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Knockdown of CaHSP60-6 confers enhanced sensitivity to heat stress in pepper (Capsicum annuum L.)

  • Saeed ul Haq
  • Abid Khan
  • Muhammad Ali
  • Wen-Xian Gai
  • Huai-Xia Zhang
  • Qing-Hui Yu
  • Sheng-Bao Yang
  • Ai-Min Wei
  • Zhen-Hui GongEmail author
Original Article


Main conclusion

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.


Abiotic stresses Gene expression Gene silencing Hormonal application HSP60 genes Pepper 





Heat stress


Heat shock protein


Methyl jasmonate


Nitro-blue tetrazolium


Phytoene desaturase


Reactive oxygen species


Salicylic acid


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.

Supplementary material

425_2019_3290_MOESM1_ESM.tif (78.7 mb)
Fig. S1 Expression pattern of pepper HSP60 genes under HS treatment (42 °C) at 0, 2, and 8 h in the TRV2:CaHSP60-6 and TRV2:00 pepper R9 plants after VIGS to check the silencing specificity of CaHSP60-6 knockdown. Mean values and SDs are for three replicates, while letters (a–c) represent the significant differences at P < 0.05 (TIFF 80574 kb)
425_2019_3290_MOESM2_ESM.docx (16 kb)
Table S1 Primers for qRT-PCR for CaHSP60 genes (DOCX 15 kb)
425_2019_3290_MOESM3_ESM.docx (16 kb)
Table S2 Primers for gene sequencing and confirmation (DOCX 15 kb)
425_2019_3290_MOESM4_ESM.docx (505 kb)
Table S3 Ten conserved motifs found in pepper HSP60 proteins (DOCX 504 kb)
425_2019_3290_MOESM5_ESM.docx (16 kb)
Table S4 List of Cpn60_TCP1 domain, e value, formulas and total number of atoms of HSP60 genes in pepper (DOCX 16 kb)
425_2019_3290_MOESM6_ESM.docx (15 kb)
Table S5 Detail of the cis-acting elements found in the promoter region of CaHSP60s (DOCX 14 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Saeed ul Haq
    • 1
  • Abid Khan
    • 1
  • Muhammad Ali
    • 1
  • Wen-Xian Gai
    • 1
  • Huai-Xia Zhang
    • 1
  • Qing-Hui Yu
    • 2
  • Sheng-Bao Yang
    • 2
  • Ai-Min Wei
    • 3
  • Zhen-Hui Gong
    • 1
    • 4
    Email author
  1. 1.College of HorticultureNorthwest A&F UniversityYanglingPeople’s Republic of China
  2. 2.Institute of Horticulture Crops, Xinjiang Academy of Agricultural SciencesUrumqiPeople’s Republic of China
  3. 3.Tianjin Vegetable Research CenterTianjinPeople’s Republic of China
  4. 4.State Key Laboratory of Vegetable Germplasm InnovationTianjinPeople’s Republic of China

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