcription of MsP5CS-1 and MsP5CS-2 in roots was studied, using northern blot analysis and a RT-PCR approach. A rapid increase in the steady-state transcript level of both genes in roots was observed by RT-PCR upon exposure of hydroponically grown 6-day old seedlings to 90 mM NaCl, suggesting that both are salt-inducible genes, yet a higher response was observed for MsP5CS-2.

Medicago sativa proline pyrroline-5-carboxylate synthase salt stress 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Aspinall D, Paleg LG: Proline accumulation: physiological aspects. In: Pleg LG, Aspinall D (eds) Physiology and Biochemistry of Drought Resistance in Plants, pp. 243–259. Academic Press, Sydney (1981).Google Scholar
  2. 2.
    Bekki A, Trinchant JC, Rigaud J: Nitrogen fixation (C2H2 reduction) by Medicago nodules and bacteroids under sodium chloride stress. Physiol Plant 71: 61–67 (1987).Google Scholar
  3. 3.
    Brown JWS: Arabidopsis intron mutations and pre-mRNA splicing. Plant J 10: 771–780 (1996).Google Scholar
  4. 4.
    Chee PP, Drong RP, Slighton JL: Using polymerase chain reaction to identify transgenic plants. In: Gelvin SB, Schilperoort RA (eds), Plant Molecular Biology Manual C3, pp. 1–28. Kluwer Academic Publishers, Dordrecht (1991).Google Scholar
  5. 5.
    Delauney AJ, Verma DPS: Proline biosynthesis and osmoregulation in plants. Plant J 4: 215–223 (1993).Google Scholar
  6. 6.
    Delauney AJ, Hu CA, Kishor PBK, Verma DPS: Cloning of ornithine δ-aminotransferase cDNA from Vigna aconitifolia by trans-complementation in Escherichia coli and regulation of proline biosynthesis. J Biol Chem 268: 18673–18678 (1993).Google Scholar
  7. 7.
    Fougère F, Le Rudulier D, Streeter GJ: Effects of salt stress on amino acid, organic acid, and carbohydrate composition of roots, bacteroids, and cytosol of alfalfa (Medicago sativa L.). Plant Physiol 96: 1228–1236 (1991).Google Scholar
  8. 8.
    Girousse C, Bournoville R, Bonnemain J-L: Water deficitinduced changes in concentration in proline and some other amino acids in the phloem sap of alfalfa. Plant Physiol 111: 109–113 (1996).Google Scholar
  9. 9.
    Hanson AD, Hitz WD: Metabolic responses of mesophytes to plant water deficits. Annu Rev Plant Physiol 33: 163–203 (1982).Google Scholar
  10. 10.
    Hu CA, Delauney AJ, Verma DPS: A bifunctional enzyme (Δ1-pyrroline-5-carboxylate synthase) catalyzes the first two steps in proline biosynthesis in plants. Proc Natl Acad Sci USA 89: 9354–9358 (1992).Google Scholar
  11. 11.
    Irigoyen JJ, Emerich DW, Sanchez-Diaz: Water stress induced changes in concentrations of proline and total soluble sugars in nodulated alfalfa (Medicago sativa) plants. Physiol Plant 84: 55–60 (1992).Google Scholar
  12. 12.
    Johnson CM, Stout PR, Beyer JC, Carlson AB: Comparative chlorine requirement of different species. Plant Soil 8: 337–345 (1957).Google Scholar
  13. 13.
    Kapulnik Y, Heuer B: Forage production of four alfalfa (Medicago sativa) cultivars under salinity. Arid Soil Res Rehabil 5: 127–135 (1991).Google Scholar
  14. 14.
    Kimura N, Kimura N, Cathala G, Baxter JD, Johnson GS: Nicotinamide and its derivatives increase growth hormone and prolactin synthase in cultured GH3 cells; role of ADPribosylation in modulating specific gene expression. DNA 2: 195–203 (1983).Google Scholar
  15. 15.
    Kishor PBK, Hong Z, Miao GH, Hu CAA, Verma DPS: Overexpression of Δ1-pyrroline-5-carboxylate synthase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol 108: 1386–1394 (1995).Google Scholar
  16. 16.
    Kiyosue T, Yoshiba Y, Yamaguchi-Shinozaki K, Shinozaki K: A nuclear gene encoding mitochondrial proline dehydrogenase, an enzyme involved in proline metabolism, is upregulated by proline but downregulated by dehydration in Arabidopsis. Plant Cell 8: 1323–1335 (1996).Google Scholar
  17. 17.
    Leisinger T: Biosynthesis of proline. In: Neidhardt FC, Ingraham JL, Low KB, Magasanik B, Schaechter M, Umbarger HE (eds) Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, pp. 346–351. American Society for Microbiology, Washington, DC (1987).Google Scholar
  18. 18.
    Meyer K, Leube MP, Grill E: A protein phosphatase 2C involved in ABA signal transduction in Arabidopsis thaliana. Science 264: 1452–1455 (1994).Google Scholar
  19. 19.
    Rayapati PJ, Stewart CR: Solubilization of a proline dehydrogenase from maize (Zea mays L.) mitochondria. Plant Physiol 95: 787–791 (1991).Google Scholar
  20. 20.
    Rentsch D, Hirner B, Schmelzer E, Frommer WB: Salt stressinduced proline transporters and salt stress-repressed broad specificity amino acid permeases identified by suppression of a yeast amino acid permease-targeting mutant. Plant Cell 8: 1437–1446 (1996).Google Scholar
  21. 21.
    Rhodes D, Handa S, Bressan RA: Metabolic changes associated with adaptation of plant cells to water stress. Plant Physiol 82: 890–903 (1986).Google Scholar
  22. 22.
    Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning, a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)Google Scholar
  23. 23.
    Savouré A, Jaoua S, Hua XJ, Ardiles W, Van Montagu M, Verbruggen N: Isolation, characterization, and chromosomal location of a gene encoding the Δ1-pyrroline-5-carboxylate synthase in Arabidopsis thaliana. FEBS Lett 372: 13–19 (1995).Google Scholar
  24. 24.
    Simpson GG, Filipowicz, W: Splicing of precursors to mRNA in higher plants: mechanism, regulation and sub-nuclear organization of the spliceosomal machinery. Plant Mol Biol 32: 1–41 (1996).Google Scholar
  25. 25.
    Stewart CR, Boggess SF, Aspinall D, Paleg LG: Inhibition of proline oxidation by water stress. Plant Physiol 59: 930–932 (1977).Google Scholar
  26. 26.
    Strizhov N, Abraham E, Okresz L, Zilberstein A, Koncz C, Szabados L: Two genes are coding for the Δ1-pyrroline-5-carboxylate synthase enzyme in Arabidopsis thaliana. Plant J 12: 557–569 (1997).Google Scholar
  27. 27.
    Szoke A, Miao GH, Hong Z, Verma DPS: Subcellular location of Δ1-pyrroline-5-carboxylate reductase in root/nodule and leaf of soybean. Plant Physiol 99: 1642–1649 (1992).Google Scholar
  28. 28.
    Verma DPS, Hu CAA, Delauney AJ, Miao GH, Hong Z: Deciphering proline biosynthesis pathways in plants by direct, trans-, and co-complementation in bacteria. In: Singh BK, Flores HE, Shannon JC (eds) Biosynthesis and Molecular Regulation of Amino Acids in Plants, vol 7, pp. 128–138. American Society of Plant Physiologists, Rockville, MD (1992).Google Scholar
  29. 29.
    Voetberg GS, Sharp RE: Growth of the maize primary root at low water potentials. III. Role of increased proline deposition in osmotic adjustment. Plant Physiol 96: 1125–1130 (1991).Google Scholar
  30. 30.
    Yoshiba Y, Kiyosue T, Katagiri T, Ueda H, Mizoguchi T, Yamaguchi-Shinozaki K, Harada Y, Shinozaki K: Correlation between the induction of a gene for Δ1-pyrroline-5-carboxylate synthase and the accumulation of proline in Arabidopsis thaliana under osmotic stress. Plant J 7: 751–760 (1995).Google Scholar
  31. 31.
    Zhang CS, Lu Q, Verma DPS: Removal of feedback inhibition of Δ1-pyrroline-5-carboxylate synthase, a bifunctional enzyme catalyzing the first two steps of proline biosynthesis in plants. J Biol Chem 270: 20491–20496 (1995).Google Scholar

Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • Idit Ginzberg
    • 1
  • Hanan Stein
    • 1
  • Yoram Kapulnik
    • 2
  • Laszlo Szabados
    • 3
  • Nicolai Strizhov
    • 4
  • Jeff Schell
    • 4
  • Csaba Koncz
    • 3
  • Aviah Zilberstein
    • 1
  1. 1.Department of BotanyTel-Aviv UniversityTel-AvivIsrael
  2. 2.Institute of Field and Garden Crops, The Volcani CenterBet DaganIsrael
  3. 3.Institute of Plant Biology, Biological Research CenterHungarian Academy of SciencesSzegedHungary
  4. 4.Max-Planck InstituteKölnGermany

Personalised recommendations