Abstract
Heat shock protein 70 kDa proteins (Hsp70 s) are among the most important molecular chaperone groups and play a significant role in the stress responses and development of plants. In the present study, the full-length cDNA of the heat shock cognate 70 protein 2 gene EjHsc70-2, which encodes a loquat Hsp70 s member, was cloned and characterized, and its expression and subcellular localization were also investigated. The full-length cDNA of EjHsc70-2 consists of an open reading frame (ORF) of 1950 bp, a 5′-UTR of 103 bp, and a 3′-UTR of 62 bp, and the ORF encodes 649 amino acid residues. The structure of the loquat Hsc70-2 protein was analysed using several bioinformatics tools, and the results showed that the protein was, indeed, a member of the Hsp70 s. Phylogenetic tree analysis suggested that the genetic evolution of Hsc70-2 genes conformed well to the morphology based taxonomic classification of seed plants. BLAST and multiple alignment analyses determined that the Hsc70-2 genes and Hsc70-2 proteins were both highly conserved among loquat and other seed plants, suggesting that the functions of EjHsc70-2 might be similar to those of other Hsc70-2 genes. The bioinformatics and experimental subcellular localization analyses both supported that EjHsc70-2 was a cytoplasmic and/or nuclear protein. Quantitative real-time RT-PCR (RT-qPCR) suggested its conserved functions involved in loquat organ development. Moreover, EjHsc70-2 were also inducible, which may contribute to the low-temperature adaptation of loquat fruits in cold storage. These results provide new insights into the characteristics and functions of Hsp70 s in Eriobotrya japonica.
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References
Bartlett AI (2009) An expanding arsenal of experimental methods yields an explosion of insights into protein folding mechanisms. Nat Struct Mol Biol 16(6):582–588
Bukau B, Weissman J, Horwich A (2011) Molecular chaperones and protein quality control. Protein Pept Lett 18(2):443–451
Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55(4):611
Cazalé AC, Clément M, Chiarenza S, Roncato MA, Pochon N, Creff A, Marin E, Leonhardt N, Noël LD (2009) Altered expression of cytosolic/nuclear HSC70-1 molecular chaperone affects development and abiotic stress tolerance in Arabidopsis thaliana. J Exp Bot 60(9):2653
Cho EK, Choi YJ (2009) A nuclear-localized HSP70 confers thermoprotective activity and drought-stress tolerance on plants. Biotech Lett 31(4):597–606
Cho EK, Hong CB (2004) Molecular cloning and expression pattern analyses of heat shock protein 70 genes from Nicotiana tabacum. J Plant Biol 47(2):149–159
Chong KY, Lai CC, Lille S, Chang C, Su CY (1998) Stable overexpression of the constitutive form of heat shock protein 70 confers oxidative protection. J Mol Cell Cardiol 30(3):599–608
Cuevas J, Pinillos V, Cañete ML, González M, Alonso F, Fernández MD, Hueso JJ (2009) Optimal levels of postharvest deficit irrigation for promoting early flowering and harvest dates in loquat (Eriobotrya japonica Lindl.). Agric Water Manag 96(5):831–838
Dong YS, Vierling E, Guy CL (2001) Comprehensive expression profile analysis of the Arabidopsis Hsp70 gene family. Plant Physiol 126(2):789
Ellis RJ, Minton AP (2006) Protein aggregation in crowded environments. Biol Chem 387(5):485–497
Finka A, Sharma SK, Goloubinoff P (2015) Multi-layered molecular mechanisms of polypeptide holding, unfolding and disaggregation by HSP70/HSP110 chaperones. Front Mol Biosci 2:29
Finka A, Mattoo RU, Goloubinoff P (2016) Experimental milestones in the discovery of molecular chaperones as polypeptide unfolding enzymes. Annu Rev Biochem 85(85):715
Goloubinoff P (2017) Editorial: the HSP70 molecular chaperone machines. Front Mol Biosci 4:1
Hartl FU, Hayer-Hartl M (2002) Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295(5561):1852–1858
Jung KH, Gho HJ, Nguyen MX, Kim SR, An G (2013) Genome-wide expression analysis of HSP70 family genes in rice and identification of a cytosolic HSP70 gene highly induced under heat stress. Funct Integr Genomics 13(3):391–402
Jungkunz I, Link K, Vogel F, Voll LM, Sonnewald S, Sonnewald U (2011) AtHsp70-15-deficient Arabidopsis plants are characterized by reduced growth, a constitutive cytosolic protein response and enhanced resistance to TuMV. Plant J Cell Mol Biol 66(6):983–995
Kanzaki H, Saitoh H, Ito A, Fujisawa S, Kamoun S, Katou S, Yoshioka H, Terauchi R (2010) Cytosolic HSP90 and HSP70 are essential components of INF1-mediated hypersensitive response and non-host resistance to Pseudomonas cichorii in Nicotiana benthamiana. Mol Plant Pathol 4(5):383–391
Kim NH, Hwang BK (2015) Pepper heat shock protein 70a interacts with the type III effector AvrBsT and triggers plant cell death and immunity. Plant Physiol 167(2):307–322
Kim YE, Hipp MS, Bracher A, Hayer-Hartl M, Hartl FU (2013) Molecular chaperone functions in protein folding and proteostasis. Annu Rev Biochem 82(8):323
Lee S, Lee DW, Lee Y, Mayer U, Stierhof YD, Lee S, Jürgens G, Hwang I (2009) Heat shock protein cognate 70-4 and an E3 ubiquitin ligase, CHIP, mediate plastid-destined precursor degradation through the ubiquitin-26S proteasome system in Arabidopsis. Plant Cell 21(12):3984–4001
Li QB, Haskell DW, Guy CL (1999) Coordinate and non-coordinate expression of the stress 70 family and other molecular chaperones at high and low temperature in spinach and tomato. Plant Mol Biol 39(1):21–34
Lin BL, Wang JS, Liu HC, Chen RW, Meyer Y, Barakat A, Delseny M (2001) Genomic analysis of the Hsp70 superfamily in Arabidopsis thaliana. Cell Stress Chaperones 6(3):201
Lin S, Huang X, Cuevas J, Janick J (2007) Loquat: an ancient fruit crop with a promising future. Chronica Horticult 47(2):12–15
Liu Y, Zou D, Wu B, Lin D, Zhang Z, Wu J (2015) Cloning and expression analysis of a CCoAOMT homolog in loquat fruit in response to low-temperature storage. Postharvest Biol Technol 105:45–50
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25(4):402–408
Martin J, Hartl FU (1996) Molecular chaperones in cellular protein folding. Nature 381(6583):571–579
Mayer MP (2010) Gymnastics of molecular chaperones. Mol Cell 39(3):321–331
Mayer MP, Bukau B (2005) Hsp70 chaperones: cellular functions and molecular mechanism. Cell Mol Life Sci Cmls 62(6):670–684
Monterobarrientos M, Hermosa R, Cardoza RE, Gutiérrez S, Nicolás C, Monte E (2010) Transgenic expression of the Trichoderma harzianum hsp70 gene increases Arabidopsis resistance to heat and other abiotic stresses. J Plant Physiol 167(8):659–665
Mulaudzi-Masuku T, Mutepe RD, Mukhoro OC, Faro A, Ndimba B (2015) Identification and characterization of a heat-inducible Hsp70 gene from Sorghum bicolor which confers tolerance to thermal stress. Cell Stress Chaperones 20(5):793
Murphy ME (2013) The HSP70 family and cancer. Carcinogenesis 34(6):1181–1188
Noël LD, Cagna G, Stuttmann J, Wirthmüller L, Betsuyaku S, Witte C, Bhat R, Pochon N, Colby T, Parker JE (2007) Interaction between SGT1 and cytosolic/nuclear HSC70 chaperones regulates arabidopsis immune responses. Plant Cell 19(12):4061–4076
Sarkar NK, Kim YK, Grover A (2009) Rice sHsp genes: genomic organization and expression profiling under stress and development. BMC Genomics 10(1):393
Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3(6):1101–1108
Shan LL, Li X, Wang P, Cai C, Zhang B, Sun CD, Zhang WS, Xu CJ, Ferguson I, Chen KS (2008) Characterization of cDNAs associated with lignification and their expression profiles in loquat fruit with different lignin accumulation. Planta 227(6):1243
Shi LX, Theg SM (2010) A stromal heat shock protein 70 system functions in protein import into chloroplasts in the moss Physcomitrella patens. Plant Cell 22(1):205
Song H, Zhao X, Hu W, Wang X, Shen T, Yang L (2016) Comparative transcriptional analysis of loquat fruit identifies major signal networks involved in fruit development and ripening process. Int J Mol Sci 17(11):1837
Srikanthbabu V, Ganeshkumar Krishnaprasad BT, Gopalakrishna R, Savitha M, Udayakumar M (2002) Identification of pea genotypes with enhanced thermotolerance using temperature induction response technique (TIR). J Plant Physiol 159(5):535–545
Su PH, Li HM (2008) Arabidopsis stromal 70-kD heat shock proteins are essential for plant development and important for thermotolerance of germinating seeds. Plant Physiol 146(3):1231
Su PH, Li HM (2010) Stromal Hsp70 is important for protein translocation into pea and Arabidopsis chloroplasts. Plant Cell 22(5):1516–1531
Sung DY, Guy CL (2003) Physiological and molecular assessment of altered expression of Hsc70-1 in Arabidopsis. Evidence for pleiotropic consequences. Plant Physiol 132(2):979–987
Swindell WR, Huebner M, Weber AP (2007) Transcriptional profiling of Arabidopsis heat shock proteins and transcription factors reveals extensive overlap between heat and non-heat stress response pathways. BMC Genomics 8(1):125
Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28(10):2731–2739
Tukaj S, Bisewska J, Roeske K, Tukaj Z (2011) Time- and dose-dependent induction of HSP70 in Lemna minor exposed to different environmental stressors. Bull Environ Contam Toxicol 87(3):226–230
Zhang Y, Wang M, Chen J, Rong J, Ding M (2014) Genome-wide analysis of HSP70 superfamily in Gossypium raimondii and the expression of orthologs in Gossypium hirsutum. Hereditas 36(9):921
Zhang L, Zhao HK, Dong QL, Zhang YY, Wang YM, Li HY, Xing GJ, Li QY, Dong YS (2015) Genome-wide analysis and expression profiling under heat and drought treatments of HSP70 gene family in soybean (Glycine max L.). Front Plant Sci 6:773
Zuiderweg ERP, Bertelsen EB, Rousaki A, Mayer MP, Gestwicki JE, Ahmad A (2013) Allostery in the Hsp70 chaperone proteins. Top Curr Chem 328:99–153
Acknowledgements
This research was supported by the Natural Science Foundation of Fujian Province (2017J01644, 2017J01645), the Education and Research Project of Young and Middle-aged Teachers of Fujian Province (JAT160434, JA15454), the College Outstanding Young Researchers Cultivation Program of Fujian education department, the Research and Innovation Special Foundation of Putian University (2016CX001, 2017081, 2018006, 2018064) and the Open Fund of Key laboratory of Loquat Germplasm Innovation and Utilization (Putian University), and Fujian Province University (2016001, 2017005). The English language of this manuscript was edited by American Journal Experts (AJE).
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Lin, S., Wu, T., Li, M. et al. Cloning, in silico characterization, subcellular localization, and expression of a heat shock cognate 70 kDa protein/gene (EjHsc70-2) from Eriobotrya japonica. Acta Physiol Plant 41, 119 (2019). https://doi.org/10.1007/s11738-019-2908-8
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DOI: https://doi.org/10.1007/s11738-019-2908-8