Figure 1 . Phylogenetic tree of CssHSPs. Phylogenetic tree of sHSP proteins in tea plant and other plant species were generated by MEGA 7 using neighbor-joining. At: Arabidopsis thaliana , Os:Oryza sativa , Gm: Glycine max , and Sl: Solanum lycopersicum ; CI–CVII: cytoplasm I–VII, ER: endoplasmic reticulum, MI–MII: mitochondria I–II, P: plastid, and Px:peroxide.
Figure 2 . Distribution of conserved motifs in CssHSP. Putative motifs are represented by a number in a colored box.CI-CVII: cytoplasm CI-CVII; ER: endoplasmic reticulum, MI: mitochondria I; P: plastid, and Px: peroxide
Figure 3 . Expression profiles of CssHSP genes. (A)Expression profiles of CssHSP genes in different tissues. (A)Cold treatment included CK, CA1 and de-acclimated (CA3);(B) MeJA treatment; (C)200mM NaCl treatment for 0, 24, 48, and 72 h; (D) Drought treatment: 25% polyethylene glycol (PEG) treatment for 0, 24, 48 and 72 h. (E) Venn diagram of CssHSP genes expressed in four treatments. The heatmap was generated by Heml software using the CssHSPgenes’ expression data, and normalized log2 transformed values were used with hierarchical clustering. The TPM values of theCssHSP genes in different tissues are listed in Table S2 and normalized log2(TPM treatment/TPM_control) transformed values were used with hierarchical clustering(A, B, C, E). Blue represents low expression, and red represents high expression. The venn diagram was finished by omicshare website.
Figure 4 Expression pattern of TEA033542 under heat, salt, drought and MeJA stress in tea plant leaves. (A) Expression levels of TEA033542 following heat stress in different time points: 40 °C treatment for 0 h, 1 h, 2 h, 4 h, 8 h, 12 h. (B) Expression levels of TEA033542 following 0.25% MeJA treatments in different time points: 0 h, 12 h, 24 h, 48 h, 72 h.(C) Expression levels of TEA033542 following salt treatment in different time points: 200 mM NaCl treatment for 0 h, 6 h, 12 h, 24 h, 36 h, 48 h, 60h. (D) Expression levels of TEA033542 following drought treatment in different time points: 25% PEG4000 treatment for 0 h, 4 h, 8 h, 12 h, 24 h, 36 h. Data are means with standard deviations of three biological replicates. Different letters denote statistical significance at p≤0.05.
Figure 5 . Interaction between CsHSP24.6 andCspTAC5 in tea plants. (A) Putative interaction network ofTEA033542 in tea plants. Homologous proteins in tea plant and Arabidopsis are shown in red and black, respectively. (B)qRT-PCR data show expression profiles of CsHSP24.6 and CspTAC5 in buds, leaves (different stages), young stems, and roots. (C)Images obtained using confocal microscopy indicated chloroplast localization of theCsHSP24.6 -GFP protein and CspTAC5 -GFP protein in leaf epidermal cells of Nicotiana benthamiana. GFP alone were used as controls. Bars = 30 μm. (D) In vivo interaction between CsHSP24.6and CspTAC5 examined by BiFC. The YFP confocal microscopy images show that Arabidopsis protoplasts express instantaneously combination encoding fusion proteins. Each image represents at least three independent experiments. Bars = 5 μm. (E) In vitro interaction betweenCsHSP24.6 and CspTAC5 analyzed by pull down. GST-CsHSP24.6 and free GST proteins coupled to GST binding resin were incubated with MBP-CspTAC5 and free MBP proteins. Bound proteins were separated by SDS-PAGE and immunoblotted with MBP or GST antibodies. The results were repeated twice.
Figure 6 Overexpression of CsHSP24.6 and CspTAC5in transgenic arabidopsis confers enhanced heat and hight tolerance. (A and B) Chl fluorescence imaging (A) and Fv/Fm (B) of transgenic lines and WT before and after the heat treatment. 6-week-old Arabidopsis plantlet in 45 °C for 6 h. Control,22 ℃;Heat,45 ℃. (C and D) Chl fluorescence imaging (C) and Fv/Fm (D) of transgenic lines and WT before and after the light treatment. 6-week-old Arabidopsis plantlet in high light (40000 lx) for 3 days. Control,8000 lx;Light,40000 lx. All date is mean of 3 biological Replicates, Bar is ±SD; Statistical significant was analyzed by SPSS software, n=3, ** P <0.01
Figure 7 Growth and biochemical analysis of CsHSP24.6and CspTAC5 overexpressing plants after 100 mM NaCl for 7 days.(A) Growth status of control and CsHSP24.6 transgenic Arabidopsis plants after 100 mM NaCl for 7 days and normal condition on the left. Corresponding to the right are the physiological indicators of the control and CsHSP24.6 transgenic plants such as main root, lateral root, and total leaf area. (B) Growth status of control andCspTAC5 transgenic Arabidopsis plants after 100 mM NaCl for 7 days and normal condition on the left. Corresponding to the right are the physiological indicators of the control and CspTAC5transgenic plants such as main root, lateral root, and total leaf area. All date is mean of 50 biological Replicates, Bar is ±SD; Statistical significant was analyzed by SPSS software, n=50, * P <0.05,** P <0.01.