Knockdown of CaHSP60-6 confers enhanced sensitivity to heat stress in pepper (Capsicum annuum L.)
HSP60 gene family in pepper was analyzed through bioinformatics along with transcriptional regulation against multiple abiotic and hormonal stresses. Furthermore, the knockdown of CaHSP60-6 increased sensitivity to heat stress.
The 60 kDa heat shock protein (HSP60) also known as chaperonin (cpn60) is encoded by multi-gene family that plays an important role in plant growth, development and in stress response as a molecular chaperone. However, little is known about the HSP60 gene family in pepper (Capsicum annuum L.). In this study, 16 putative pepper HSP60 genes were identified through bioinformatic tools. The phylogenetic tree revealed that eight of the pepper HSP60 genes (50%) clustered into group I, three (19%) into group II, and five (31%) into group III. Twelve (75%) CaHSP60 genes have more than 10 introns, while only a single gene contained no introns. Chromosomal mapping revealed that the tandem and segmental duplication events occurred in the process of evolution. Gene ontology enrichment analysis predicted that CaHSP60 genes were responsible for protein folding and refolding in an ATP-dependent manner in response to various stresses in the biological processes category. Multiple stress-related cis-regulatory elements were found in the promoter region of these CaHSP60 genes, which indicated that these genes were regulated in response to multiple stresses. Tissue-specific expression was studied under normal conditions and induced under 2 h of heat stress measured by RNA-Seq data and qRT-PCR in different tissues (roots, stems, leaves, and flowers). The data implied that HSP60 genes play a crucial role in pepper growth, development, and stress responses. Fifteen (93%) CaHSP60 genes were induced in both, thermo-sensitive B6 and thermo-tolerant R9 lines under heat treatment. The relative expression of nine representative CaHSP60 genes in response to other abiotic stresses (cold, NaCl, and mannitol) and hormonal applications [ABA, methyl jasmonate (MeJA), and salicylic acid (SA)] was also evaluated. Knockdown of CaHSP60-6 increased the sensitivity to heat shock treatment as documented by a higher relative electrolyte leakage, lipid peroxidation, and reactive oxygen species accumulation in silenced pepper plants along with a substantial lower chlorophyll content and antioxidant enzyme activity. These results suggested that HSP60 might act as a positive regulator in pepper defense against heat and other abiotic stresses. Our results provide a basis for further functional analysis of HSP60 genes in pepper.
KeywordsAbiotic stresses Gene expression Gene silencing Hormonal application HSP60 genes Pepper
Heat shock protein
Reactive oxygen species
Tobacco rattle virus
Virus-induced gene silencing
This work was supported through funding from the National Natural Science Foundation of China (no. U1603102) and National Key R&D Program of China (no. 2016YFD0101900)
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
- Ali M, Luo D-X, Khan A et al (2018) Classification and genome-wide analysis of chitin-binding proteins gene family in pepper (Capsicum annuum L.) and transcriptional regulation to phytophthora capsici, abiotic stresses and hormonal applications. Int J Mol Sci 19:2216. https://doi.org/10.3390/ijms19082216 CrossRefPubMedCentralGoogle Scholar
- Kim H-J, Hwang NR, Lee K-J (2007) Heat shock responses for understanding diseases of protein denaturation. Mol Cells (Springer Sci Bus Media BV) 23:123–131Google Scholar
- Liu RH, Meng JL (2003) MapDraw: a microsoft excel macro for drawing genetic linkage maps based on given genetic linkage data. Yi Chuan Hered 25:317–321Google Scholar
- Miller G, Suzuki N, Rizhsky L et al (2007) Double mutants deficient in cytosolic and thylakoid ascorbate peroxidase reveal a complex mode of interaction between reactive oxygen species, plant development, and response to abiotic stresses. Plant Physiol 144:1777–1785. https://doi.org/10.1104/pp.107.101436 CrossRefPubMedPubMedCentralGoogle Scholar
- Sergiev I, Alexieva V, Karanov E (1997) Effect of spermine, atrazine and combination between them on some endogenous protective systems and stress markers in plants. Proc Bulg Acad Sci 51:121–124Google Scholar
- Török Z, Horváth I, Goloubinoff P et al (1997) Evidence for a lipochaperonin: association of active protein-folding GroESL oligomers with lipids can stabilize membranes under heat shock conditions. Proc Natl Acad Sci USA 94:2192–2197. https://doi.org/10.1073/pnas.94.6.2192 CrossRefPubMedPubMedCentralGoogle Scholar
- Yamauchi N, Funamoto Y, Shigyo M (2004) Peroxidase-mediated chlorophyll degradation in horticultural crops. Phytochem Rev 3:221–228. https://doi.org/10.1023/B:PHYT.0000047796.98784.06 CrossRefGoogle Scholar