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.