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Oxidative stress induced in chloroplasts or mitochondria promotes proline accumulation in leaves of pea (Pisum sativum): another example of chloroplast-mitochondria interactions


Oxidative stress can occur in different parts of plant cells. We employed two oxidants that induce reactive oxygen species (ROS) in different intracellular compartments: methyl viologen (MV, in chloroplasts) and menadione (MD, in mitochondria). The responses of pea (Pisum sativum) leaf discs to MV or MD after 4-h incubation in dark or moderate (300 μE m−2 s−1) or high light (1200 μE m−2 s−1) were examined. Marked increase in ROS levels was observed, irrespective of compartment targeted. The levels of proline, a compatible solute, increased markedly much more than that of ascorbate or glutathione during oxidative/photo-oxidative stress, emphasizing the importance of proline. Further, the activities and transcripts of enzymes involved in biosynthesis or oxidation of proline were studied. An upregulation of biosynthesis and downregulation of oxidation was the basis of proline accumulation. Pyrroline-5-carboxylate synthetase (P5CS, involved in biosynthesis) and proline dehydrogenase (PDH, involved in oxidation) were the key enzymes regulated under oxidative stress. Since these two enzymes—P5CS and PDH—are located in chloroplasts and mitochondria, respectively, we suggest that proline metabolism can help to mediate inter-organelle interactions and achieve redox homeostasis under photo-oxidative stress.

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High light






Moderate light


Methyl viologen


Nitro blue tetrazolium chloride


Pyrroline-5-carboxylate dehydrogenase


Pyrroline-5-carboxylate reductase


Pyrroline-5-carboxylate synthetase


Proline dehydrogenase


Polyvinylidene difluoride


Reactive oxygen species


  • Armengaud P, Thiery L, Buhot N, Grenier-DeMarch G, 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 

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

    Article  CAS  Google Scholar 

  • Bradford MM (1976) A 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  PubMed  CAS  Google Scholar 

  • Cecchini NM, Monteoliva MI, Alvarez ME (2011) Proline dehydrogenase contributes to pathogen defense in Arabidopsis. Plant Physiol 155:1947–1959

    Article  PubMed  PubMed Central  CAS  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  PubMed  CAS  Google Scholar 

  • Donahue JL, Okpodu CM, Cramer CL, Grabau EA, Alscher RG (1997) Responses of antioxidants to paraquat in pea leaves. Plant Physiol 113:249–257

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Foyer CH (2001) Prospects for enhancement of the soluble antioxidants, ascorbate and glutathione. BioFactors 15:75–78

    Article  PubMed  CAS  Google Scholar 

  • Foyer CH, Noctor G (2003) Redox sensing and signalling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria. Physiol Plant 119:355–364

    Article  CAS  Google Scholar 

  • Garcia-Rios 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 U S A 94:8249–8254

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Gill SS, Tuteja N (2010) Reactive oxygen species and anti-oxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol 148:909–930

    Google Scholar 

  • Gillespie KM, Ainsworth EA (2007) Measurement of reduced, oxidizied and total ascorbate content in plants. Nat Protoc 2:871–874

    Article  PubMed  CAS  Google Scholar 

  • Griffith OW (1980) Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinyl-pyridine. Anal Biochem 106:207–212

    Article  PubMed  CAS  Google Scholar 

  • Hawkes TR (2014) Mechanisms of resistance to paraquat in plants. Pest Manag Sci 70:1316–1323

    Article  PubMed  CAS  Google Scholar 

  • Hayat S, Hayat Q, Alyemeni MN, Wani AS, Pichtel J, Ahmad A (2012) Role of proline under changing environments. Plant Signal Behav 7:1456–1466

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Hebbelmann I, Selinski J, Wehmeyer C, Goss T, Voss I, Mulo P, Kangasjarvi S, Aro EM, Oelze ML, Dietz KJ, Nunes-Nesi A, Do PT, Fernie AR, Talla SK, Raghavendra AS, Linke V, Scheibe R (2012) Multiple strategies to prevent oxidative stress in Arabidopsis plants lacking the malate valve enzyme NADP-malate dehydrogenase. J Exp Bot 63:1445–1459

    Article  PubMed  CAS  Google Scholar 

  • Hemavathi UCP, Akula N, Kim HS, Jeon JH, Ho OM, Chun SC, Kim DH, Park SW (2011) Biochemical analysis of enhanced tolerance in transgenic potato plants overexpressing D-galacturonic acid reductase gene in response to various abiotic stresses. Mol Breed 28:105–115

    Article  CAS  Google Scholar 

  • Hodges DM, DeLong JM, Forney CF, Prange RK (1999) Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta 207:604–611

    Article  CAS  Google Scholar 

  • Huang Z, Zhao L, Chen D, Liang M, Liu Z (2013) Salt stress encourages proline accumulation by regulating proline biosynthesis and degradation in Jerusalem artichoke plantlets. PLoS One 8:e62085

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Kishor PBK, 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

    Article  CAS  Google Scholar 

  • Kishor PBK, Sangam S, Amrutha RN, SriLaxmi P, Naidu KR, Rao KRSS (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

    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

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Kulaeva OA, Zhernakov AI, Afonin AM, Boikov SS, Sulima AS, Tikhonovich IA, Zhukov VA (2017) Pea marker database (PMD) - a new online database combining known pea (Pisum sativum L.) gene-based markers. PLoS One 12:e018713

    Article  CAS  Google Scholar 

  • Kwon KC, Verma D, Jin S, Singh ND, Daniell H (2013) Release of proteins from intact chloroplasts induced by reactive oxygen species during biotic and abiotic stress. PLoS One 8:e67106

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Lee S, Seo PJ, Lee HJ, Park C (2012) A NAC transcription factor NTL4 promotes reactive oxygen species production during drought-induced leaf senescence in Arabidopsis. Plant J 70:831–844

    Article  PubMed  CAS  Google Scholar 

  • Lehmann M, Schwarzlander M, Obata T, Sirikantaramas S, Burow M, Olsen CE, Tohge T, Fricker MD, Møller BL, Fernie AR, Sweetlove LJ, Laxa M (2009) The metabolic response of arabidopsis roots to oxidative stress is distinct from that of heterotrophic cells in culture and highlights a complex relationship between the levels of transcripts, metabolites, and flux. Mol Plant 2:390–406

    Article  PubMed  CAS  Google Scholar 

  • Li J, Mu J, Bai J, Fu F, Zou T, An F, Zhang J, Jing H, Wang Q, Li Z, Yang S, Zuo J (2013) Paraquat resistant 1, a golgi-localized putative transporter protein, is involved in intracellular transport of paraquat. Plant Physiol 162:470–483

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Li Z, Yu J, Peng Y, Huang B (2016) Metabolic pathways regulated by γ-aminobutyric acid (GABA) contributing to heat tolerance in creeping bentgrass (Agrostis stolonifera). Sci Rep 6:30338

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Liang X, Zhang L, Natarajan SK, Becker DF (2013) Proline mechanisms of stress survival. Antioxid Redox Signal 19:998–1011

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Moustaka J, Tanou G, Adamakis ID, Elefteriou EP, Moustakas M (2015) Leaf age-dependent photoprotective and antioxidative response mechanisms to paraquat-induced oxidative stress in Arabidopsis thaliana. Int J Mol Sci 16:13989–14006

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Noctor G, Mhamdi A, Chaouch S, Han Y, Neukermans J, Marquez-Garcia B, Queval G, Foyer CH (2012) Glutathione in plants: an integrated overview. Plant Cell Environ 35:454–484

    Article  PubMed  CAS  Google Scholar 

  • Obata T, Matthes A, Koszior S, Lehmann M, Araujo WL, Bock R, Sweetlove LJ, Fernie AR (2011) Alteration of mitochondrial protein complexes in relation to metabolic regulation under short-term oxidative stress in Arabidopsis seedlings. Phytochemistry 72:1081–1091

    Article  PubMed  CAS  Google Scholar 

  • Ozden M, Demirel U, Kahraman A (2009) Effects of proline on antioxidant system in leaves of grapevine (Vitis vinifera L.) exposed to oxidative stress by H2O2. Sci Hort 119:163–168

    Article  CAS  Google Scholar 

  • Parida AK, Dagaonkar VS, Phalak MS, Aurangabadkar LP (2008) Differential responses of the enzymes involved in proline biosynthesis and degradation in drought tolerant and sensitive cotton genotypes during drought stress and recovery. Acta Physiol Plant 30:619–627

    Article  CAS  Google Scholar 

  • Raghavendra AS, Padmasree K (2003) Beneficial interactions of mitochondrial metabolism with photosynthetic carbon assimilation. Trends Plant Sci 8:546–553

    Article  PubMed  CAS  Google Scholar 

  • Rejeb KB, Vos LD, Le Disquet I, Leprince AS, Bordenave M, Maldiney R, Asma J, Abdelly C, Savour A (2015) Hydrogen peroxide produced by NADPH oxidases increases proline accumulation during salt or mannitol stress in Arabidopsis thaliana. New Phytol 208:1138–1148

    Article  PubMed  CAS  Google Scholar 

  • Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132:365–386

    PubMed  CAS  Google Scholar 

  • Sánchez E, López-Lefebre LR, García PC, Rivero RM, Ruiz JM, Romero L (2001) Proline metabolism in response to highest nitrogen dosages in green bean plants (Phaseolus vulgaris L. cv. Strike). J Plant Physiol 158:593–598

    Article  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  PubMed  PubMed Central  CAS  Google Scholar 

  • Shinde S, Villamor JG, Lin W, Sharma S, Verslues PE (2016) Proline coordination with fatty acid synthesis and redox metabolism of chloroplast and mitochondria. Plant Physiol 172:1074–1088

    PubMed  PubMed Central  CAS  Google Scholar 

  • Signorelli S (2016) The fermentation analogy: a point of view for understanding the intriguing role of proline accumulation in stressed plants. Front Plant Sci 7:1339

    Article  PubMed  PubMed Central  Google Scholar 

  • Sperdouli I, Moustakas M (2012) Interaction of proline, sugars, and anthocyanins during photosynthetic acclimation of Arabidopsis thaliana to drought stress. J Plant Physiol 169:577–585

    Article  PubMed  CAS  Google Scholar 

  • Sunil B, Talla SK, Aswani V, Raghavendra AS (2013) Optimization of photosynthesis by multiple metabolic pathways involving interorganelle interactions: resource sharing and ROS maintainance as the bases. Photosynth Res 117:61–71

    Article  PubMed  CAS  Google Scholar 

  • Sweetlove LJ, Heazlewood JL, Herald V, Holtzapffel R, Day DA, Leaver CJ, Millar AH (2002) The impact of oxidative stress on Arabidopsis mitochondria. Plant J 32:891–904

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Szalai G, Kellos T, Galiba G, Kocsy G (2009) Glutathione as an antioxidant and regulatory molecule in plants under abiotic stress conditions. J Plant Growth Regul 28:66–80

    Article  CAS  Google Scholar 

  • Szarka A, Banhegyi G, Asard H (2013) The inter-relationship of ascorbate transport, metabolism and mitochondrial, plastidic respiration. Antioxid Redox Signal 19:1036–1044

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Talla SK, Riazunnisa K, Padmavathi L, Sunil B, Rajsheel P, Raghavendra AS (2011) Ascorbic acid is a key participant during the interactions between chloroplasts and mitochondria to optimize photosyhthesis and protect against photoinhibition. J Biosci 36:163–173

    Article  PubMed  CAS  Google Scholar 

  • Towbin H, Staehlin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. Procedure and some applications. Proc Natl Acad Sci U S A 76:4350–4354

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Wang H, Tang X, Wang H, Shao HB (2015) Proline accumulation and metabolism-related genes expression profiles in Kosteletzkya virginica seedlings under salt stress. Front Plant Sci 6:792

    PubMed  PubMed Central  Google Scholar 

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Authors thank Prof. Maria Elena Alvarez, Faculty of Chemistry, National University of Cordoba, Argentina for kindly providing us with proline dehydrogenase antibodies for our experiments.


This work was supported by grants to ASR from Council of Scientific and Industrial Research (No. 38(1404)/15/EMR-II), JC Bose National Fellowship (No. SR/S2/JCB-06/2006). VA, PR, and RBB were all supported by Research Fellowships from University Grants Commission, New Delhi, India. We also thank grants from DST-FIST, UGC-SAP-CAS, and DBT-CREBB, from New Delhi, India for support of infrastructure in Department/School.

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ASR designed the study. VA performed most of the experiments. PR, RBB, and BS performed some experiments. VA and ASR analyzed results and wrote the manuscript. VA, PR, RBB, BS, and ASR revised and finalized the manuscript. All the authors read and approved the final manuscript.

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Correspondence to Agepati S. Raghavendra.

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Aswani, V., Rajsheel, P., Bapatla, R.B. et al. Oxidative stress induced in chloroplasts or mitochondria promotes proline accumulation in leaves of pea (Pisum sativum): another example of chloroplast-mitochondria interactions. Protoplasma 256, 449–457 (2019).

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