Skip to main content

Modulation of proline metabolic gene expression in Arabidopsis thaliana under water-stressed conditions by a drought-mitigating Pseudomonas putida strain

Abstract

Although amelioration of drought stress in plants by plant growth promoting rhizobacteria (PGPR) is a well reported phenomenon, the molecular mechanisms governing it are not well understood. We have investigated the role of a drought ameliorating PGPR strain, Pseudomonas putida GAP-P45 on the regulation of proline metabolic gene expression in Arabidopsis thaliana under water-stressed conditions. Indeed, we found that Pseudomonas putida GAP-P45 alleviates the effects of water-stress in A. thaliana by drastic changes in proline metabolic gene expression profile at different time points post stress induction. Quantitative real-time expression analysis of proline metabolic genes in inoculated plants under water-stressed conditions showed a delayed but prolonged up-regulation of the expression of genes involved in proline biosynthesis, i.e., ornithine-Δ-aminotransferase (OAT), Δ 1 -pyrroline-5-carboxylate synthetase1 (P5CS1), Δ 1 -pyrroline-5-carboxylate reductase (P5CR), as well as proline catabolism, i.e., proline dehydrogenase1 (PDH1) and Δ 1 -pyrroline-5-carboxylate dehydrogenase (P5CDH). These observations were positively correlated with morpho-physiological evidences of water-stress mitigation in the plants inoculated with Pseudomonas putida GAP-P45 that showed better growth, increased fresh weight, enhanced plant water content, reduction in primary root length, enhanced chlorophyll content in leaves, and increased accumulation of endogenous proline. Our observations point towards PGPR-mediated enhanced proline turnover rate in A. thaliana under dehydration conditions.

This is a preview of subscription content, access via your institution.

Fig. 1a–p
Fig. 2a–d
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

References

  • 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. https://doi.org/10.1111/j.0031-9317.2004.00251.x

  • Barnes JD, Balaguer L, Manrique E, Elvira S, Davison AW (1992) A reappraisal of the use of DMSO for the extraction and determination of chlorophylls a and b in lichens and higher plants. Environ Exp Bot 32:85–100. https://doi.org/10.1016/0098-8472(92)90034-Y

  • Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free proline for water-stress studies. Plant Soil 39:205–207. https://doi.org/10.1007/BF00018060

  • Ben Rejeb K, Abdelly C, Savouré A (2014) How reactive oxygen species and proline face stress together. Plant Physiol Biochem 80:278–284. https://doi.org/10.1016/j.plaphy.2014.04.007

  • Bhaskara GB, Yang T-H, Verslues PE (2015) Dynamic proline metabolism: importance and regulation in water limited environments. Front Plant Sci 6:484. https://doi.org/10.3389/fpls.2015.00484

  • Bhattacharyya PN, Jha DK (2012) Plant growth-promoting rhizobacteria (PGPR): emergence in agriculture. World J Microbiol Biotechnol 28:1327–1350. https://doi.org/10.1007/s11274-011-0979-9

  • Bishnoi U (2015) PGPR Interaction: an ecofriendly approach promoting the sustainable agriculture system. Adv Bot Res 75: 81–113

  • Borsani O, Zhu J, Verslues PE, Sunkar R, Zhu JK (2005) Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis. Cell 123:1279–1291. https://doi.org/10.1016/j.cell.2005.11.035

  • Cho SM, Kang BR, Han SH, Anderson AJ, Park JY, Lee YH, Cho BH, Yang KY, Ryu CM, Kim YC (2008) 2R,3R-butanediol, a bacterial volatile produced by Pseudomonas chlororaphis O6, is involved in induction of systemic tolerance to drought in Arabidopsis thaliana. Mol Plant-Microbe Interact 21:1067–1075. https://doi.org/10.1094/MPMI-21-8-1067

  • Choudhary NL, Sairam RK, Tyagi A (2005) Expression of delta1-pyrroline-5-carboxylate synthetase gene during drought in rice (Oryza sativa L.) Indian J Biochem Biophys 42:366–370

    CAS  PubMed  Google Scholar 

  • Delauney AJ, Hu CA, Kishor PB, Verma DP (1993) Cloning of ornithine delta-aminotransferase cDNA from Vigna aconitifolia by trans-complementation in Escherichia coli and regulation of proline biosynthesis. J Biol Chem 268:18673–18678

    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 [Delta]1-pyrroline-5-carboxylate dehydrogenase in proline degradation. Plant Cell 16:3413–3425. https://doi.org/10.1105/tpc.104.023622

  • Fabro G, Kovács I, Pavet V, Szabados L, Alvarez ME (2004) Proline accumulation and AtP5CS2 gene activation are induced by plant-pathogen incompatible interactions in Arabidopsis. Mol Plant-Microbe Interact 17:343–350. https://doi.org/10.1094/MPMI.2004.17.4.343

  • 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. https://doi.org/10.1186/1471-2229-8-40

  • Funck D, Eckard S, Müller G (2010) Non-redundant functions of two proline dehydrogenase isoforms in Arabidopsis. BMC Plant Biol 10:70. https://doi.org/10.1186/1471-2229-10-70

  • Funck D, Winter G, Baumgarten L, Forlani G (2012) Requirement of proline synthesis during Arabidopsis reproductive development. BMC Plant Biol 12:191. https://doi.org/10.1186/1471-2229-12-191

  • Giberti S, Funck D, Forlani G (2014) Δ 1 -pyrroline-5-carboxylate reductase from Arabidopsis thaliana : stimulation or inhibition by chloride ions and feedback regulation by proline depend on whether NADPH or NADH acts as co-substrate. New Phytol 202:911–919. https://doi.org/10.1111/nph.12701

  • Grossnickle SC (2005) Importance of root growth in overcoming planting stress. New For 30:273–294. https://doi.org/10.1007/s11056-004-8303-2

  • Hare PD, Cress WA (1996) Tissue-specific accumulation of transcript encoding Δ 1-pyrrolline-5-carboxylate reductase in Arabidopsis thaliana. Plant Growth Regul 19:249–256. https://doi.org/10.1007/BF00037798

  • Hayat R, Ali S, Amara U, Khalid R, Ahmed I (2010) Soil beneficial bacteria and their role in plant growth promotion: a review. Ann Microbiol 60:579–598. https://doi.org/10.1007/s13213-010-0117-1

  • Hong Z, Lakkineni K, Zhang Z, Verma DP (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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Hu X, Tanaka A, Tanaka R (2013) Simple extraction methods that prevent the artifactual conversion of chlorophyll to chlorophyllide during pigment isolation from leaf samples. Plant Methods 9:19. https://doi.org/10.1186/1746-4811-9-19

  • Jacobs DF, Rose R, Haase DL, Alzugaray PO (2004) Fertilization at planting impairs root system development and drought avoidance of Douglas-fir ( Pseudotsuga menziesii ) seedlings. Ann For Sci 61:643–651. https://doi.org/10.1051/forest:2004065

  • Kaplan F, Kopka J, Sung DY, Zhao W, Popp M, Porat R, Guy CL (2007) Transcript and metabolite profiling during cold acclimation of Arabidopsis reveals an intricate relationship of cold-regulated gene expression with modifications in metabolite content. Plant J 50:967–981. https://doi.org/10.1111/j.1365-313X.2007.03100.x

  • Kavi Kishor PB, Sreenivasulu N (2014) Is proline accumulation per se correlated with stress tolerance or is proline homeostasis a more critical issue? Plant Cell Environ 37:300–311. https://doi.org/10.1111/pce.12157

  • Kavi Kishor PB, Sangam S, Amrutha RN, Sri Laxmi P, Naidu KR, Rao KRSS, Rao S, Reddy KJ, Theriappan P, 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 

  • Krasensky J, Jonak C (2012) Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. J Exp Bot 63:1593–1608. https://doi.org/10.1093/jxb/err460

  • Liang X, Zhang L, Natarajan SK, Becker DF (2013) Proline mechanisms of stress survival. Antioxid Redox Signal 19:998–1011. https://doi.org/10.1089/ars.2012.5074

  • Liu F, Xing S, Ma H, Du Z, Ma B (2013) Cytokinin-producing, plant growth-promoting rhizobacteria that confer resistance to drought stress in Platycladus orientalis container seedlings. Appl Microbiol Biotechnol 97:9155–9164. https://doi.org/10.1007/s00253-013-5193-2

  • Mattioli R, Falasca G, Sabatini S, Attamura MM, Costantino P, Trovato M (2009) The proline biosynthetic genes P5CS1 and P5CS2 play overlapping roles in Arabidopsis flower transition but not in embryo development. Physiol Plant 137:72–85. https://doi.org/10.1111/j.1399-3054.2009.01261.x

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Planta 15:473–497

  • Ngumbi E, Kloepper J (2016) Bacterial-mediated drought tolerance: current and future prospects. Appl Soil Ecol 105:109–125

    Article  Google Scholar 

  • Pace PF, Cralle HT, El-Halawany SHM, Cothern JT, Senseman SA (1999) PHYSIOLOGY drought-induced changes in shoot and root growth of young cotton plants. J Cotton Sci 3:183–187

    Google Scholar 

  • Reddy PS, Jogeswar G, Rasineni GK, Maheswari M, Reddy AR, Varshney RK, Kavi Kishor PB (2015) Proline over-accumulation alleviates salt stress and protects photosynthetic and antioxidant enzyme activities in transgenic sorghum [Sorghum bicolor (L.) Moench]. Plant Physiol Biochem 94:104–113. https://doi.org/10.1016/j.plaphy.2015.05.014

  • Rizzi YS, Monteoliva MI, Fabro G, Grosso CL, Laróvere LE, Alvarez ME (2015) P5CDH affects the pathways contributing to pro synthesis after ProDH activation by biotic and abiotic stress conditions. Front Plant Sci 6:572. https://doi.org/10.3389/fpls.2015.00572

  • Roosens NH, Thu TT, Iskandar HM, Jacobs M (1998) Isolation of the ornithine-delta-aminotransferase cDNA and effect of salt stress on its expression in Arabidopsis thaliana. Plant Physiol 117:263–271

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Roosens NH, Bitar FA, Loenders K, Angenon G, Jacobs M (2002) Overexpression of ornithine-δ-aminotransferase increases proline biosynthesis and confers osmotolerance in transgenic plants. Mol Breed 9:73–80. https://doi.org/10.1023/A:1026791932238

  • Saharan BS, Nehra V (2011) Plant growth promoting Rhizobacteria: a critical review. Life Sci Med Res 21:1–30

    Google Scholar 

  • Sandhya V, Ali SKZ, Grover M, Reddy G, Venkateswarlu B (2009) Alleviation of drought stress effects in sunflower seedlings by the exopolysaccharides producing Pseudomonas putida strain GAP-P45. Biol Fertil Soils 46:17–26. https://doi.org/10.1007/s00374-009-0401-z

  • Sandhya V, Ali SZ, Grover M, Reddy G, Venkateswarlu B (2010a) Effect of plant growth promoting Pseudomonas spp. on compatible solutes, antioxidant status and plant growth of maize under drought stress. Plant Growth Regul 62:21–30. https://doi.org/10.1007/s10725-010-9479-4

  • Sandhya V, Ali SZ, Venkateswarlu B, Reddy G, Grover M (2010b) Effect of osmotic stress on plant growth promoting Pseudomonas spp. Arch Microbiol 192:867–876. https://doi.org/10.1007/s00203-010-0613-5

  • Savouré A, Jaoua S, Hua XJ, Ardiles W, Van Montagu M, Verbruggen N (1995) Isolation, characterization, and chromosomal location of a gene encoding the delta 1-pyrroline-5-carboxylate synthetase in Arabidopsis thaliana. FEBS Lett 372:13–19

    Article  PubMed  Google Scholar 

  • Sharma S, Verslues PE (2010) Mechanisms independent of abscisic acid (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. https://doi.org/10.1111/j.1365-3040.2010.02188.x

  • 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. https://doi.org/10.1104/pp.111.183210

  • Sharma S, Shinde S, Verslues PE (2013) Functional characterization of an ornithine cyclodeaminase-like protein of Arabidopsis thaliana. BMC Plant Biol 13:182. https://doi.org/10.1186/1471-2229-13-182

  • Szabados L, Savouré A (2010) Proline: a multifunctional amino acid. Trends Plant Sci 15:89–97. https://doi.org/10.1016/j.tplants.2009.11.009

  • Székely G, Abrahám E, Cséplo A, Rigó G, Zsigmond L, Csiszár J, Ayaydin F, 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. https://doi.org/10.1111/j.1365-313X.2007.03318.x

  • Timmusk S, Wagner EGH (1999) The plant-growth-promoting rhizobacterium Paenibacillus polymyxa induces changes in Arabidopsis thaliana gene expression: a possible connection between biotic and abiotic stress responses. Mol Plant-Microbe Interact 12:951–959. https://doi.org/10.1094/MPMI.1999.12.11.951

  • Timmusk S, Abd El-Daim IA, Copolovici L, Tanilas T, Kännaste A, Behers L, Nevo E, Seisenbaeva G, Stenström E, Niinemets Ü (2014) Drought-tolerance of wheat improved by rhizosphere bacteria from harsh environments: enhanced biomass production and reduced emissions of stress volatiles. PLoS One 9:e96086. https://doi.org/10.1371/journal.pone.0096086

  • Toka I, Planchais S, Cabassa C, Justin AM, De Vos D, Richard L, Savouré A, Carol P (2010) Mutations in the hyperosmotic stress-responsive mitochondrial BASIC AMINO ACID CARRIER2 enhance proline accumulation in Arabidopsis. Plant Physiol 152:1851–1862. https://doi.org/10.1104/pp.109.152371

  • Turner NC (1981) Techniques and experimental approaches for the measurement of plant water status. Plant Soil 58:339–366. https://doi.org/10.1007/BF02180062

  • van der Weele CM (2000) Growth of Arabidopsis thaliana seedlings under water deficit studied by control of water potential in nutrient-agar media. J Exp Bot 51:1555–1562. https://doi.org/10.1093/jexbot/51.350.1555

  • Vardharajula S, Zulfikar Ali S, Grover M, Reddy G, Bandi V (2011) Drought-tolerant plant growth promoting Bacillus spp.: effect on growth, osmolytes, and antioxidant status of maize under drought stress. J Plant Interact 6:1–14. https://doi.org/10.1080/17429145.2010.535178

  • Verbruggen N, Hermans C (2008) Proline accumulation in plants: a review. Amino Acids 35:753–759. https://doi.org/10.1007/s00726-008-0061-6

  • 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 93:8787–8791

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Verslues PE, Sharp RE (1999) Proline accumulation in maize (Zea mays L.) primary roots at low water potentials. II. Metabolic source of increased proline deposition in the elongation zone. Plant Physiol 119:1349–1360

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  • Verslues PE, Kim YS, Zhu JK (2007) Altered ABA, proline and hydrogen peroxide in an Arabidopsis glutamate:glyoxylate aminotransferase mutant. Plant Mol Biol 64:205–217. https://doi.org/10.1007/s11103-007-9145-z

  • Wu L (2003) Over-expression of an Arabidopsis d -OAT gene enhances salt and drought tolerance in transgenic rice. Chinese Sci Bull 48:2594. https://doi.org/10.1360/03wc0218

  • Yancey PH, Clark ME, Hand SC, Bowlus RD, Somero GN (1982) Living with water stress: evolution of osmolyte systems. Science 217:1214–1222

    CAS  Article  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 delta 1-pyrroline-5-carboxylate synthetase and the accumulation of proline in Arabidopsis thaliana under osmotic stress. Plant J 7:751–760

    CAS  Article  PubMed  Google Scholar 

  • Zhang C, Lu Q, Verma DPS (1997) Characterization of Δ1-pyrroline-5-carboxylate synthetase gene promoter in transgenic Arabidopsis thaliana subjected to water stress. Plant Sci 129:81–89. https://doi.org/10.1016/S0168-9452(97)00174-X

  • Zhang H, Murzello C, Sun Y, Kim MS, Xie X, Jeter RM, Zak JC, Dowd SE, Paré PW (2010) Choline and osmotic-stress tolerance induced in Arabidopsis by the soil microbe Bacillus subtilis (GB03). Mol Plant-Microbe Interact 23:1097–1104. https://doi.org/10.1094/MPMI-23-8-1097

  • Zlatev Z, Lidon FC (2012) An overview on drought induced changes in plant growth, water relations and photosynthesis. Emir J Food Agric Plant Sci 24:57–72

    Article  Google Scholar 

Download references

Acknowledgements

The authors thank Minakshi Grover (Principal Scientist, Indian Agricultural Research Institute, New Delhi, India) for help in procuring the rhizobacterial strain used in this study.

Funding

This work was funded by BITS-Pilani Seed Grant Scheme and Science and Engineering Research Board, Govt. of India.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Sridev Mohapatra.

Electronic supplementary material

supplementary 1

(DOCX 4029 kb)

supplementary 2

(DOCX 4805 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ghosh, D., Sen, S. & Mohapatra, S. Modulation of proline metabolic gene expression in Arabidopsis thaliana under water-stressed conditions by a drought-mitigating Pseudomonas putida strain. Ann Microbiol 67, 655–668 (2017). https://doi.org/10.1007/s13213-017-1294-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s13213-017-1294-y

Keywords

  • Arabidopsis thaliana
  • Pseudomonas putida GAP-P45
  • Water-stress
  • Proline
  • Gene expression