Molecular Biology Reports

, Volume 43, Issue 8, pp 861–870 | Cite as

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

  • Rahul B. Nitnavare
  • Richa K. Yeshvekar
  • Kiran K. Sharma
  • Vincent Vadez
  • Malireddy K. Reddy
  • Palakolanu Sudhakar Reddy
Original Article


Heat shock proteins (Hsp10) belong to the ubiquitous family of heat-shock molecular chaperones found in the organelles of both prokaryotes and eukaryotes. Chaperonins assist the folding of nascent and stress-destabilized proteins. A cDNA clone encoding a 10 kDa Hsp was isolated from pearl millet, Pennisetum glaucum (L.) by screening a heat stress cDNA library. The fulllength PgHsp10 cDNA consisted of 297 bp open reading frame (ORF) encoding a 98 amino acid polypeptide with a predicted molecular mass of 10.61 kDa and an estimated isoelectric point (pI) of 7.95. PgHsp10 shares 70–98 % sequence identity with other plant homologs. Phylogenetic analysis revealed that PgHsp10 is evolutionarily close to the maize Hsp10 homolog. The predicted 3D model confirmed a conserved eight-stranded ß-barrel with active site between the ß-barrel comprising of eight-strands, with conserved domain VLLPEYGG sandwiched between two ß-sheets. The gene consisted of 3 exons and 2 introns, while the position and phasing of these introns were conserved similar to other plant Hsp10 family genes. In silico analysis of the promoter region of PgHsp10 presented several distinct set of cis-elements and transcription factor binding sites. Quantitative RT-PCR analysis showed that PgHsp10 gene was differentially expressed in response to abiotic stresses with the highest level of expression under heat stress conditions. Results of this study provide useful information regarding the role of chaperonins in stress regulation and generated leads for further elucidation of their function in plant stress tolerance.


ABA Heat stress cDNA library Chaperonin Pennisetum glaucum 



Pennisetum glaucum


Pennisetum glaucum heat shock protein 10


Abscisic acid


Salicylic acid





This work was supported partially by the Department of Biotechnology (Ministry of Science and Technology, Government of India) to MKR. PSR acknowledges the Department of Science and Technology, Govt. of India for the fellowship and research grant through the INSPIRE Faculty Award No. IFALSPA-06 and Young Scientist Scheme SB/YS/LS-12/2013. This work has been undertaken as part of the CGIAR Research Program on Dryland Cereals.

Author contribution

Conceived and designed the experiments: PSR, VV and MKR. Performed the experiments: RN, RY, PSR. Analyzed the data: PSR, KKR, VV. Wrote the manuscript: PSR, RN, RY, MKR and KKS.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.


  1. 1.
    Spiess C, Meyer AS, Reissmann S, Frydman J (2004) Mechanism of the eukaryotic chaperonin: protein folding in the chamber of secrets. Trends Cell Biol 14(11):598–604CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Hemmingsen SM, Woolford C, van der Vies SM, Tilly K, Dennis DT, Georgopoulos CP, Hendrix RW, Ellis RJ (1988) Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature 333(6171):330–334CrossRefPubMedGoogle Scholar
  3. 3.
    Hartl FU (1996) Molecular chaperones in cellular protein folding. Nature 381(6583):571–580CrossRefPubMedGoogle Scholar
  4. 4.
    Ranson NA, White HE, Saibil HR (1998) Chaperonins. Biochem J 333(Pt2):233–242CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Schroda M (2004) The Chlamydomonas genome reveals its secrets: chaperone genes and the potential roles of their gene products in the chloroplast. Photosynth Res 82(3):221–240CrossRefPubMedGoogle Scholar
  6. 6.
    Hill JE, Hemmingsen SM (2001) Arabidopsis thaliana type I and II chaperonins. Cell Stress Chaperones 6(3):190CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Jung KH, Ko HJ, Nguyen MX, Kim SR, Ronald P, An G (2012) Genome-wide identification and analysis of early heat stress responsive genes in rice. J Plant Biol 55(6):458–468CrossRefGoogle Scholar
  8. 8.
    Hartman DJ, Dougan D, Hoogenraad NJ, Høj PB (1992) Heat shock proteins of barley mitochondria and chloroplasts Identification of organellar hsp 10 and 12: putative chaperonin 10 homologues. FEBS Lett 305(2):147–150CrossRefPubMedGoogle Scholar
  9. 9.
    Viitanen PV, Schmidt M, Buchner J, Suzuki T, Vierling E, Dickson R, Soll J (1995) Functional characterization of the higher plant chloroplast chaperonins. J Biol Chem 270(30):18158–18164CrossRefPubMedGoogle Scholar
  10. 10.
    Trosch R, Muhlhaus T, Schroda M, Willmund F (2015) ATP-dependent molecular chaperones in plastids—more complex than expected. Biochim Biophys Acta 9:872–888CrossRefGoogle Scholar
  11. 11.
    Bukau B, Horwich AL (1998) The Hsp70 and Hsp60 chaperone machines. Cell 92(3):351–366CrossRefPubMedGoogle Scholar
  12. 12.
    Parsell DA, Lindquist S (1993) The function of heat-shock proteins in stress tolerance: degradation and reactivation of damaged proteins. Annu Rev Genet 27(1):437–496CrossRefPubMedGoogle Scholar
  13. 13.
    Wang WX, Vinocur B, Shoseyov O, Altman A (2004) Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci 9(5):244–252CrossRefPubMedGoogle Scholar
  14. 14.
    Zou J, Liu A, Chen X, Zhou X, Gao G, Wang W, Zhang X (2009) Expression analysis of nine rice heat shock protein genes under abiotic stresses and ABA treatment. J Plant Physiol 166(8):851–861CrossRefPubMedGoogle Scholar
  15. 15.
    Sun L, Liu Y, Kong X, Zhang D, Pan J, Zhou Y, Wang L, Li D, Yang X (2012) ZmHSP16. 9, a cytosolic class I small heat shock protein in maize (Zea mays), confers heat tolerance in transgenic tobacco. Plant Cell Rep 31(8):1473–1484CrossRefPubMedGoogle Scholar
  16. 16.
    Islam SM, Tuteja N (2012) Enhancement of androgenesis by abiotic stress and other pretreatments in major crop species. Plant Sci 182:134–144CrossRefPubMedGoogle Scholar
  17. 17.
    Xu Q, Qin Y (2012) Molecular cloning of heat shock protein 60 (PtHSP60) from Portunus trituberculatus and its expression response to salinity stress. Cell Stress Chaperones 17(5):589–601CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    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(3):243–254CrossRefPubMedGoogle Scholar
  19. 19.
    Rao JL, Reddy PS, Mishra RN, Gupta D, Sahal D, Tuteja N, Sopory SK, Reddy MK (2010) Thermo and pH stable ATP-independent chaperone activity of heat-inducible Hsp70 from Pennisetum glaucum. Plant Signal Behav 5(2):110–121CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Reddy PS, Thirulogachandar V, Vaishnavi CS, Aakrati A, Sopory SK, Reddy MK (2011) Molecular characterization and expression of a gene encoding cytosolic Hsp90 from Pennisetum glaucum and its role in abiotic stress adaptation. Gene 474(1–2):29–38CrossRefPubMedGoogle Scholar
  21. 21.
    Reddy PS, Reddy DS, Bhatnagar-Mathur P, Sharma KK, Vadez V (2015) Cloning and validation of reference genes for normalization of gene expression studies in pearl millet [Pennisetum glaucum (L.) R. Br.] by quantitative real-time PCR. Plant Gene 1:35–42CrossRefGoogle Scholar
  22. 22.
    Serraj R, Hash CT, Rizvi SMH, Sharma A, Yadav RS, Bidinger FR (2005) Recent advances in marker-assisted selection for drought tolerance in pearl millet. Plant Prod Sci 8:334–337CrossRefGoogle Scholar
  23. 23.
    Vadez V, Kholova J, Zaman-Allah M, Belko N (2013) Water: the most important ‘molecular’ component of water stress tolerance research. Funct Plant Biol 40:1310–1322CrossRefGoogle Scholar
  24. 24.
    Kholová J, Nepolean T, Hash CT, Supriya A, Rajaram V, Senthilvel S, Kakkera A, Yadav R, Vadez V (2012) Water saving traits co-map with a major terminal drought tolerance quantitative trait locus in pearl millet [Pennisetum glaucum (L.) R. Br.]. Mol Breed 30(3):1337–1353CrossRefGoogle Scholar
  25. 25.
    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(6):713–732CrossRefPubMedGoogle Scholar
  26. 26.
    Reddy PS, Nair S, Mallikarjuna G, Kaul T, Markandeya G, Sopory SK, Reddy MK (2008) A high-throughput, low-cost method for the preparation of ‘‘sequencing-ready’’ phage DNA template. Anal Biochem 376:258–261CrossRefPubMedGoogle Scholar
  27. 27.
    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 381:248–253CrossRefPubMedGoogle Scholar
  28. 28.
    Higo K, Ugawa Y, Iwamoto M, Korenaga T (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27(1):297–300CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Lescot M, Dehais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouze P, 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(1):325–327CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22(2):195–201CrossRefPubMedGoogle Scholar
  31. 31.
    Jones DT (1999) Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292(2):195–202CrossRefPubMedGoogle Scholar
  32. 32.
    Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30(9):e36CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Hellemans J, Mortier G, De Paepe A, Speleman F, Vandesompele J (2007) qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data. Genome Biol 8(2):R19CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Wang W, Vinocur B, Altman A (2003) Plant responses to drought, salinity and extreme temperatures: towards genetic engineering for stress tolerance. Planta 218(1):1–14CrossRefPubMedGoogle Scholar
  35. 35.
    Zhu JK (2001) Plant salt tolerance. Trends Plant Sci 6(2):66–71CrossRefPubMedGoogle Scholar
  36. 36.
    Ashraf M, Hafeez M (2004) Thermotolerance of pearl millet and maize at early growth stages: growth and nutrient relations. Biol Plant 48(1):81–86CrossRefGoogle Scholar
  37. 37.
    Reddy RA, Kumar B, Reddy PS, Mishra RN, Mahanty S, Kaul T, Nair S, Sopory SK, 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(15):1646–1659CrossRefPubMedGoogle Scholar
  38. 38.
    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 4:55–63CrossRefGoogle Scholar
  39. 39.
    Pareek A, Singla SL, Grover A (1995) Immunological evidence for accumulation of two high-molecular-weight (104 and 90 kDa) HSPs in response to different stresses in rice and in response to high temperature stress in diverse plant genera. Plant Mol Biol 29(2):293–301CrossRefPubMedGoogle Scholar
  40. 40.
    Prasad TK, Stewart CR (1992) cDNA clones encoding Arabidopsis thaliana and Zea mays mitochondrial chaperonin HSP60 and gene expression during seed germination and heat shock. Plant Mol Biol 18(5):873–885CrossRefPubMedGoogle Scholar
  41. 41.
    Shimamura T, Koike-Takeshita A, Yokoyama K, Masui R, Murai N, Yoshida M, Taguchi H, Iwata S (2004) Crystal structure of the native chaperonin complex from Thermus thermophilus revealed unexpected asymmetry at the cis-cavity. Structure 12(8):1471–1480CrossRefPubMedGoogle Scholar
  42. 42.
    Yamaguchi-Shinozaki K, Shinozaki K (2005) Organization of cis-acting regulatory elements in osmotic- and cold-stress-responsive promoters. Trends Plant Sci 10(2):88–94CrossRefPubMedGoogle Scholar
  43. 43.
    Jain D, Chattopadhyay D (2010) Analysis of gene expression in response to water deficit of chickpea (Cicer arietinum L.) varieties differing in drought tolerance. BMC Plant Biol 10:24CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Maurel C, Chrispeels MJ (2001) Aquaporins. A molecular entry into plant water relations. Plant Physiol 125(1):135–138CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2016

Authors and Affiliations

  • Rahul B. Nitnavare
    • 1
  • Richa K. Yeshvekar
    • 1
  • Kiran K. Sharma
    • 1
  • Vincent Vadez
    • 1
  • Malireddy K. Reddy
    • 2
  • Palakolanu Sudhakar Reddy
    • 1
    • 2
  1. 1.International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)HyderabadIndia
  2. 2.Plant Molecular Biology GroupInternational Centre for Genetic Engineering and Biotechnology (ICGEB)New DelhiIndia

Personalised recommendations