Skip to main content
SpringerLink
Log in

Physiological and metabolomic analysis of a knockout mutant suggests a critical role of MtP5CS3 gene in osmotic stress tolerance of Medicago truncatula

  • Published:
Euphytica Aims and scope Submit manuscript

Abstract

In the model legume Medicago truncatula, Δ1-pyrroline-5-carboxylate synthetase (P5CS), the rate-limiting enzyme of proline biosynthesis, is encoded by three closely related genes, MtP5CS1, MtP5CS2, and MtP5CS3. While MtP5CS1 is constitutively expressed, MtP5CS2 and MtP5CS3 are induced by adverse environmental conditions, of which MtP5CS3 is prevalently expressed during drought and salinity stresses. Mtp5cs3, a transposon (Tnt1) insertion mutant of MtP5CS3 that cannot synthesize a mature protein, showed decreased proline accumulation and increased sensitivity to salinity, drought, and low water potential stresses, as evidenced by decreased seedling growth and chlorophyll content and increased hydrogen peroxide content. These defective phenotypes were complemented by externally supplied proline or ectopically expressed cDNA to the wild-type gene (MtP5CS3). Gas chromatography–mass spectrometry-based analysis of soluble metabolites revealed that some major metabolites contributing to osmotolerance, including certain amino acids, sugars, and polyols, accumulated more abundantly in the Mtp5cs3 roots than in the wild type, whereas a few other amino acids accumulated less during drought and salinity stresses. While such metabolic reconfiguration apparently fell short of compensating for proline deficiency in Mtp5cs3, overexpression of MtP5CS3 significantly increased tolerance of M. truncatula to salinity and low water potential stress. Thus, MtP5CS3 plays a crucial role in proline accumulation and osmotic stress tolerance of M. truncatula. Manipulation of this predominant proline biosynthetic gene will facilitate the development of environmentally stable legume crops.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Ábrahám E, Rigó G, Székely 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 51:363–372

    Google Scholar 

  • Alexieva V, Sergiev I, Mapelli S, Karanov E (2001) The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant, Cell Environ 24:1337–1344

    Article  CAS  Google Scholar 

  • Aranjuelo I, Molero G, Erice G, Avice JC, Nogués S (2011) Plant physiology and proteomics reveals the leaf response to drought in alfalfa (Medicago sativa L.). J Exp Bot 62:111–123

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Barker DG, Pfaff T, Moreau D, Groves E, Ruffel S, Lepetit M, Whitehand S, Maillet F, Nair RM, Journet EP (2006) Growing M. truncatula: choice of substrates and growth conditions. In: Mathesius U (ed) The Medicago truncatula Handbook. The Samuel Roberts Noble Foundation, Ardmore

    Google Scholar 

  • Boisson-Dernier A, Chabaud M, Garcia F, Bécard G, Rosenberg C, Barker DG (2001) Hairy roots of Medicago truncatula as tools for studying nitrogen-fixing and endomycorrhizal symbioses. Mol Plant-Microbe Interact 14:639–700

    Article  Google Scholar 

  • Bradford MM (1976) Rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159

    Article  CAS  PubMed  Google Scholar 

  • Cook D, Fowler S, Fiehn O, Thomashow MF (2004) A prominent role for the CBF cold response pathway in configuring the low-temperature metabolome of Arabidopsis. Proc Natl Acad Sci USA 101:15243–15248

    Article  CAS  PubMed  Google Scholar 

  • Debouba M, Maâroufi-Dghimi MH, Suzuki A, Ghorbel MH, Gouia H (2007) Changes in growth and activity of enzymes involved in nitrate reduction and ammonium assimilation in tomato seedlings in response to NaCl stress. Ann Bot 99:1143–1151

    Google Scholar 

  • Degenkolbe T, Do PT, Zuther E, Repsilber D, Walther D, Hincha DK, Kohl KI (2009) Expression profiling of rice cultivars differing in their tolerance to long-term drought stress. Plant Mol Biol 69:133–153

    Article  CAS  PubMed  Google Scholar 

  • Delauney A, Verma DPS (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  CAS  PubMed  Google Scholar 

  • Deuschle K, Funck D, Forlani G, Stransky H, Biehl A, Leister D, van der Graaff E, Kunze R, Frommer WB (2004) The role of Δ1 -pyrroline-5-carboxylate dehydrogenase in proline degradation. Plant Cell 16:3413–3425

    Article  CAS  PubMed  Google Scholar 

  • Fujita T, Maggio A, García-Ríos M, Bressan RA, Csonka LN (1998) Comparative analysis of the regulation of expression and structures of two evolutionarily divergent genes for Δ1-pyrroline-5-carboxylate synthetase from tomato. Plant Physiol 118:661–674

    Article  CAS  PubMed  Google Scholar 

  • Funck D, Stadelhofer B, Koch W (2008) Ornithine-δ-aminotransferase is essential for arginine catabolism but not for proline biosynthesis. BMC Plant Biol 8:40

    Article  PubMed  Google Scholar 

  • Gagneul D, Aïnouche A, Duhazé C, Lugan R, Larher FR, Bouchereau A (2007) A reassessment of the function of the so-called compatible solutes in the halophytic plumbaginaceae Limonium latifolium. Plant Physiol 144:1598–1611

    Article  CAS  PubMed  Google Scholar 

  • García-Ríos M, Fujita T, LaRosa PC, Locy RD, Clithero JM, Bressan RA, Csonka LN (1997) Cloning of a polycistronic cDNA from tomato encoding γ-glutamyl kinase and γ-glutamyl phosphate reductase. Proc Natl Acad Sci USA 94:8249–8254

    Article  PubMed  Google Scholar 

  • 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 Δ1-pyrroline-5-carboxylate synthase in alfalfa, transcriptionally induced upon salt stress. Plant Mol Biol 38:755–764

    Article  CAS  PubMed  Google Scholar 

  • Handa S, Handa A, Hasegawa PM, Bressan RA (1986) Proline accumulation and the adaptation of cultured plant cells to water stress. Plant Physiol 80:938–945

    Article  CAS  PubMed  Google Scholar 

  • Hare PD, Cress WA (1997) Metabolic implications of stress-induced proline accumulation in plants. Plant Growth Regul 21:79–102

    Article  CAS  Google Scholar 

  • Hare PD, Cress WA, Van Staden J (1988) Dissecting the roles of osmolyte accumulation during stress. Plant, Cell Environ 21:535–553

    Article  Google Scholar 

  • Hellmann H, Funck D, Rentsch D, Frommer WB (2000) Hypersensitivity of an Arabidopsis sugar signaling mutant toward exogenous proline application. Plant Physiol 123:779–789

    Article  CAS  PubMed  Google Scholar 

  • Hiscox JD, Israelstam GF (1979) A method for the extraction of chlorophyll from leaf tissue without maceration. Can J Bot 51:1332–1334

    Article  Google Scholar 

  • Hong Z, Lakkineni K, Zhang Z, Verma DP (2000) Removal of feedback inhibition of Δ1-pyrroline-5-carboxylate synthetase results in increased proline accumulation and protection of plants from osmotic stress. Plant Physiol 122:1129–1136

    Article  CAS  PubMed  Google Scholar 

  • Hu CAA, Delauney AJ, Verma DPS (1992) A bifunctional enzyme (Δ1-pyrroline-5-carboxylate synthetase) catalyses the first two steps in proline biosynthesis in plants. Proc Natl Acad Sci USA 89:9354–9358

    Article  CAS  PubMed  Google Scholar 

  • Joshi V, Jander G (2009) Arabidopsis methionine gamma-lyase is regulated according to isoleucine biosynthesis needs but plays a subordinate role to threonine deaminase. Plant Physiol 151:367–378

    Article  CAS  PubMed  Google Scholar 

  • Kang Y, Han Y, Torres-Jerez I, Wang M, Tang Y, Monteros M, Udvardi M (2011) System responses to long-term drought and re-watering of two contrasting alfalfa varieties. Plant J 68:871–889

    Article  CAS  PubMed  Google Scholar 

  • Karimi M, Inzé D, Depicker A (2002) GATEWAY vectors for Agrobacterium-mediated plant transformation. Trends Plant Sci 7:193–195

    Article  CAS  PubMed  Google Scholar 

  • Kesari R, Lasky JR, Villamor JG, Marais DLD, Chen YJC, Liu TW, Lin W, Juenger TE, Verslues PE (2012) Intron-mediated alternative splicing of Arabidopsis P5CS1 and its association with natural variation in proline and climate adaptation. Proc Natl Acad Sci USA 109:9197–9202

    Article  CAS  PubMed  Google Scholar 

  • Kim GB, Nam YW (2013) A novel Δ1-pyrroline-5-carboxylate synthetase gene of Medicago truncatula plays a predominant role in stress-induced proline accumulation during symbiotic nitrogen fixation. J Plant Physiol 170:291–302

    Article  CAS  PubMed  Google Scholar 

  • Kishor KPB (1989) Salt stress in cultured rice cells: effects of proline and abscisic acid. Plant, Cell Environ 12:629–633

    Article  CAS  Google Scholar 

  • 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 8:1323–1335

    CAS  PubMed  Google Scholar 

  • Kohl DH, Schubert KR, Carter MB, Hagedorn CH, Shearer G (1988) Proline metabolism in N2-fixing root nodules: energy transfer and regulation of purine synthesis. Proc Natl Acad Sci USA 85:2036–2040

    Article  CAS  PubMed  Google Scholar 

  • Last RL, Jones AD, Shachar-Hill Y (2007) Towards the plant metabolome and beyond. Nature Rev Mol Cell Biol 8:167–174

    Article  CAS  Google Scholar 

  • Less H, Galili G (2008) Principal transcriptional programs regulating plant amino acid metabolism in response to abiotic stresses. Plant Physiol 147:316–330

    Article  CAS  PubMed  Google Scholar 

  • Lugan R, Niogret MF, Leport L, Guégan JP, Larher FR, Savouré A, Kopka J, Bouchereau A (2010) A metabolome and water homeostasis analysis of Thellungiella salsuginea suggests that dehydration tolerance is a key response to osmotic stress in this halophyte. Plant J 64:215–229

    Article  CAS  PubMed  Google Scholar 

  • 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 31:699–712

    Article  CAS  PubMed  Google Scholar 

  • Mattioli R, Falasca G, Sabatini S, Altamura M, Costantino P, Trovato M (2009) The proline biosynthesis genes P5CS1 and P5CS2 play overlapping roles in Arabidopsis flower transition but not in embryo development. Physiol Plant 137:72–85

    Article  CAS  PubMed  Google Scholar 

  • Matysik J, Alia Bhalu B, Mohanty P (2002) Molecular mechanisms of quenching of reactive oxygen species by proline under stress in plants. Curr Sci 82:525–532

    CAS  Google Scholar 

  • Merchan F, de Lorenzo L, Rizzo SG, Niebel A, Manyani H, Frugier F, Sousa C, Crespi M (2007) Analysis of regulatory pathways involved in the reacquisition of root growth after salt stress in Medicago truncatula. Plant J 51:1–17

    Google Scholar 

  • Michel BE, Kaufmann MR (1973) The osmotic potential of polyethelene glycol 6000. Plant Physiol 51:914–916

    Article  CAS  PubMed  Google Scholar 

  • Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol 59:651–681

    Article  CAS  PubMed  Google Scholar 

  • 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 44:541–548

    Article  CAS  PubMed  Google Scholar 

  • Parre E, de Virville J, Cochet F, Leprince AS, Richard L, Lefebvre-De Vos D, Ghars MA, Bordenave M, Zachowski A, Savouré A (2010) A new method for accurately measuring Delta(1)-pyrroline-5-carboxylate synthetase activity. Methods Mol Biol 639:333–340

    Article  CAS  PubMed  Google Scholar 

  • Planchet E, Rannou O, Ricoult C, Mercey BS, Grondard MA, Limami AM (2011) Nitrogen metabolism responses to water deficit act through both abscisic acid (ABA)-dependent and independent path ways in Medicago truncatula during post-germination. J Exp Bot 62:605–615

    Article  CAS  PubMed  Google Scholar 

  • Quandt HJ, Pühler A, Broer I (1993) Transgenic root nodules of Vicia hirsuta: a fast and efficient system for the study of gene expression in indeterminate-type nodules. Mol Plant Microbe Interact 6:699–706

    Article  Google Scholar 

  • Rizhsky L, Liang H, Mittler R (2002) The combined effect of drought stress and heat shock on gene expression in tobacco. Plant Physiol 130:1143–1151

    Article  CAS  PubMed  Google Scholar 

  • Saito K, Matsuda F (2010) Metabolomics for functional genomics, systems biology, and biotechnology. Annu Rev Plant Biol 61:463–489

    Article  CAS  PubMed  Google Scholar 

  • Sanchez DH, Lippold F, Redestig H, Hannah MA, Erban A, Krämer U, Kopka J, Udvardi MK (2008a) Integrative functional genomics of salt acclimatization in the model legume Lotus japonicus. Plant J 53:973–987

    Article  CAS  PubMed  Google Scholar 

  • Sanchez DH, Siahpoosh MR, Roessner U, Udvardi M, Kopka J (2008b) Plant metabolomics reveals conserved and divergent metabolic responses to salinity. Physiol Plant 132:209–219

    CAS  PubMed  Google Scholar 

  • Sanchez DH, Pieckenstain FL, Szymanski J, Erban A, Bromke M, Hannah MA, Kraemer U, Kopka J, Udvardi MK (2011) Comparative functional genomics of salt stress in related model and cultivated plants identifies and overcomes limitations to translational genomics. PLoS ONE 6:e17094

    Article  CAS  PubMed  Google Scholar 

  • Sanchez DH, Schwabe F, Erbani A, Udvardi MK, Kopka J (2012) Comparative metabolomics of drought acclimation in model and forage legumes. Plant, Cell Environ 35:136–149

    Article  CAS  Google Scholar 

  • Schmidt R, Stransky H, Koch W (2007) The amino acid permease AAP8 is important for early seed development in Arabidopsis thaliana. Planta 226:805–813

    Article  CAS  PubMed  Google Scholar 

  • Sharma S, Verslues PE (2010) Mechanisms independent of ABA or proline feedback have a predominant role in transcriptional regulation of proline metabolism during low water potential and stress recovery. Plant, Cell Environ 33:1838–1851

    Article  CAS  Google Scholar 

  • Sharma S, Villamor JG, Verslues PE (2011) Essential role of tissue-specific proline synthesis and catabolism in growth and redox balance at low water potential. Plant Physiol 157:292–304

    Article  CAS  PubMed  Google Scholar 

  • Strizhov N, Ábrahám E, Ökrész L, Blickling S, Zilberstein A, Schell J, Koncs 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 12:557–569

    Google Scholar 

  • Su J, Wu R (2004) Stress-inducible synthesis of proline in transgenic rice confers faster growth under stress conditions than that with constitutive synthesis. Plant Sci 166:941–948

    Article  CAS  Google Scholar 

  • Szabados L, Savouré A (2010) Proline: a multifunctional amino acid. Trends Plant Sci 15:89–97

    Article  CAS  PubMed  Google Scholar 

  • Székely G, Ábrahám E, Cséplö A, Rigó G, Zsigmond L, Csiszár J, Ayaydin F, Nicolai Strizhov N, Jásik J, Schmelzer E, Koncz C, 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 

  • Thapa B, Arora R, Knapp AD, Brummer EC (2008) Applying freezing test to quantify cold acclimation in Medicago truncatula Gaertn. J Am Soc Hortic Sci 133:684–691

    Google Scholar 

  • Urano K, Maruyama K, Ogata Y, Morishita Y, Takeda M, Sakurai N, Suzuki H, Saito K, Shibata D, Kobayashi M, Yamaguchi-Shinozaki K, Shinozaki K (2009) Characterization of the ABA-regulated global responses to dehydration in Arabidopsis by metabolomics. Plant J 57:1065–1078

    Article  CAS  PubMed  Google Scholar 

  • Vendruscolo ACG, Schuster I, Pileggi M, Scapim CA, Molinari HBC, Marur CJ, Vieira LGC (2007) Stress-induced synthesis of proline confers tolerance to water deficit in transgenic wheat. J Plant Physiol 164:1367–1376

    Article  CAS  PubMed  Google Scholar 

  • Venekamp JH, Koot JTM (1984) The distribution of free amino acids, especially of proline, in the organs of field bean plants, Vicia faba L., during development in the field. J Plant Physiol 116:343–349

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Verdoy D, de la Peńa TC, Redondo FJ, Lucas MM, Pueyo JJ (2006) Transgenic Medicago truncatula plants that accumulate proline display nitrogen-fixing activity with enhanced tolerance to osmotic stress. Plant, Cell Environ 29:1913–1923

    Article  CAS  Google Scholar 

  • Verslues PE, Agarwal M, Katiyar-Agarwal S, Zhu J, Zhu JK (2006) Methods and concepts in quantifying resistance to drought, salt and freezing, abiotic stresses that affect plant water status. Plant J 45:523–539

    Article  CAS  PubMed  Google Scholar 

  • Yoshiba Y, Kiyosue T, Katagiri T, Ueda H, Mizoguchi T, Yamaguchi-Shinozaki K, Wada K, Harada Y, Shinozaki K (1995) Correlation between the induction of a gene for Δ1-pyrroline-5-carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana under osmotic stress. Plant J 7:751–760

    Article  CAS  PubMed  Google Scholar 

  • Zhang CS, Lu Q, Verma DPS (1995) Removal of feedback inhibition of Δ1-pyrroline-5-carboxylate synthetase, a bifunctional enzyme catalyzing the first two steps of proline biosynthesis in plants. J Biol Chem 270:20491–20496

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Life Science, Sogang University, Seoul, 121-742, Korea

    Minh Luan Nguyen, Goon-Bo Kim & Young-Woo Nam

  2. College of Pharmacy, Chung-Ang University, Seoul, 156-756, Korea

    Sun-Hee Hyun, Seok-Young Lee & Hyung-Kyoon Choi

  3. Department of Genetic Engineering, College of Natural Resources and Life Science, Dong-A University, Pusan, 604-714, Korea

    Chae-Young Lee & Hong-Kyu Choi

Authors
  1. Minh Luan Nguyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Goon-Bo Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sun-Hee Hyun
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Seok-Young Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chae-Young Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hong-Kyu Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hyung-Kyoon Choi
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Young-Woo Nam
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Young-Woo Nam.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOC 1437 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nguyen, M.L., Kim, GB., Hyun, SH. et al. Physiological and metabolomic analysis of a knockout mutant suggests a critical role of MtP5CS3 gene in osmotic stress tolerance of Medicago truncatula . Euphytica 193, 101–120 (2013). https://doi.org/10.1007/s10681-013-0957-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10681-013-0957-4

Keywords

Search

Navigation