Insights into Salt Stress-Induced Biochemical, Molecular and Epigenetic Regulation of Spatial Responses in Pigeonpea (Cajanus cajan L.)

  • Monika Awana
  • Karmveer Yadav
  • Kirti Rani
  • Kishor Gaikwad
  • Shelly Praveen
  • Suresh Kumar
  • Archana SinghEmail author


Pigeonpea (Cajanus cajan L.) is a rich source of nutritionally good quality proteins, carbohydrates, minerals, and vitamins. However, environmental stresses adversely affect its productivity. Only limited reports are available on biochemical/physiological responses of pigeonpea under salt stress. The objectives of the present study were to screen pigeonpea germplasm accessions for salt stress tolerance, followed by understanding their biochemical, epigenetic and molecular responses. Based on germination, growth, and vigor of seedlings under salt stress, the most contrasting pair of salt-responsive genotypes (ICP1071- most salt-sensitive, and ICP7- most salt-tolerant) were selected. Three-week-old seedlings subjected to 250 mM NaCl stress for 7 days showed a significant increase in proline and reducing sugar contents in the case of ICP7, whereas a considerable increase in cell wall-degrading enzyme activity and protein oxidation was observed in ICP1071. Superoxide dismutase, peroxidase, and glutathione reductase activity increased considerably in shoots of ICP7. We observed the CcCYP gene to be upregulated in root, whereas CcCDR was upregulated in shoots of the salt-tolerant genotype to provide protection against the stress. The extent of DNA hypomethylation in the contrasting pigeonpea genotypes under salt stress was correlated with their salt tolerance level. Bisulfite sequencing of CcCDR revealed that methylation of three cytosine residues in CHH context in shoots of the ICP7 genotype due to salt stress results in 2.6-fold upregulated expression of the gene. With a 6.8% increase in methylation of the coding region of CcCDR, its expression level increased by 22%. To the best of our knowledge, this is the first report on a comprehensive study of salt-induced biochemical, epigenetic and molecular responses of pigeonpea, which might be useful in the development of improved salt-tolerant variety.


Antioxidant activity Cajanus cajan Cell wall-degrading enzyme Epigenetics Pigeonpea Salinity 



MA and KY acknowledge Fellowship from the Department of Biotechnology (DBT), Government of India. AS and KG acknowledge the financial support from the DBT (BT/Bio-CARe/02/45/2012) to carry out the research work. The authors acknowledge Director, IARI, New Delhi, for the facilities and supports provided. AS sincerely acknowledge the ICRISAT, Hyderabad, for providing salt-responsive Mini-core collections of pigeonpea, and Dr. R.S. Raje for providing part of the pigeonpea germplasm collections from Pulse Research Laboratory, Division of Genetics, IARI, New Delhi.

Compliance with Ethical Standards

Conflict of interest

The authors declare that there is no conflict of interest.

Supplementary material

344_2019_9955_MOESM1_ESM.xlsx (16 kb)
Supplementary material 1 (XLSX 15 KB)
344_2019_9955_MOESM2_ESM.xlsx (17 kb)
Supplementary material 2 (XLSX 16 KB)
344_2019_9955_MOESM3_ESM.xlsx (13 kb)
Supplementary material 3 (XLSX 12 KB)


  1. Abbas SR, Ahmad SD, Sabir SM, Shah AH (2014) Detection of drought tolerant sugarcane genotypes (Saccharum officinarum) using lipid peroxidation, antioxidant activity, glycine-betaine and proline contents. J Soil Sci Plant Nutr 14:233–243Google Scholar
  2. AbdElgawad H, Zinta G, Hegab MM, Pandey R, Asard H, Abuelsoud W (2016) High salinity induces different oxidative stress and antioxidant responses in maize seedlings organs. Front Plant Sci 7:e276Google Scholar
  3. Abid G, Mingeot D, Muhovski Y, Mergeai G, Aouida M, Abdelkarim S, Aroua I, El Ayed M, M’hamdi M, Sassi K, Jebara M (2017) Analysis of DNA methylation patterns associated with drought stress response in faba bean (Vicia faba L.) using methylation-sensitive amplification polymorphism (MSAP). Environ Exp Bot 142:34–44CrossRefGoogle Scholar
  4. Arefian M, Vessal S, Shafaroudi SM, Bagheri A (2018) Comparative analysis of the reaction to salinity of different chickpea (Cicer aretinum L.) genotypes: a biochemical, enzymatic and transcriptional study. J Plant Growth Regul 37:91–402CrossRefGoogle Scholar
  5. Arzani A (2008) Improving salinity tolerance in crop plants: a biotechnological view. In Vitro Cell Dev Biol–Plant 44:373–383CrossRefGoogle Scholar
  6. Aysin F, Erson-Bensan AE, Eyidogan F, Öktem HA (2015) Generating salt-tolerant Nicotiana tabacum and identification of stress-responsive miRNAs in transgenics. Turk J Bot 39:757–768CrossRefGoogle Scholar
  7. Bates LS, Waldran R, Teare ID (1973) Rapid determination of free proline for water studies. Plant Soil 39:205–208CrossRefGoogle Scholar
  8. Bewick AJ, Ji L, Niederhuth CE, Willing E-M, Hofmeister BT et al (2016) On the origin and evolutionary consequences of gene body DNA methylation. Proc Natl Acad Sci USA 113:9111–9116CrossRefGoogle Scholar
  9. Chaves MM, Oliveira MM (2004) Mechanisms underlying plant resilience to water deficits: prospects for water-saving agriculture. J Exp Bot 55:2365–2384CrossRefGoogle Scholar
  10. Chawla S, Jain S, Jain V (2013) Salinity induced oxidative stress and antioxidant system in salt-tolerant and salt-sensitive cultivars of rice (Oryza sativa L.). J Plant Biochem Biotechnol 22:27–34CrossRefGoogle Scholar
  11. Chikha MB, Hessini K, Ourteni RM, Ghorbel A, Zoghlami N (2016) Identification of barley landrace genotypes with contrasting salinity tolerance at vegetative growth stage. Plant Biotechnol. Google Scholar
  12. Cokus SJ, Feng S, Zhang X, Chen Z, Merriman B et al (2008) Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature 452:215–219CrossRefGoogle Scholar
  13. Danekar P, Tyagi A, Mahto A, Krishna KG, Singh A, Raje RS, Gaikwad K, Singh NK (2014) Genome wide characterization of Hsp 100 family genes from pigeonpea. Indian J Genet Plant Breed 74:325–334CrossRefGoogle Scholar
  14. de Freitas PA, de Souza Miranda R, Marques EC, Prisco JT, Gomes-Filho E (2018) Salt tolerance induced by exogenous proline in maize is related to low oxidative damage and favorable ionic homeostasis. J Plant Growth Regul 1–4Google Scholar
  15. Deaton AM, Bird A (2011) CpG islands and the regulation of transcription. Genes Dev 25:1010–1022CrossRefGoogle Scholar
  16. Demiral T, Türkan I (2004) Does exogenous glycinebetaine affect antioxidative system of rice seedlings under NaCl treatment? J Plant Physiol 161:1089–1100CrossRefGoogle Scholar
  17. Demiral T, Türkan I (2005) Comparative lipid peroxidation, antioxidant defense systems and proline content in roots of two rice cultivars differing in salt tolerance. Environ Exp Bot 53:247–257CrossRefGoogle Scholar
  18. Foyer CH, Noctor G (2003) Redox sensing and signaling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria. Physiol Plant 119:355–364CrossRefGoogle Scholar
  19. Garratt LC, Janagoudar BS, Lowe KC, Anthony P, Power JB, Davey MR (2002) Salinity tolerance and antioxidant status in cotton cultures. Free Radic Biol Med 33:502–511CrossRefGoogle Scholar
  20. Gharsallah C, Fakhfakh H, Grubb D, Gorsane F (2016) Effect of salt stress on ion concentration, proline content, antioxidant enzyme activities and gene expression in tomato cultivars. AoB Plants 8:plw055. CrossRefGoogle Scholar
  21. Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930CrossRefGoogle Scholar
  22. Gupta B, Huang B (2014) Mechanism of salinity tolerance in Plants: Physiological, biochemical, and molecular characterization. Int J Genomics. Google Scholar
  23. Hernandez JA, Jimenez A, Mullineaux P, Sevilla F (2000) Tolerance of pea (Pisum sativum L.) to long-term salt stress is associated with induction of antioxidant defences. Plant Cell Environ 23:853–862CrossRefGoogle Scholar
  24. Javed S, Bukhari SA, Ashraf MY, Mahmood S, Iftikhar T (2014) Effect of salinity on growth, biochemical parameters and fatty acid composition in safflower (Carthamus tinctorius L.). Pak J Bot 46:115–358Google Scholar
  25. Kerepesi I, Galiba G (2000) Osmotic and salt stress-induced alteration in soluble carbohydrate content in wheat seedlings. Crop Sci 40:482–487CrossRefGoogle Scholar
  26. Konate M, Wilkinson MJ, Mayne BT, Pederson SM, Scott ES, Berger B, Lopez CM (2018) Salt stress induces non-CG methylation in coding regions of barley seedlings (Hordeum vulgare). Epigenomes 2:12. CrossRefGoogle Scholar
  27. Krishnan HB, Natarajan SS, Oehrle NW, Garrett WM, Darwish O (2017) Proteomic analysis of pigeonpea (Cajanus cajan) seeds reveals the accumulation of numerous stress-related proteins. J Agric Food Chem 65:4572–4581CrossRefGoogle Scholar
  28. Kudapa H, Bharti AK, Cannon SB, Farmer AD, Mulaosmanovic B, Kramer R et al (2012) A comprehensive transcriptome assembly of pigeonpea (Cajanus cajan L.) using sanger and second-generation sequencing platforms. Mol Plant 5:1020–1028CrossRefGoogle Scholar
  29. Kumar S (2018a) Epigenetic memory of stress responses in plants. J Phytochem Biochem 2:e102Google Scholar
  30. Kumar S (2018b) Epigenomics of plant responses to environmental stress. Epigenomes 2:e6. CrossRefGoogle Scholar
  31. Kumar S, Singh A (2016) Epigenetic regulation of abiotic stress tolerance in plants. Adv Plants Agric Res 5:00179. Google Scholar
  32. Kumar M, Hassan M, Arora A, Gaikwad K, Kumar S, Rai RD, Singh A (2015) Sodium chloride-induced spatial and temporal manifestation in membrane stability index and protein profiles of contrasting wheat (Triticum aestivum L.) genotypes under salt stress. Indian J Plant Physiol 20:271–275CrossRefGoogle Scholar
  33. Kumar S, Beena AS, Awana M, Singh A (2017a) Salt-induced tissue-specific cytosine methylation downregulates expression of HKT genes in contrasting wheat (Triticum aestivum L.) genotypes. DNA Cell Biol 36:283–294CrossRefGoogle Scholar
  34. Kumar S, Beena AS, Awana M, Singh A (2017b) Physiological, biochemical, epigenetic and molecular analyses of wheat (Triticum aestivum) genotypes with contrasting salt tolerance. Front Plant Sci 8:e1151. CrossRefGoogle Scholar
  35. Kumar S, Singh AK, Mohapatra T (2017c) Epigenetics: history, present status and future perspective. Indian J Genet 77:445–463Google Scholar
  36. Kumar S, Chinnusamy V, Mohapatra T (2018) Epigenetics of modified DNA bases: 5-methylcytosine and beyond. Front Genet 9:e640. CrossRefGoogle Scholar
  37. Levin DE (2011) Regulation of cell wall biogenesis in Saccharomyces cerevisiae: the cell wall integrity signaling pathway. Genetics 189:1145–1175CrossRefGoogle Scholar
  38. Li Y, Kumar S, Qian W (2018) Active DNA demethylation: mechanism and role in plant development. Plant Cell Rep 37:77–85. CrossRefGoogle Scholar
  39. Lira-Medeiros CF, Parisod C, Fernandes RA, Mata CS, Cardoso MA (2010) Epigenetic variation in mangrove plants occurring in contrasting natural environment. PLoS ONE 5:e10326CrossRefGoogle Scholar
  40. Mellacheruvu S, Tamirisa S, Vudem DS, Khareedu VR (2016) Pigeonpea hybrid-Proline-rich protein (CcHyPRP) confers biotic and abiotic stress tolerance in transgenic rice. Front Plant Sci 6:e1167. CrossRefGoogle Scholar
  41. Meneguzzo S, Navam-Izzo F, Izzo R (1999) Antioxidative responses of shoots and roots of wheat to increasing NaCI concentrations. J Plant Physiol 155:274–280CrossRefGoogle Scholar
  42. Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol 59:651–681CrossRefGoogle Scholar
  43. Nelson N (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem 153:375–380Google Scholar
  44. Nigam D, Saxena S, Ramakrishna G, Singh A, Singh NK, Gaikwad K (2017) De novo assembly and characterization of Cajanus scarabaeoides (L.) thouars transcriptome by pair-end sequencing. Front Mol Biosci 4:e48. doi.10.3389/fmolb.2017.00048CrossRefGoogle Scholar
  45. Parida AK, Das AB, Mohanty P (2004) Investigations on the antioxidative defence responses to NaCl stress in a mangrove, Bruguiera parviflora: differential regulations of isoforms of some antioxidative enzymes. Plant Growth Regul 42:213–226CrossRefGoogle Scholar
  46. Priyanka B, Sekhar K, Sunitha T, Reddy VD, Rao KV (2010) Characterization of expressed sequence tags (ESTs) of pigeonpea (Cajanus cajan L.) and functional validation of selected genes for abiotic stress tolerance in Arabidopsis thaliana. Mol Genet Genomics 283:273–287CrossRefGoogle Scholar
  47. Randhawa N, Kaur J (2015) Antioxidant responses of chickpea genotypes exposed to moisture stress. Int J Adv Res 3:50–955Google Scholar
  48. Romero-Aranda MR, Jurado O, Cuartero J (2006) Silicon alleviates the deleterious salt effect on tomato plant growth by improving plant water status. J Plant Physiol 63:847–855CrossRefGoogle Scholar
  49. Samota MK, Sasi M, Awana M, Yadav OP, Mithra SVA, Tyagi A, Kumar S, Singh A (2017) Elicitor-induced biochemical and molecular manifestations to improve drought tolerance in rice (Oryza sativa L.) through seed-priming. Front Plant Sci 8:e934. CrossRefGoogle Scholar
  50. Seifert GJ, Blaukopf C (2010) Irritable walls: The plant extracellular matrix and signaling. Plant Physiol 153:467–478CrossRefGoogle Scholar
  51. Singh A, Singh M (1993) Cell wall degrading enzymes in Orobanche aegyptiaca Pers. and its host Brassica campestris. Physiol Plant 89:177–181CrossRefGoogle Scholar
  52. Singh A, Singh M (1997) Incompatibility of Cuscuta haustoria with the resistant hosts – Ipomoea batatas L. and Lycopersicon esculentum Mill. J Plant Physiol 150:592–596CrossRefGoogle Scholar
  53. Singh NK, Gupta DK, Jayaswal PK, Mahato AK, Dutta S, Singh S et al (2012) The first draft of the pigeonpea genome sequence. J Plant Biochem Biotechnol 21:98–112CrossRefGoogle Scholar
  54. Singh A, Bhushan B, Gaikwad K, Yadav OP, Kumar S, Rai RD (2015) Induced defence response of contrasting bread wheat genotype under differential salt stress imposition. Ind J Biochem Biophys 52:78–85Google Scholar
  55. Somogyi M (1952) Notes on sugar determination. J Biol Chem 195:19–23Google Scholar
  56. Srivastava N, Vadez V, Upadhyaya HD, Saxena KB (2006) Screening for intra and inter-specific variability for salinity tolerance in pigeonpea (Cajanus cajan L. Millsp.) and its related wild species. Crop Improv 2:1–24Google Scholar
  57. Srivastava MK, Yadav C, Bhat Y, Kumar S (2011) Cloning and characterization of cDNA encoding xyloglucan endotransglucosylase in Pennisetum glaucum L. Afr J Biotechnol 10:9242–9252CrossRefGoogle Scholar
  58. Sunitha M, Tamirisa S, Vudem DR, Khareedu VR (2017) Expression of cold and drought regulatory protein (CcCDR) of pigeonpea imparts enhanced tolerance to major abiotic stresses in transgenic rice plants. Planta 245:1137–1148. CrossRefGoogle Scholar
  59. Surekha CH, Kumari KN, Aruna LV, Suneetha G, Arundhati A, Kishor PK (2014) Expression of the Vigna aconitifolia P5CSF129A gene in transgenic pigeonpea enhances proline accumulation and salt tolerance. Plant Cell Tissue Organ Cult 116:27–36CrossRefGoogle Scholar
  60. Tamirisa S, Reddy VD, Rao KV (2014) Ectopic expression of pigeonpea cold and drought regulatory protein (CcCDR) in yeast and tobacco affords multiple abiotic stress tolerance. Plant Cell Tissue Org Cult 119:489–499CrossRefGoogle Scholar
  61. Valifard M, Mohsenzadeh S, Kholdebarin B, Rowshan V (2014) Effects of salt stress on volatile compounds, total phenolic content and antioxidant activities of Salvia mirzayanii. South Afr J Bot 93:92–97CrossRefGoogle Scholar
  62. VazPatto MC, Amarowicz R, Aryee ANA, Boye JI, Chung HJ, Martín-Cabrejas MA, Domoney C (2014) Achievements and challenges in improving the nutritional quality of food legumes. Crit Rev Plant Sci 34:105–143CrossRefGoogle Scholar
  63. Waheed A, Ishfaq AH, Ghulam Q, Ghulam M, Tariq M, Muhammad A (2006) Effect of salinity on germination, growth, yield, ionic balance and solute composition of pigeonpea (Cajanus cajan (L.) Millsp). Pak J Bot 38:1103–1117Google Scholar
  64. Wang M, Qin L, Xie C, Li W, Yuan J, Kong L, Yu W, Xia G, Liu S (2014) Induced and constitutive DNA methylation in a salinity-tolerant wheat introgression line. Plant Cell Physiol 55:1354–1365CrossRefGoogle Scholar
  65. Wang X, Li Q, Yuan W, Cao Z, Qi B, Kumar S, Li Y, Qian W (2016) The cytosolic Fe–S cluster assembly component MET18 is required for the full enzymatic activity of ROS1 in active DNA demethylation. Sci Rep 6:e26443CrossRefGoogle Scholar
  66. Wu W, Zhang Q, Ervin EH, Yang Z, Zhang X (2017) Physiological mechanism of enhancing salt stress tolerance of perennial ryegrass by 24-epibrassinolide. Front Plant Sci 8:e1017. CrossRefGoogle Scholar
  67. Xia H, Huang W, Xiong J, Yan S, Tao T, Li J, Wu J, Luo L (2017) Differentially methylated epiloci generated from numerous genotypes of contrasting tolerances are associated with osmotic-tolerance in rice seedlings. Front Plant Sci 8:e11Google Scholar
  68. Zemach A, McDaniel IE, Silva P, Zilberman D (2010) Genome-wide evolutionary analysis of eukaryotic DNA methylation. Science 328:916–919CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Monika Awana
    • 1
    • 2
  • Karmveer Yadav
    • 1
  • Kirti Rani
    • 2
  • Kishor Gaikwad
    • 3
  • Shelly Praveen
    • 1
  • Suresh Kumar
    • 1
  • Archana Singh
    • 1
    Email author
  1. 1.Division of BiochemistryICAR-Indian Agricultural Research InstituteNew DelhiIndia
  2. 2.Amity Institute of BiotechnologyAmity UniversityNoidaIndia
  3. 3.National Research Centre on Plant BiotechnologyNew DelhiIndia

Personalised recommendations