Genome-wide identification and expression analysis of the molecular chaperone binding protein BiP genes in Citrus
- 128 Downloads
Here, we report for the first time the genome-wide identification and expression analysis of the molecular chaperone BiP genes in Citrus. Six genes encoding the conserved protein domain family GPR78/BiP/KAR2 were identified in the genome of Citrus sinensis and C. clementina. Two of them, named here as CsBiP1 and CsBiP2, were classified as true BiPs based on their deduced amino acid sequences. Alignment of the deduced amino acid sequences of CsBiP1 and CsBiP2 with BiP homologs from soybean and Arabidopsis showed that they contain all the conserved functional motifs of BiPs. Analysis of the promoter region of CsBiPs revealed the existence of cis-acting regulatory sequences involved in abiotic, heat-shock, and endoplasmic reticulum (ER) stress responses. Publicly available RNA-seq data indicated that CsBiP1 is abundantly expressed in leaf, flower, fruit, and callus, whereas CsBiP2 expression is rarely detected in any tissues under normal conditions. Comparative quantitative real-time PCR (qPCR) analysis of expression of these genes between C. sinensis grafted on the drought-tolerant “Rangpur” lime (C. limonia) and -sensitive “Flying Dragon” trifoliate orange (Poncirus trifoliata) rootstocks showed that CsBiP1 was upregulated by drought stress on the former but downregulated on the latter, whereas the CsBiP2 mRNA levels were downregulated on drought-stressed “Flying Dragon,” but remained constant on “Rangpur.” CsBiP2 upregulation was only observed in C. sinensis seedlings subjected to osmotic and cold treatments. Taken together, these results indicate the existence of two highly conserved BiP genes in Citrus that are differentially regulated in the different tissues and in response to abiotic stresses.
KeywordsAbiotic stress Drought tolerance Endoplasmic reticulum HSP70 Immunoglobulin-heavy-chain-binding protein Mandarin Sweet orange UPR pathway
R.F.G., L.P.G., J.C.C.M., and G.S.S. conducted the experiments. R.F.G., L.R.C., A.O.S., and A.M.M.B. analyzed the data and drafted the manuscript. F.C.A., M.G.C.C., and C.P.P. supported the project and designed the experiments. M.G.C.C. polished the manuscript. All authors read and approved the manuscript.
This work was supported by research grants from CNPq (Process # 306667/2014-2), Instituto Nacional de Ciência e Tecnologia (INCT) de Genômica para Melhoramento de Citros (CNPq Process # 465440/2014-2, and FAPESP Process # 2008/2014/50880-0) and Embrapa (Macroprograma 2). L.R.C. is recipient of a CAPES/PNPD postdoctoral fellowship. M.G.C.C. and C.P.P. are CNPq Research Fellows. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
- Anderson JV, Li Q, Haskell DW, Guy CL (1994) Structural organization of the spinach endoplasmic reticulum-luminal 70-kilodalton heat-shock cognate gene and expression of 70-kilodalton heat-shock genes during cold acclimation. Plant Physiol 104:1359–1370. https://doi.org/10.1104/pp.104.4.1359 CrossRefPubMedPubMedCentralGoogle Scholar
- Buzeli RAA, Cascardo JCM, Rodrigues LAZ, Andrade MO, Almeida RS, Loureiro ME, Otoni WC, Fontes EPB (2002) Tissue-specific regulation of BiP genes: a cis-acting regulatory domain is required for BiP promoter activity in plant meristems. Plant Mol Biol 50:757–771. https://doi.org/10.1023/A:1019994721545 CrossRefPubMedGoogle Scholar
- Cascardo JCM, Almeida RS, Buzeli RAA, Carolino SMB, Otoni WC, Fontes EPB (2000) The phosphorylation state and expression of soybean BiP isoforms are differentially regulated following abiotic stresses. J Biol Chem 275:14494–14500. https://doi.org/10.1074/jbc.275.19.14494 CrossRefPubMedGoogle Scholar
- Figueiredo JEF, Cascardo JCM, Carolino SMB, Alvim F, Fontes EPB (1997) Water-stress regulation and molecular analysis of the soybean BiP gene family. Rev Bras Fisiol Veg 9:103–110Google Scholar
- Gonçalves LP, Alves TF, Martins CP, Sousa AO, Santos IC, Pirovani CP, Almeida A-AF, Coelho Filho MA, Gesteira AS, Soares Filho WS, Girardi EA, Costa MGC (2016) Rootstock-induced physiological and biochemical mechanisms of drought tolerance in sweet orange. Acta Physiol Plant 38:174. https://doi.org/10.1007/s11738-016-2198-3 CrossRefGoogle Scholar
- Korennykh A, Walter P (2012) Structural basis of the unfolded protein response. Annu Rev Cell Dev Biol 28:251–277. https://doi.org/10.1146/annurev-cellbio-101011-155826 CrossRefPubMedGoogle Scholar
- Liu JX, Srivastava R, Che P, Howell SH (2007) An endoplasmic reticulum stress response in Arabidopsis is mediated by proteolytic processing and nuclear relocation of a membrane-associated transcription factor, bZIP28. Plant Cell 19:4111–4119. https://doi.org/10.1105/tpc.106.050021 CrossRefPubMedPubMedCentralGoogle Scholar
- Mafra V, Kubo KS, Alves-Ferreira M, Ribeiro-Alves M, Stuart RM, Boava LP, Rodrigues CM, Machado MA (2012) Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions. PLoS One 7:e31263. https://doi.org/10.1371/journal.pone.0031263 CrossRefPubMedPubMedCentralGoogle Scholar
- Muench DG, Wu Y, Zhang Y, Li X, Boston RS, Okita TW (1997) Molecular cloning, expression and subcellular localization of a BiP homolog from rice endosperm tissue. Plant Cell Physiol 38:404–412. https://doi.org/10.1093/oxfordjournals.pcp.a029183 CrossRefPubMedGoogle Scholar
- Naved AF, Ozawa M, Yu S, Miyauchi T, Muramatsu H, Muramatsu T (1995) CBP-140, a novel endoplasmic reticulum resident Ca(2+)-binding protein with a carboxy-terminal NDEL sequence showed partial homology with 70-kDa heat shock protein (hsp70). Cell Struct Funct 20:133–141. https://doi.org/10.1247/csf.20.133 CrossRefPubMedGoogle Scholar
- Reis PAA, Rosado GL, Silva LAC, Oliveira LC, Oliveira LB, Costa MDL, Alvim FC, Fontes EPB (2011) The binding protein BiP attenuates stress induced cell death in soybean via modulation of the N-rich protein mediated signaling pathway. Plant Physiol 157:1853–1865. https://doi.org/10.1104/pp.111.179697 CrossRefPubMedPubMedCentralGoogle Scholar
- Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425. https://doi.org/10.1093/oxfordjournals.molbev.a040454 CrossRefPubMedGoogle Scholar
- Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. https://doi.org/10.1093/nar/22.22.4673 CrossRefPubMedPubMedCentralGoogle Scholar
- Valente MAS, Faria JAQA, Soares-Ramos JRL, Reis PAB, Pinheiro GL, Piovesan ND, Morais AT, Menezes CC, Cano MAO, Fietto LG, Loureiro ME, Aragão FJL, Fontes EPB (2009) The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought-induced leaf senescence in soybean and tobacco. J Exp Bot 60:533–546. https://doi.org/10.1093/jxb/ern296 CrossRefPubMedGoogle Scholar
- Xu Q, Chen LL, Ruan X, Chen D, Zhu A, Chen C, Bertrand D, Jiao WB, Hao BH, Lyon MP, Chen J, Gao S, Xing F, Lan H, Chang JW, Ge X, Lei Y, Hu Q, Miao Y, Wang L, Xiao S, Biswas MK, Zeng W, Guo F, Cao H, Yang X, Xu XW, Cheng YJ, Xu J, Liu JH, Luo OJ, Tang Z, Guo WW, Kuang H, Zhang HY, Roose ML, Nagarajan N, Deng XX, Ruan Y (2013) The draft genome of sweet orange (Citrus sinensis). Nat Genet 45:59–66. https://doi.org/10.1038/ng.2472 CrossRefPubMedGoogle Scholar
- Yu HY, Ziegelhoffer T, Osipiuk J, Ciesielski SJ, Baranowski M, Zhou M, Joachimiak A, Craig EA (2015) Roles of intramolecular and intermolecular interactions in functional regulation of the Hsp70 J-protein co-chaperone Sis1. J Mol Biol 427:1632–1643. https://doi.org/10.1016/j.jmb.2015.02.007 CrossRefPubMedPubMedCentralGoogle Scholar
- Zhang L, Zhao H-K, Dong Q-L, Zhang Y-Y, Wang YM, Li H-Y, Xing G-J, Li Q-Y, Dong Y-S (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. https://doi.org/10.3389/fpls.2015.00773 CrossRefPubMedPubMedCentralGoogle Scholar
- Zhu J, Hao P, Chen G, Han C, Li X, Zeller FJ, Hsam SLK, Hu Y, Yan Y (2014) Molecular cloning, phylogenetic analysis, and expression profiling of endoplasmic reticulum molecular chaperone BiP genes from bread wheat (Triticum aestivum L.). BMC Plant Biol 14(260):260. https://doi.org/10.1186/s12870-014-0260-0 CrossRefPubMedPubMedCentralGoogle Scholar