, Volume 222, Issue 1, pp 70–79 | Cite as

Responsive modes of Medicago sativa proline dehydrogenase genes during salt stress and recovery dictate free proline accumulation

  • Gadi Miller
  • Hanan Stein
  • Arik Honig
  • Yoram Kapulnik
  • Aviah ZilbersteinEmail author
Original Article


Free proline accumulation is an innate response of many plants to osmotic stress. To characterize transcriptional regulation of the key proline cycle enzymes in alfalfa (Medicago sativa), two proline dehydrogenase (MsPDH) genes and a partial sequence of Δ 1 -pyrroline-5-carboxylate dehydrogenase (MsP5CDH) gene were identified and cloned. The two MsPDH genes share a high nucleotide sequence homology and a similar exon/intron structure. Estimation of transcript levels during salt stress and recovery revealed that proline accumulation during stress was linearly correlated with a strong decline in MsPDH transcript levels, while Δ 1 -pyrroline-5-carboxylate synthetase (MsP5CS) and MsP5CDH steady-state transcript levels remained essentially unchanged. MsPDH transcript levels dramatically decreased in a fast, salt concentration-dependent manner. The extent of salt-induced proline accumulation also correlated with salt concentrations. Salt-induced repression of MsPDH1 promoter linked to the GUS reporter gene confirmed that the decline in MsPDH transcript levels was due to less transcription initiation. Contrary to the salt-dependent repression, a rapid induction of MsPDH transcription occurred at a very early stage of the recovery process, independently of earlier salt treatments. Hence our results suggest the existence of two different regulatory modes of MsPDH expression; the repressing mode that quantifies salt concentration in an as yet unknown mechanism and the ”rehydration”-enhancing mode that responds to stress relief in a maximal induction of MsPDH transcription. As yet the components of salt sensing as well as those that might interact with MsPDH promoter to reduce transcription are still unknown.


Medicago sativa Proline Proline dehydrogenase P5C dehydrogenase Salt stress 



Abscisic acid


ABA-responsive element


Proline dehydrogenase


P5C dehydrogenase


Δ 1 -Pyrroline-5-carboxylate synthetase




4-Methylumbelliferyl β-D-glucuronide


Δ 1 -Pyrroline-5-carboxylate




5-Bromo-4-chloro-3-indolyl glucuronide



We thank H. Eilenberg, Department of Plant Sciences, Tel Aviv University, for critically reading the manuscript. This study was supported by the European Commission Research grant (Contract OLK5-CT-2002-0084) and the Israeli Ministry of Agriculture, Chief Scientist grant (891-0155-01).


  1. Abraham E, Rigo G, Szekely G, Nagy R, Koncz C, Szabados L (2003) Light-dependent induction of proline biosynthesis by abscisic acid and salt stress is inhibited by brassinosteroid in Arabidopsis. Plant Mol Biol 51:363–372PubMedCrossRefGoogle Scholar
  2. Alia, Mohanty P, Matysik J (2001) Effect of proline on the production of singlet oxygen. Amino Acids 21:195–200PubMedCrossRefGoogle Scholar
  3. Armengaud P, Thiery L, Buhot N, Grenier-De March G, Savoure A (2004) Transcriptional regulation of proline biosynthesis in Medicago truncatula reveals developmental and environmental specific features. Physiol Plant 120:442–450PubMedCrossRefGoogle Scholar
  4. Bates LS, Waldren IP, Teare ID (1973) Rapid determination of free proline for water-stress studies. Plant Soil 39:205–207CrossRefGoogle Scholar
  5. Cai XL, Wang ZY, Xing YY, Zhang JL, Hong MM (1998) Aberrant splicing of intron 1 leads to the heterogeneous 5’ UTR and decreased expression of waxy gene in rice cultivars of intermediate amylose content. Plant J 14: 459–465PubMedCrossRefGoogle Scholar
  6. Chen TH, Murata N (2002) Enhancement of tolerance of abiotic stress by metabolic engineering of betaines and other compatible solutes. Curr Opin Plant Biol 5:250–257PubMedCrossRefGoogle Scholar
  7. Csonka LN (1989) Physiological and genetic responses of bacteria to osmotic stress. Microbiol Rev 53:121–147PubMedGoogle Scholar
  8. Delauney AJ, Verma DPS (1993) Proline biosynthesis and osmoregulation in plants. Plant J 4:215–223CrossRefGoogle Scholar
  9. Deruere J, Bouvier F, Steppuhn J, Klein A, Camara B, Kuntz M (1994) Structure and expression of two plant genes encoding chromoplast-specific proteins: occurrence of partially spliced transcripts. Biochem Biophys Res Commun 199:1144–1150PubMedCrossRefGoogle Scholar
  10. Deuschle K, Funck D, Hellmann H, Daschner K, Binder S, Frommer WB (2001) A nuclear gene encoding mitochondrial delta-pyrroline-5-carboxylate dehydrogenase and its potential role in protection from proline toxicity. Plant J 27:345–356PubMedCrossRefGoogle Scholar
  11. Fujita T, Maggio A, Garcia-Rios M, Bressan RA, Csonka LN (1998) Comparative analysis of the regulation of expression and structures of two evolutionarily divergent genes for delta1-pyrroline-5-carboxylate synthetase from tomato. Plant Physiol 118:661–674PubMedCrossRefGoogle Scholar
  12. Fujita T, Maggio A, Garcia-Rios M, Stauffacher C, Bressan RA, Csonka LN (2003) Identification of regions of the tomato gamma-glutamyl kinase that are involved in allosteric regulation by proline. J Biol Chem 278: 14203–14210PubMedCrossRefGoogle Scholar
  13. Ginzberg I, Stein H, Kapulnik Y, Szabados L, Strizhov N, Schell J, Koncz C, Zilberstein A (1998) Isolation and characterization of two different cDNAs of delta-1-pyrroline-5-carboxylate synthase in alfalfa, transcriptionally induced upon salt stress. Plant Mol Biol 38:755–764PubMedCrossRefGoogle Scholar
  14. Girousse C, Bournoville R, Bonnemain JL (1996) Water deficit-induced changes in concentrations in proline and some other amino acids in the phloem sap of alfalfa. Plant Physiol 111:109–113PubMedGoogle Scholar
  15. Hare PD, Cress WA (1997) Metabolic implications of stress-induced proline accumulation in plants. Plant Growth Reg 21:79–102CrossRefGoogle Scholar
  16. Hare PD, Cress WA, van Staden J (1999) Proline synthesis and degradation: a model system for elucidating stress-related signal transduction. J Exp Bot 50:413–434CrossRefGoogle Scholar
  17. Hua XJ, Van de Cotte B, Van Montagu M, Verbruggen N (2001) The 5’ untranslated region of the At-P5R gene is involved in both transcriptional and post-transcriptional regulation. Plant J 26: 157–169PubMedCrossRefGoogle Scholar
  18. Jefferson RA, Burgess SM, Hirsh D (1986) ß-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci USA 83: 8447–8451PubMedCrossRefGoogle Scholar
  19. Johnson CM, Stout, PR, Broyer TC, Carlton, AB (1957) Comparative chlorine requirement of different plant species. Plant Soil 4:337CrossRefGoogle Scholar
  20. Kiegerl S, Cardinale F, Siligan C, Gross A, Baudouin E, Liwosz A, Eklof S, Till S, Bogre L, Hirt H, Meskiene I (2000) SIMKK, a mitogen-activated protein kinase (MAPK) kinase, is a specific activator of the salt stress-induced MAPK, SIMK. Plant Cell 12:2247–2258PubMedCrossRefGoogle Scholar
  21. Kishor PBK, Hong ZL, Miao GH, Hu CAA, Verma DPS (1995) Overexpression of delta-pyrroline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol 108: 1387–1394PubMedGoogle Scholar
  22. Kiyosue T, Yoshiba Y, Yamaguchi-Shinozaki K, Shinozaki K (1996) 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: 1323–1335PubMedCrossRefGoogle Scholar
  23. Koncz C, Martini N, Mayerhofer R, Koncz-Kalman Z, Korber H, Redei GP, Schell J (1989) High-frequency T-DNA-mediated gene tagging in plants. Proc Natl Acad Sci USA 86:8467–8471PubMedCrossRefGoogle Scholar
  24. Maggio A, Miyazaki S, Veronese P, Fujita T, Ibeas JI, Damsz B, Narasimhan ML, Hasegawa PM, Joly RJ, Bressan RA (2002) Does proline accumulation play an active role in stress-induced growth reduction?. Plant J 31:699–712PubMedCrossRefGoogle Scholar
  25. Mani S, Van De Cotte B, Van Montagu M, Verbruggen N (2002) Altered levels of proline dehydrogenase cause hypersensitivity to proline and its analogs in Arabidopsis. Plant Physiol 128:73–83PubMedCrossRefGoogle Scholar
  26. Munnik T, Ligterink W, Meskiene I, Calderini O, Beyerly J, Musgrave A, Hirt H (1999) Distinct osmo-sensing protein kinase pathways are involved in signalling moderate and severe hyper-osmotic stress. Plant J 20: 381–388PubMedCrossRefGoogle Scholar
  27. Nakashima K, Satoh R, Kiyosue T, Yamaguchi-Shinozaki K, Shinozaki K (1998) A gene encoding proline dehydrogenase is not only induced by proline and hypoosmolarity, but is also developmentally regulated in the reproductive organs of Arabidopsis. Plant Physiol 118:1233–1241PubMedCrossRefGoogle Scholar
  28. Nanjo T, Fujita M, Seki M, Kato T, Tabata S, Shinozaki K (2003) Toxicity of free proline revealed in an Arabidopsis T-DNA-tagged mutant deficient in proline dehydrogenase. Plant Cell Physiol 44:541–548PubMedCrossRefGoogle Scholar
  29. Oono Y, Seki M, Nanjo T, Narusaka M, Fujita M, Satoh R, Satou M, Sakurai T, Ishida J, Akiyama K, Iida K, Maruyama K, Satoh S, Yamaguchi-Shinozaki K, Shinozaki K (2003) Monitoring expression profiles of Arabidopsis gene expression during rehydration process after dehydration using ca 7000 full-length cDNA microarray. Plant J 34: 868–887PubMedCrossRefGoogle Scholar
  30. Peng Z, Lu Q, Verma, DPS (1996) Reciprocal regulation of delta(1)-pyrroline-5-carboxylate synthetase and proline dehydrogenase genes controls proline levels during and after osmotic-stress in plants. Mol Gen Genet 253:334–341PubMedGoogle Scholar
  31. Rayapati PJ, Stewart CR (1991) Solubilization of a proline dehydrogenase from maize (Zea Mays L.) mitochondria. Plant Physiol 95:787–791PubMedCrossRefGoogle Scholar
  32. Rentsch D, Hirner B, Schmelzer E, Frommer WB (1996). Salt stress-induced proline transporters and salt stress-repressed broad specificity amino acid permeases identified by suppression of a yeast amino acid permease-targeting mutant. Plant Cell 8:1437–1446PubMedCrossRefGoogle Scholar
  33. Roosens NH, Willem R, Li Y, Verbruggen II, Biesemans M, Jacobs, M (1999) Proline metabolism in the wild-type and in a salt-tolerant mutant of Nicotiana plumbaginifolia studied by (13)C-nuclear magnetic resonance imaging. Plant Physiol 121:1281–1290PubMedCrossRefGoogle Scholar
  34. Russo AT, Rosgen J, Bolen DW (2003) Osmolyte effects on kinetics of FKBP12 C22A folding coupled with prolyl isomerization. J Mol Biol 330: 851–866PubMedCrossRefGoogle Scholar
  35. Satoh R, Nakashima K, Seki M, Shinozaki K, Yamaguchi-Shinozaki K (2002) ACTCAT, a novel cis-acting element for proline- and hypoosmolarity-responsive expression of the ProDH gene encoding proline dehydrogenase in Arabidopsis. Plant Physiol 130:709–719PubMedCrossRefGoogle Scholar
  36. Satoh R, Fujita Y, Nakashima K, Shinozaki K, Yamaguchi-Shinozaki, K. (2004) A novel subgroup of bZIP proteins functions as transcriptional activators in hypoosmolarity-responsive expression of the ProDH gene in Arabidopsis. Plant Cell Physiol 45:309–317PubMedCrossRefGoogle Scholar
  37. Strizhov N, Keller M, Mathur J, Koncz-Kalman Z, Bosch D, Prudovsky E, Schell J, Sneh B, Koncz C, Zilberstein A (1996) A synthetic cryIC gene, encoding a Bacillus thuringiensis delta-endotoxin, confers Spodoptera resistance in alfalfa and tobacco. Proc Natl Acad Sci USA 93:15012–15017PubMedCrossRefGoogle Scholar
  38. Strizhov N, Abraham E, Okresz L, Blickling S, Zilberstein A, Schell J, Koncz C, Szabados L (1997) Differential expression of two P5CS genes controlling proline accumulation during salt-stress requires ABA and is regulated by ABA1, ABI1 and AXR2 in Arabidopsis. Plant J 12:557–569PubMedCrossRefGoogle Scholar
  39. Szoke A, Miao GH, Hong ZL, Verma DPS (1992) Subcellular location of delta-1-pyrroline-5-carboxylate reductase in root nodule and leaf of soybean. Plant Physiol 99:1642–1649PubMedCrossRefGoogle Scholar
  40. Taniguchi M, Sugiyama T (1997) The expression of 2-oxoglutarate/malate translocator in the bundle-sheath mitochondria of Panicum miliaceum, a NAD-malic enzyme-type C4 plant, is regulated by light and development. Plant Physiol 114:285–293PubMedGoogle Scholar
  41. Ueda A, Shi W, Sanmiya K, Shono M, Takabe T (2001). Functional analysis of salt-inducible proline transporter of barley roots. Plant Cell Physiol 42:1282–1289PubMedCrossRefGoogle Scholar
  42. Vancanneyt G, Rosahl S, Willmitzer L (1990) Translatability of a plant-mRNA strongly influences its accumulation in transgenic plants. Nucleic Acids Res 18:2917–2921PubMedCrossRefGoogle Scholar
  43. Verbruggen N, Hua XJ, May M, Van Montagu M (1996) Environmental and developmental signals modulate proline homeostasis: evidence for a negative transcriptional regulator. Proc Natl Acad Sci USA 93: 8787–8791PubMedCrossRefGoogle Scholar
  44. Yoshiba Y, Kiyosue T, Katagiri T, Ueda H, Mizoguchi T, Yamaguchishinozaki K, Wada K, Harada Y, Shinozaki, K (1995) Correlation between the induction of a gene for delta(1)-pyrroline-5-carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana under osmotic stress. Plant J 7:751–760PubMedCrossRefGoogle Scholar
  45. Yoshiba Y, Kiyosue T, Nakashima K, Yamaguchi-Shinozaki K, Shinozaki K (1997) Regulation of levels of proline as an osmolyte in plants under water stress. Plant Cell Physiol 38:1095–1102PubMedGoogle Scholar
  46. Zhang CS, Lu Q, Verma DPS (1995) Removal of feedback inhibition of delta(1)-pyrroline-5-carboxylate synthetase, a bifunctional enzyme catalyzing the first 2 steps of proline biosynthesis in plants. J Biol Chem 270:20491–20496PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Gadi Miller
    • 1
  • Hanan Stein
    • 1
  • Arik Honig
    • 1
  • Yoram Kapulnik
    • 2
  • Aviah Zilberstein
    • 1
    Email author
  1. 1.Department of Plant ScienceTel Aviv UniversityTel-AvivIsrael
  2. 2.Institute of Field and Garden CropsThe Volcani CenterBeit DaganIsrael

Personalised recommendations