Skip to main content
SpringerLink
Log in

Methods for Determination of Proline in Plants

  • Protocol
  • First Online:
Book cover Plant Stress Tolerance

Part of the book series: Methods in Molecular Biology ((MIMB,volume 639))

Abstract

Accumulation of proline in higher plants is an indication of disturbed physiological condition, triggered by biotic or abiotic stress condition. Free proline content can increase upon exposure of plants to drought, salinity, cold, heavy metals, or certain pathogens. Determination of free proline levels is a useful assay to monitor physiological status and to assess stress tolerance of higher plants. Here we describe three methods suitable for determination of free proline content. The isatin paper assay is simple and is suitable to assay proline content in large number of samples. The colorimetric measurement is quantitative and provides reliable data about proline content. The HPLC-based amino acid analysis can be employed when concentration of all amino acids has to be compared.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Kemble, A.R. and MacPherson, H.T. (1954) Liberation of amino acids in perennial ray grass during wilting. Biochem J 58, 46–59.

    PubMed  CAS  Google Scholar 

  2. Rhodes, D., Handa, S., and Bressan, R.A. (1986) Metabolic changes associated with adaptation of plant cells to water stress. Plant Physiol 82, 890–903.

    Article  PubMed  CAS  Google Scholar 

  3. Delauney, A.J. and Verma, D.P.S. (1993) Proline biosynthesis and osmoregulation in plants. Plant J 4, 215–223.

    Article  CAS  Google Scholar 

  4. Kavi Kishor, P.B., Sangam, S., Amrutha, R.N., Laxmi, P.S., Naidu, K.R., Rao, K.R.S.S., Rao, S., Reddy, K.J., Theriappan, P., and Sreenivasulu, N. (2005) Regulation of proline biosynthesis, degradation, uptake and transport in higher plants: its implications in plant growth and abiotic stress tolerance. Curr Sci 88, 424–438.

    Google Scholar 

  5. Verbruggen, N. and Hermans, C. (2008) Proline accumulation in plants: a review. Amino Acids 35, 753–759.

    Article  PubMed  CAS  Google Scholar 

  6. Ahmad, I., Wainwright, S.J., and Stewart, G.R. (1981) The solute and water relations of Agrostis stolonifera ecotypes differing in their salt tolerance. New Phytol 87, 615–629.

    Article  CAS  Google Scholar 

  7. Fougere, F., Le Rudulier, D., and Streeter, J.G. (1991) 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.

    Article  PubMed  CAS  Google Scholar 

  8. Armengaud, P., Thiery, L., Buhot, N., Grenier-De March, G., and Savouré, A. (2004) Transcriptional regulation of proline biosynthesis in Medicago truncatula reveals developmental and environmental specific features. Physiol Plant 120, 442–450.

    Article  PubMed  CAS  Google Scholar 

  9. Newton, R.J., Sen, S., and Puryear, J.D. (1986) Free proline changes in Pinus taeda L. callus in response to drought stress. Tree Physiol 1, 325–332.

    PubMed  CAS  Google Scholar 

  10. Yamada, M., Morishita, H., Urano, K., Shiozaki, N., Yamaguchi-Shinozaki, K., Shinozaki, K., and Yoshiba, Y. (2005) Effects of free proline accumulation in petunias under drought stress. J Exp Bot 56, 1975–1981.

    Article  PubMed  CAS  Google Scholar 

  11. Huang, A.H. and Cavalieri, A.J. (1979) Proline oxidase and water stress-induced proline accumulation in spinach leaves. Plant Physiol 63, 531–535.

    Article  PubMed  CAS  Google Scholar 

  12. Mehta, S.K. and Gaur, J.P. (1999) Heavy-metal-induced proline accumulation and its role in ameliorating metal toxicity in Chlorella vulgaris. New Phytol 143, 253–259.

    Article  CAS  Google Scholar 

  13. Chen, C.T., Chen, L., Lin, C.C., and Kao, C.H. (2001) Regulation of proline accumulation in detached rice leaves exposed to excess copper. Plant Sci 160, 283–290.

    Article  PubMed  CAS  Google Scholar 

  14. Draper, S.R. (1972) Amino acid changes associated with low temperature treatment of Lolium perenne. Phytochemistry 11, 639–641.

    Article  CAS  Google Scholar 

  15. Naidu, B.P., Paleg, L.G., Aspinall, D., Jennings, A.C., and Jones, G.P. (1991) Amino acid and glycine betaine accumulation in cold-stressed wheat seedlings. Phytochemistry, 30, 407–409.

    Article  CAS  Google Scholar 

  16. Kuo, C.G. and Chen, B.W. (1980) Physiological responses of tomato cultivars to flooding. J Am Soc Hort Sci 105, 751–755.

    CAS  Google Scholar 

  17. Aloni, B. and Rosenshtein, G. (1982) Effect of flooding on tomato cultivars: the relationship between proline accumulation and other morphological and physiological changes. Physiol Plant 56, 513–517.

    Article  CAS  Google Scholar 

  18. Saradhi, P.P., Alia Arora, A.S., and Prasad, K.V.S.K (1995) Proline accumulates in plants exposed to UV radiation and protects them against UV induced peroxidation. Biochem Biophys Res Commun, 209, 1–5.

    Article  PubMed  CAS  Google Scholar 

  19. Meon, S., Fisher, J.M., and Wallace, H.R. (1978) Changes in free proline following infection of plants with either Meloidogyne javanica or Agrobacterium tumefaciens. Physiol Plant Pathol, 12, 251–256.

    Article  CAS  Google Scholar 

  20. Fabro, G., Kovács, I., Pavet, V., Szabados, L., and Alvarez, M.E. (2004) Proline accumulation and AtP5CS2 gene activation are induced by plant–pathogen incompatible interactions in Arabidopsis. Mol Plant Microbe Interact 17, 343–350.

    Article  PubMed  CAS  Google Scholar 

  21. Samuel, D., et al. (2000) Proline inhibits aggregation during protein refolding. Protein Sci 9(2), 344–352.

    Article  PubMed  CAS  Google Scholar 

  22. Terao, Y., Nakamori, S., and Takagi, H. (2003) Gene dosage effect of l-proline biosynthetic enzymes on l-proline accumulation and freeze tolerance in Saccharomyces cerevisiae. Appl Environ Microbiol 69, 6527–6532.

    Article  PubMed  CAS  Google Scholar 

  23. Sharma, S.S., Schat, H., and Vooijs, R. (1998) In vitro alleviation of heavy metal-induced enzyme inhibition by proline. Phytochemistry 49, 1531–1535.

    Article  PubMed  CAS  Google Scholar 

  24. Smirnoff, N. and Cumbes, Q.J. (1989) Hydroxyl radical scavenging activity of compatible solutes. Phytochemistry 28, 1057–1060.

    Article  CAS  Google Scholar 

  25. Hong, Z., Lakkineni, K., Zhang, Z., and Verma, D.P.S. (2000) Removal of feedback inhibition of delta(1)-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress. Plant Physiol. 122, 1129–1136.

    Article  PubMed  CAS  Google Scholar 

  26. Alia, Mohanty, P., and Matysik, J. (2001) Effect of proline on the production of singlet oxygen. Amino Acids 21, 195–200.

    Article  PubMed  CAS  Google Scholar 

  27. Székely, G., Abrahám, E., Cséplo, A., Rigó, G., Zsigmond, L., Csiszár, J., Ayaydin, F., Strizhov, N., Jásik, J., Schmelzer, E., Koncz, C., and Szabados, L. (2008) Duplicated P5CS genes of Arabidopsis play distinct roles in stress regulation and developmental control of proline biosynthesis. Plant J 53, 11–28.

    Article  PubMed  Google Scholar 

  28. Hare, P. and Cress, W. (1997) Metabolic implications of stress induced proline accumulation in plants. Plant Growth Reg 21, 79–102.

    Article  CAS  Google Scholar 

  29. Hu, C.A., Delauney, A.J., and Verma, D.P.S. (1992) A bifunctional enzyme (delta 1-pyrroline-5-carboxylate synthetase) catalyzes the first two steps in proline biosynthesis in plants. Proc Natl Acad Sci USA, 89, 9354–9358.

    Article  PubMed  CAS  Google Scholar 

  30. Delauney, A.J. and Verma, D.P.S. (1990) A soybean gene encoding delta 1-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia coli and is found to be osmoregulated. Mol Gen Genet 221, 299–305.

    Article  PubMed  CAS  Google Scholar 

  31. Verbruggen, N., Villarroel, R., and Van Montagu, M. (1993) Osmoregulation of a pyrroline-5-carboxylate reductase gene in Arabidopsis thaliana. Plant Physiol 103, 771–781.

    Article  PubMed  CAS  Google Scholar 

  32. Zhang, C.S., Lu, Q., and Verma, D.P.S. (1995) Removal of feedback inhibition of delta 1-pyrroline-5-carboxylate synthetase, a bifunctional enzyme catalyzing the first two steps of proline biosynthesis in plants. J Biol Chem 270, 20491–20496.

    Article  PubMed  CAS  Google Scholar 

  33. Yoshiba, Y., Kiyosue, T., Katagiri, T., Ueda, H., Mizoguchi, T., Yamaguchi-Shinozaki, K., Wada, K., Harada, Y., and 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, 751–760.

    Article  PubMed  CAS  Google Scholar 

  34. Strizhov, N., Abrahám, E., Okrész, L., Blickling, S., Zilberstein, A., Schell, J., Koncz, C., and 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, 557–569.

    Article  PubMed  CAS  Google Scholar 

  35. Peng, Z., Lu, Q., and Verma, D.P.S. (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, 334–341.

    PubMed  CAS  Google Scholar 

  36. Verbruggen, N., Hua, X.J., May, M., and Van Montagu, M. (1996) Environmental and developmental signals modulate proline homeostasis: evidence for a negative transcriptional regulator. Proc Natl Acad Sci USA, 93, 8787–8791.

    Article  PubMed  CAS  Google Scholar 

  37. Forlani, G., Scainelli, D. and Nielsen, E. (1997) [delta]1-Pyrroline-5-carboxylate dehydrogenase from cultured cells of potato (purification and properties). Plant Physiol 113, 1413–1418.

    PubMed  CAS  Google Scholar 

  38. Deuschle, K., Funck, D., Forlani, G., Stransky, H., Biehl, A., Leister, D., van der Graaff, E., Kunze, R., and Frommer, W.B. (2004) The role of [delta]1-pyrroline-5-carboxylate dehydrogenase in proline degradation. Plant Cell 16, 3413–3425.

    Article  PubMed  CAS  Google Scholar 

  39. Kiyosue, T., Yoshiba, Y., Yamaguchi-Shinozaki, K., and 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, 1323–1335.

    Article  PubMed  CAS  Google Scholar 

  40. Kishor, P., Hong, Z., Miao, G.H., Hu, C.A.A., and Verma, D.P.S. (1995) Overexpression of [delta]-pyrroline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol 108, 1387–1394.

    PubMed  CAS  Google Scholar 

  41. Verdoy, D., Coba de la Peña, T., Redondo, F.J., Lucas, M.M., and Pueyo, J.J. (2006) Transgenic Medicago truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced tolerance to osmotic stress. Plant Cell Environ 29, 1913–1923.

    Google Scholar 

  42. Nanjo, T., Kobayashi, M., Yoshiba, Y., Kakubari, Y., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1999) Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana. FEBS Lett 461, 205–210.

    Article  PubMed  CAS  Google Scholar 

  43. Nanjo, T., et al. (1999) Biological functions of proline in morphogenesis and osmotolerance revealed in antisense transgenic Arabidopsis thaliana. Plant J 18, 185–193.

    Article  PubMed  CAS  Google Scholar 

  44. De Ronde, J.A., et al. (2004) Photosynthetic response of transgenic soybean plants, containing an Arabidopsis P5CR gene, during heat and drought stress. J Plant Physiol 161, 1211–1224.

    Article  PubMed  Google Scholar 

  45. Chen, Z., et al. (2007) Compatible solute accumulation and stress-mitigating effects in barley genotypes contrasting in their salt tolerance. J Exp Bot 58, 4245–4255.

    Article  PubMed  CAS  Google Scholar 

  46. Xin, Z. and Browse, J. (1998) Eskimo1 mutants of Arabidopsis are constitutively freezing-tolerant. Proc Natl Acad Sci USA 95, 7799–7804.

    Article  PubMed  CAS  Google Scholar 

  47. Liu, J. and Zhu, J.K. (1997) Proline accumulation and salt-stress-induced gene expression in a salt-hypersensitive mutant of Arabidopsis. Plant Physiol 114, 591–596.

    Article  PubMed  CAS  Google Scholar 

  48. Mani, S., et al. (2002) Altered levels of proline dehydrogenase cause hypersensitivity to proline and its analogs in Arabidopsis. Plant Physiol 128, 73–83.

    Article  PubMed  CAS  Google Scholar 

  49. Nanjo, T., et al. (2003) Toxicity of free proline revealed in an Arabidopsis T-DNA-tagged mutant deficient in proline dehydrogenase. Plant Cell Physiol 44, 541–548.

    Article  PubMed  CAS  Google Scholar 

  50. Hare, P., Cress, W., and van Staden, J. (1999) Proline synthesis and degradation: a model system for elucidating stress-related signal transduction. J Exp Bot 50, 413–434.

    Article  CAS  Google Scholar 

  51. Maggio, A., et al. (2002) Does proline accumulation play an active role in stress-induced growth reduction? Plant J 31, 699–712.

    Article  PubMed  CAS  Google Scholar 

  52. Taji, T., et al. (2004) Comparative genomics in salt tolerance between Arabidopsis and Arabidopsis-related halophyte salt cress using Arabidopsis microarray. Plant Physiol 135, 1697–1709.

    Article  PubMed  CAS  Google Scholar 

  53. Inan, G., et al. (2004) Salt cress. A halophyte and cryophyte Arabidopsis relative model system and its applicability to molecular genetic analyses of growth and development of extremophiles. Plant Physiol 135, 1718–1737.

    Article  PubMed  CAS  Google Scholar 

  54. Kant, S., et al. (2006) Evidence that differential gene expression between the halophyte, Thellungiella halophila, and Arabidopsis thaliana is responsible for higher levels of the compatible osmolyte proline and tight control of Na+ uptake in T. halophila. Plant Cell Environ 29, 1220–1234.

    Article  PubMed  CAS  Google Scholar 

  55. Ghars, M.A., et al. (2008) Comparative salt tolerance analysis between Arabidopsis thaliana and Thellungiella halophila, with special emphasis on K(+)/Na(+) selectivity and proline accumulation. J Plant Physiol 165, 588–599.

    Article  PubMed  CAS  Google Scholar 

  56. Smith, I. (1953) Colour reactions on paper chromatograms by a dipping technique. Nature 171, 43–44.

    Article  PubMed  CAS  Google Scholar 

  57. Boctor, F.N. (1971) An improved method for colorimetric determination of proline with isatin. Anal Biochem 43, 66–70.

    Article  PubMed  CAS  Google Scholar 

  58. Elliott, R.J. and Gardner, D.L. (1976) Proline determination with isatin, in the presence of amino acids. Anal Biochem 70, 268–273.

    Article  PubMed  CAS  Google Scholar 

  59. Erdei, L. (2003) Final Report to the PHARE CBC Project No. HU-0009-03-01-014 L. (Erdei, ed.). University of Szeged, Szeged, pp. 37–63.

    Google Scholar 

  60. Grainger, D.J. and S. Aitken (2004) A microtitre format assay for proline in human serum or plasma. Clin Chim Acta 343, 113–118.

    Article  PubMed  CAS  Google Scholar 

  61. Chinard, F.P. (1952) Photometric estimation of proline and ornithine. J Biol Chem 199, 91–95.

    PubMed  CAS  Google Scholar 

  62. Bates, L.S., Waldren, R.P., and Teare, I.D. (1973) Rapid determination of free proline for water-stress studies. Plant Soil 39, 205–207.

    Article  CAS  Google Scholar 

  63. Noctor, G. and Foyer, C.H. (1998) Simultaneous measurement of foliar glutathione, gamma-glutamylcysteine, and amino acids by high-performance liquid chromatography: comparison with two other assay methods for glutathione. Anal Biochem 264, 98–110.

    Article  PubMed  CAS  Google Scholar 

  64. Hanczko, R., et al. (2007) Advances in the o-phthalaldehyde derivatizations. Comeback to the o-phthalaldehyde–ethanethiol reagent. J Chromatogr A 1163, 25–42.

    Article  PubMed  CAS  Google Scholar 

  65. Benson, J.R. and Hare, P.E. (1975) o-Phthalaldehyde: fluorogenic detection of primary amines in the picomole range. Comparison with fluorescamine and ninhydrin. Proc Natl Acad Sci USA, 72, 619–22.

    Article  PubMed  CAS  Google Scholar 

  66. Cooper, J.D.H., Lewis, M.T., and Turnell, D.C. (1984) Pre-column o-phthalaldehyde derivatization of amino acids and their separation using reversed-phase high-performance liquid chromatography. I: Detection of the imino acids hydroxyproline and proline. J Chromatography 285, 484–489.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by OTKA grant no. F-68598, TéT grant no. ZA-16/2007, and EU grant no. FP6-020232-2. Edit Abrahám was supported by the Janos Bolyai Fellowship of the Hungarian Academy of Sciences.

Author information

Authors and Affiliations

  1. Institute of Plant Biology, Biological Research Center, Szeged, Hungary

    Edit Ábrahám & László Szabados

  2. UPMC-Univ Paris 06, PCMP, Paris, France

    Cecile Hourton-Cabassa

  3. Department of Plant Physiology, University of Szeged, Szeged, Hungary

    László Erdei

Authors
  1. Edit Ábrahám
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Cecile Hourton-Cabassa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. László Erdei
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. László Szabados
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. 246 NRC-D, Department of Biochemistry and Molecular, Oklahoma State University, Stillwater, 74078, Oklahoma, USA

    Ramanjulu Sunkar

Rights and permissions

Reprints and permissions

Copyright information

© 2010 Humana Press

About this protocol

Cite this protocol

Ábrahám, E., Hourton-Cabassa, C., Erdei, L., Szabados, L. (2010). Methods for Determination of Proline in Plants. In: Sunkar, R. (eds) Plant Stress Tolerance. Methods in Molecular Biology, vol 639. Humana Press. https://doi.org/10.1007/978-1-60761-702-0_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-60761-702-0_20

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-60761-701-3

  • Online ISBN: 978-1-60761-702-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation