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Molecular Biology Reports

, Volume 46, Issue 6, pp 6039–6052 | Cite as

Isolation and functional characterization of three abiotic stress-inducible (Apx, Dhn and Hsc70) promoters from pearl millet (Pennisetum glaucum L.)

  • Kummari Divya
  • P. B. Kavi Kishor
  • Pooja Bhatnagar-Mathur
  • Prashanth Singam
  • Kiran K. Sharma
  • Vincent Vadez
  • Palakolanu Sudhakar ReddyEmail author
Original Article
  • 160 Downloads

Abstract

Pearl millet is a C4 cereal crop that grows in arid and semi-arid climatic conditions with the remarkable abiotic stress tolerance. It contributed to the understanding of stress tolerance not only at the physiological level but also at the genetic level. In the present study, we functionally cloned and characterized three abiotic stress-inducible promoters namely cytoplasmic Apx1 (Ascorbate peroxidase), Dhn (Dehydrin), and Hsc70 (Heat shock cognate) from pearl millet. Sequence analysis revealed that all three promoters have several cis-acting elements specific for temporal and spatial expression. PgApx pro, PgDhn pro and PgHsc70 pro were fused with uidA gene in Gateway-based plant transformation pMDC164 vector and transferred into tobacco through leaf-disc method. While PgApx pro and PgDhn pro were active in seedling stages, PgHsc70 pro was active in stem and root tissues of the T2 transgenic tobacco plants under control conditions. Higher activity was observed under high temperature and drought, and less in salt and cold stress conditions. Further, all three promoters displayed higher GUS gene expression in the stem, moderate expression in roots, and less expression in leaves under similar conditions. While RT-qPCR data showed that PgApx pro and PgDhn pro were expressed highly in high temperature, salt and drought, PgHsc70 pro was fairly expressed during high temperature stress only. Histochemical and RT-qPCR assays showed that all three promoters are inducible under abiotic stress conditions. Thus, these promoters appear to be immediate candidates for developing abiotic stress tolerant crops as these promoter-driven transgenics confer high degree of tolerance in comparison with the wild-type (WT) plants.

Keywords

Abiotic stress-inducible promoters Pearl millet Cis-acting elements PgApx pro PgDhn pro PgHsc70 pro 

Abbreviations

Pg

Pennisitum glaucum

Apx

Ascorbate peroxidase

Dhn

Dehydrin

Hsc70

Heat shock cognate 70

GE

Genetic engineering

SOD

Superoxide dismutase

BAP

6-benzylaminopurine

NAA

Napthaleneacetic acid

GUS

ß-glucuronidase

MS

Murashige and Skoog

Notes

Acknowledgements

PSR acknowledges the Department of Science and Technology, New Delhi, Government of India for the research grant and award through INSPIRE Faculty (Award No. IFA11-LSPA-06). This work was undertaken as part of the CGIAR Research Program on GLDC.

Author contributions

PSR and VV designed the experiments, KD, PSR, PBM and PS executed the study, PSR, KD and PBK analyzed data. PSR, KKS, PBK and KD wrote the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary material

11033_2019_5039_MOESM1_ESM.docx (15 kb)
Supplementary material 1 (DOCX 15 kb)

References

  1. 1.
    Grayson M (2013) Agriculture and drought. Nature 501:S1.  https://doi.org/10.1038/501S1a CrossRefPubMedGoogle Scholar
  2. 2.
    Christensen JH, Christensen OB (2007) A summary of the PRUDENCE model projections of changes in European climate by the end of this century. Clim Chang 81:7–30Google Scholar
  3. 3.
    Cramer GR, Urano K, Delrot S, Pezzotti M, Shinozaki K (2011) Effects of abiotic stress on plants: a systems biology perspective. BMC Plant Biol 11:163PubMedPubMedCentralGoogle Scholar
  4. 4.
    Krasensky J, Jonak C (2012) Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. J Exp Bot 63:1593–1608PubMedPubMedCentralGoogle Scholar
  5. 5.
    Mishra RN, Reddy PS, Nair S, Markandeya G, Reddy AR, Sopory SK, Reddy MK (2007) Isolation and characterization of expressed sequence tags (ESTs) from subtracted cDNA libraries of Pennisetum glaucum seedlings. Plant Mol Biol 64:713–732PubMedGoogle Scholar
  6. 6.
    Yamaguchi-Shinozaki K, Shinozaki K (2006) Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu Rev Plant Biol 57:781–803PubMedGoogle Scholar
  7. 7.
    Morozova O, Marra MA (2008) Applications of next-generation sequencing technologies in functional genomics. Genomics 92:255–264PubMedGoogle Scholar
  8. 8.
    Schuster SC (2007) Next-generation sequencing transforms today’s biology. Nat Methods 5:16PubMedGoogle Scholar
  9. 9.
    Hirt H, Shinozaki K (2003) Plant responses to abiotic stress. Springer Science, BerlinGoogle Scholar
  10. 10.
    Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399PubMedGoogle Scholar
  11. 11.
    Bihmidine S, Lin J, Stone JM, Awada T, Specht JE, Clemente TE (2013) Activity of the Arabidopsis RD29A and RD29B promoter elements in soybean under water stress. Planta 237:55–64PubMedGoogle Scholar
  12. 12.
    Mur LA, Sturgess FJ, Farrell GG, Draper J (2004) The AoPR10 promoter and certain endogenous PR10 genes respond to oxidative signals in Arabidopsis. Mol Plant Pathol 5:435–451PubMedGoogle Scholar
  13. 13.
    Srivastava VK, Raikwar S, Tuteja N (2014) Cloning and functional characterization of the promoter of PsSEOF1 gene from Pisum sativum under different stress conditions using Agrobacterium-mediated transient assay. Plant Signal Behav 9(9):e29626PubMedPubMedCentralGoogle Scholar
  14. 14.
    Rerksiri W, Zhang X, Xiong H, Chen X (2013) Expression and promoter analysis of six heat stress-inducible genes in rice. Sci W J.  https://doi.org/10.1155/2013/397401 CrossRefGoogle Scholar
  15. 15.
    Chakravarthi M, Syamaladevi DP, Harunipriya P, Augustine SM, Subramonian N (2016) A novel PR10 promoter from Erianthus arundinaceus directs high constitutive transgene expression and is enhanced upon wounding in heterologous plant systems. Mol Biol Rep 43:17–30PubMedGoogle Scholar
  16. 16.
    Hou J, Jiang P, Qi S, Zhang K, He Q, Xu C, Li K (2016) Isolation and functional validation of salinity and osmotic stress inducible promoter from the maize type-II H + - pyrophosphatase gene by deletion analysis in transgenic tobacco plants. PLoS ONE 11:e0154041PubMedPubMedCentralGoogle Scholar
  17. 17.
    Bhuria M, Goel P, Kumar S, Singh AK (2016) The promoter of AtUSP is co-regulated by phytohormones and abiotic stresses in Arabidopsis thaliana. Front Plant Sci 7:1957PubMedPubMedCentralGoogle Scholar
  18. 18.
    Pellegrineschi A, Reynolds M, Pacheco M, Brito RM, Almeraya R, Yamaguchi- Shinozaki K, Hoisington D (2004) Stress-induced expression in wheat of the Arabidopsis thaliana DREB1A gene delays water stress symptoms under greenhouse conditions. Genome 47:493–500PubMedGoogle Scholar
  19. 19.
    Nakashima K, Tran LSP, Van Nguyen D, Fujita M, Maruyama K, Todaka D, Yamaguchi- Shinozaki K (2007) Functional analysis of a NAC type transcription factor OsNAC6 involved in abiotic and biotic stress responsive gene expression in rice. Plant J 51:617–630PubMedGoogle Scholar
  20. 20.
    Li F, Han Y, Feng Y, Xing S, Zhao M, Chen Y, Wang W (2013) Expression of wheat expansin driven by the RD29 promoter in tobacco confers water-stress tolerance without impacting growth and development. J Biotechnol 163:281–291PubMedGoogle Scholar
  21. 21.
    Shafi A, Pal AK, Sharma V, Kalia S, Kumar S, Ahuja PS, Singh AK (2017) Transgenic potato plants overexpressing SOD and APX exhibit enhanced lignification and starch biosynthesis with improved salt stress tolerance. Plant Mol Biol Rep 35:504–518Google Scholar
  22. 22.
    Wang Y, Wisniewski M, Meilan R, Cui M, Webb R, Fuchigami L (2005) Overexpression of cytosolic ascorbate peroxidase in tomato confers tolerance to chilling and salt stress. J Am Soc Hortic Sci 130:167–173Google Scholar
  23. 23.
    Wang Y, Wisniewski M, Meilan R, Cui M, Fuchigami L (2006) Transgenic tomato (Lycopersicon esculentum) overexpressing cAPX exhibits enhanced tolerance to UV-B and heat stress. J Appl Hort 8:87–90Google Scholar
  24. 24.
    Yabuta Y, Motoki T, Yoshimura K, Takeda T, Ishikawa T, Shigeoka S (2002) Thylakoid membrane-bound ascorbate peroxidase is a limiting factor of antioxidative systems under photo-oxidative stress. Plant J 32:915–925PubMedGoogle Scholar
  25. 25.
    Sato Y, Masuta Y, Saito K, Murayama S, Ozawa K (2011) Enhanced chilling tolerance at the booting stage in rice by transgenic overexpression of the ascorbate peroxidase gene, OsAPXa. Plant Cell Rep 30:399–406PubMedGoogle Scholar
  26. 26.
    Ishikawa T, Morimoto Y, Madhusudhan R, Sawa Y, Shibata H, Yabuta Y, Shigeoka S (2005) Acclimation to diverse environmental stresses caused by a suppression of cytosolic ascorbate peroxidase in tobacco BY-2 cells. Plant Cell Physiol 46:1264–1271PubMedGoogle Scholar
  27. 27.
    Sun WH, Duan M, Li F, Shu DF, Yang S, Meng QW (2010) Overexpression of tomato tAPX gene in tobacco improves tolerance to high or low temperature stress. Biol Plant 54:614–620Google Scholar
  28. 28.
    Graether SP, Boddington KF (2014) Disorder and function: a review of the dehydrin protein family. Front Plant Sci 5:576PubMedPubMedCentralGoogle Scholar
  29. 29.
    Singh J, Reddy PS, Reddy CS, Reddy MK (2015) Molecular cloning and characterization of salt inducible dehydrin gene from the C4 plant Pennisetum glaucum. Plant Gene 4:55–63Google Scholar
  30. 30.
    Nagaraju M, Reddy PS, Kumar SA, Kumar A, Suravajhala P, Ali A, Rao DM (2018) Genome-wide in silico analysis of dehydrins in Sorghum bicolor, Setaria italica and Zea mays and quantitative analysis of dehydrin gene expressions under abiotic stresses in Sorghum bicolor. Plant Gene 13:64–75Google Scholar
  31. 31.
    Peng Y, Reyes JL, Wei H, Yang Y, Karlson D, Covarrubias AA, Arora R (2008) RcDhn5, a cold acclimation-responsive dehydrin from Rhododendron catawbiense rescues enzyme activity from dehydration effects in vitro and enhances freezing tolerance in RcDhn5 overexpressing Arabidopsis plants. Physiol Plant 134:583–597PubMedGoogle Scholar
  32. 32.
    Ganguly M, Datta K, Roychoudhury A, Gayen D, Sengupta DN, Datta SK (2012) Overexpression of Rab16A gene in indica rice variety for generating enhanced salt tolerance. Plant Signal Behav 7:502–509PubMedPubMedCentralGoogle Scholar
  33. 33.
    Cheng WH, Endo A, Zhou L, Penney J, Chen HC, Arroyo A, Koshiba T (2002) A unique short-chain dehydrogenase/reductase in Arabidopsis glucose signaling and abscisic acid biosynthesis and functions. Plant Cell 14:2723–2743PubMedPubMedCentralGoogle Scholar
  34. 34.
    Brini F, Hanin M, Lumbreras V, Amara I, Khoudi H, Hassairi A, Masmoudi K (2007) Overexpression of wheat dehydrin DHN-5 enhances tolerance to salt and osmotic stress in Arabidopsis thaliana. Plant Cell Rep 26:2017–2026PubMedGoogle Scholar
  35. 35.
    Roy Choudhury A, Roy C, Sengupta DN (2007) Transgenic tobacco plants overexpressing the heterologous lea gene Rab16A from rice during high salt and water deficit display enhanced tolerance to salinity stress. Plant Cell Rep 26:1839–1859Google Scholar
  36. 36.
    Reddy PS, Mallikarjuna G, Kaul T, Chakradhar T, Mishra RN, Sopory SK, Reddy MK (2010) Molecular cloning and characterization of gene encoding for cytoplasmic Hsc70 from Pennisetum glaucum may play a protective role against abiotic stresses. Mol Genet Genomics 283:243–254PubMedGoogle Scholar
  37. 37.
    Reddy PS, Kishor PBK, Seiler C, Kuhlmann M, Eschen-Lippold L, Lee J, Sreenivasulu N (2014) Unraveling regulation of the small heat shock proteins by the heat shock factor HvHsfB2c in barley: its implications in drought stress response and seed development. PLoS ONE 9(3):e89125PubMedPubMedCentralGoogle Scholar
  38. 38.
    Reddy PS, Sharma KK, Vadez V, Reddy MK (2015) Molecular cloning and differential expression of cytosolic class I small Hsp gene family in Pennisetum glaucum (L.). Appl Biochem Biotechnol 176:598–612PubMedGoogle Scholar
  39. 39.
    Reddy PS, Chakradhar T, Reddy RA, Nitnavare RB, Mahanty S, Reddy MK (2016) Role of heat shock proteins in improving heat stress tolerance in crop plants. Heat shock proteins and plants. Springer, Cham, pp 283–307Google Scholar
  40. 40.
    Nitnavare RB, Yeshvekar RK, Sharma KK, Vadez V, Reddy MK, Reddy PS (2016) Molecular cloning, characterization and expression analysis of a heat shock protein 10 (Hsp10) from Pennisetum glaucum (L.), a C4 cereal plant from the semi-arid tropics. Mol Biol Rep 43:861–870PubMedGoogle Scholar
  41. 41.
    Wang Y, Lin S, Song Q, Li K, Tao H, Huang J, He H (2014) Genome-wide identification of heat shock proteins (Hsps) and Hsp interactors in rice: Hsp70 s as a case study. BMC Genomics 15:344PubMedPubMedCentralGoogle Scholar
  42. 42.
    Zhang XP, Glaser E (2002) Interaction of plant mitochondrial and chloroplast signal peptides with the Hsp70 molecular chaperone. Trends Plant Sci 7:14–21PubMedGoogle Scholar
  43. 43.
    Hartl FU, Hayer-Hartl M (2002) Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295:1852–1858PubMedGoogle Scholar
  44. 44.
    Mirus O (1803) Schleiff E (2009) The evolution of tetratricopeptide repeat domain containing receptors involved in protein translocation-Review. Endocytobiosis Cell Res 10:1115–1130Google Scholar
  45. 45.
    Yu A, Yu A, Li P, Tang T, Wang J, Chen Y, Liu L (2015) Roles of Hsp70 s in stress responses of microorganisms, plants, and animals. BioMed Res Int.  https://doi.org/10.1155/2015/510319 CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Reddy PS, Mahanty S, Kaul T, Nair S, Sopory SK, Reddy MK (2008) A high-throughput genome-walking method and its use for cloning unknown flanking sequences. Anal Biochem 381:248–253PubMedGoogle Scholar
  47. 47.
    Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rombauts S (2002) PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res 30:325–327PubMedPubMedCentralGoogle Scholar
  48. 48.
    Chang WC, Lee TY, Huang HD, Huang HY, Pan RL (2008) PlantPAN: plant promoter analysis navigator, for identifying combinatorial cis-regulatory elements with distance constraint in plant gene groups. BMC Genomics 9:561PubMedPubMedCentralGoogle Scholar
  49. 49.
    Horsch RB, Fry J, Hoffmann N, Neidermeyer J, Rogers SG, Fraley RT (1989) Leaf disc transformation. Plant molecular biology manual. Springer, Dordrecht, pp 63–71Google Scholar
  50. 50.
    Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedPubMedCentralGoogle Scholar
  51. 51.
    Schmidt GW, Delaney SK (2010) Stable internal reference genes for normalization of real-time RT-PCR in tobacco (Nicotiana tabacum) during development and abiotic stress. Mol Genet Genomics 283:233–241PubMedGoogle Scholar
  52. 52.
    Vijayalakshmi T, Varalaxmi Y, Jainender S, Yadav SK, Vanaja M, Jyothilakshmi N, Maheswari M (2012) Physiological and biochemical basis of water-deficit stress tolerance in pearl millet hybrid and parents. Am J Plant Sci 3(12):1730Google Scholar
  53. 53.
    Pan J, Li Z, Wang Q, Garrell AK, Liu M, Guan Y, Liu W (2018) Comparative proteomic investigation of drought responses in foxtail millet. BMC Plant Biol 18(1):315PubMedPubMedCentralGoogle Scholar
  54. 54.
    Hasanuzzaman M, Nahar K, Alam M, Roychowdhury R, Fujita M (2013) Physiological, biochemical, and molecular mechanisms of heat stress tolerance in plants. Int J Mol Sci 14(5):9643–9684PubMedPubMedCentralGoogle Scholar
  55. 55.
    Reddy RA, Kumar B, Reddy PS, Mishra RN, Mahanty S, Kaul T, Reddy MK (2009) Molecular cloning and characterization of genes encoding Pennisetum glaucum ascorbate peroxidase and heat-shock factor: interlinking oxidative and heat-stress responses. J Plant Physiol 166:1646–1659PubMedGoogle Scholar
  56. 56.
    Tóth SZ, Nagy V, Puthur JT, Kovács L, Garab G (2011) The physiological role of ascorbate as photosystem II electron donor: protection against photo inactivation in heat- stressed leaves. Plant Physiol 156(1):382–392PubMedPubMedCentralGoogle Scholar
  57. 57.
    Ortner V, Ludwig A, Riegel E, Dunzinger S, Czerny T (2015) An artificial HSE promoter for efficient and selective detection of heat shock pathway activity. Cell Stress Chaperones 20:277–288PubMedGoogle Scholar
  58. 58.
    Sato Y, Murakami T, Funatsuki H, Matsuba S, Saruyama H, Tanida M (2001) Heat shock-mediated APX gene expression and protection against chilling injury in rice seedlings. J Exp Bot 52:145–151PubMedGoogle Scholar
  59. 59.
    Yang Y, He M, Zhu Z, Li S, Xu Y, Zhang C, Wang Y (2012) Identification of the dehydrin gene family from grapevine species and analysis of their responsiveness to various forms of abiotic and biotic stress. BMC Plant Biol 12:140PubMedPubMedCentralGoogle Scholar
  60. 60.
    Hu WH, Song XS, Shi K, Xia XJ, Zhou YH, Yu JQ (2008) Changes in electron transport, superoxide dismutase and ascorbate peroxidase isoenzymes in chloroplasts and mitochondria of cucumber leaves as influenced by chilling. Photosynthetica 46:581Google Scholar
  61. 61.
    Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930PubMedGoogle Scholar
  62. 62.
    Shafi A, Dogra V, Gill T, Ahuja PS, Sreenivasulu Y (2014) Simultaneous over- expression of PaSOD and RaAPX in transgenic Arabidopsis thaliana confers cold stress tolerance through increase in vascular lignifications. PLoS ONE 9:e110302PubMedPubMedCentralGoogle Scholar
  63. 63.
    Halder T, Upadhyaya G, Basak C, Das A, Chakraborty C, Ray S (2018) Dehydrins impart protection against oxidative stress in transgenic tobacco plants. Front Plant Sci 9:136PubMedPubMedCentralGoogle Scholar
  64. 64.
    Lu Z, Liu D, Liu S (2007) Two rice cytosolic ascorbate peroxidases differentially improve salt tolerance in transgenic Arabidopsis. Plant Cell Rep 26:1909–1917PubMedGoogle Scholar
  65. 65.
    Wisniewski ME, Bassett CL, Renaut J, JrR Farrell, Tworkoski T, Artlip TS (2006) Differential regulation of two dehydrin genes from peach (Prunus persica) by photoperiod, low temperature and water deficit. Tree Physiol 26:575–584PubMedGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)Patancheru, HyderabadIndia
  2. 2.Department of GeneticsOsmania UniversityHyderabadIndia

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