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Figure 1. 

Phylogeny and classification of CsWRKY genes in the tea plants. The phylogenetic tree was generated from the conserved WRKY domains obtained from tea plants and A. thaliana using the neighbor-joining method. The circles and triangles represent tea plants and Arabidopsis, respectively. Different colors represent the classes and subclasses of each WRKY class. Green: group I; blue: II-a; purple: II-b; orange: II-c; dark blue: II-d; light green: II-e; red: III. The bootstrap values for some key nodes are presented beside the branches.

Figure 2. 

Chromosome distribution and evolution of CsWRKY genes of tea plants. (a) Chromosome distribution of WRKY genes of tea plants. Only the 74 CsWRKY genes that reside on the tea chromosomes are shown. The outer circles represent the 15 chromosomes of the tea plant genome, while the inner circles exhibit the 725 genomic collinear blocks (green links) within the tea plant genome. (b) Distribution of the synonymous mutation rate (Ks) of 8,208 gene pairs located in 725 collinear blocks. The peak of Ks distribution represents the recent whole genome duplication (WGD) event that occurred in the tea plant genome. (c) Ks distribution of CsWRKY gene pairs. The red bar indicates the total number of CsWRKY gene pairs duplicated at the time of the WGD event.

Figure 3. 

Cloning and subcellular localization of two cold-responsive WRKY genes, CsWRKY29 and CsWRKY37, in tea plants. (a) Expression patterns of the 11 differentially expressed genes of tea plant during cold acclimation. CK: non-acclimated (25 °C day, 20 °C night); CA 1-6 h: fully acclimated (10 °C for 6 h); CA 1-7 d: 10 °C day and 4 °C night for 7 d; CA 2-7 d: cold response (4 °C day, 0 °C night for 7 d); DA-7 d: recovering (25 °C day, 20 °C night for 7 d). (b) Multiple sequence alignment of CsWRKY29, CAWRKY37, and their homologous in Arabidopsis (AtWRKY6). The red and blue boxes represent the WRKY domain and C2H2 type zinc-finger motif, respectively. (c) Relative expression levels of CsWRKY29 and (d) CsWRKY37 genes under cold treatments (4 °C) were revealed by qRT-PCR experiments. (e) Subcellular localization of CsWRKY29 and CsWRKY37 proteins. Expression patterns of CsWRKY29 (f) and CsWRKY37 (g) among different tissues of tea plants.

Figure 4. 

Antisense oligodeoxynucleotides- based gene silencing of CsWRKY29 and CsWRKY37 in tender leaves of tea plants. (a) A schematic diagram of AsODN silencing of CsWRKY29 and CsWRKY37. (b) Relative expression levels of CsWRKY29 and (c) CsWRKY37 feeding with AsODN compared to control. (d) MDA contents in CsWRKY29 and (e) CsWRKY37-silenced tea plant seedlings compared to control. (f) Determination of the net photosynthetic rate and maximum photochemical efficiency of photosystem II (Fv/Fm) in CsWRKY29- and (g) CsWRKY37-silenced and control tea plants. (h), (i) Fv/Fm values in CsWRKY29/37-silenced and control tea plants.

Figure 5. 

Overexpression of the CsWRKY29 and CsWRKY37 in Arabidopsis plants. (a) Screening and PCR validation of CsWRKY29/37 transgenic Arabidopsis plants. OE1-6: overexpression line 1−6; PC: positive control; WT: wild-type. (b, c) Expression levels of CsWRKY29/37 genes in six transgenic Arabidopsis plants. (d) The top panel shows the phenotypes of wild-type and transgenic plants growing normally for 2 weeks, while the bottom panel illustrates the phenotypes of wild-type and transgenic plants after cold treatments at −6 °C for 2 h. (e, f) Survival rates of wild-type and transgenic Arabidopsis plants (p < 0.05) after cold treatments. (g, h) MDA contents of wild-type and transgenic Arabidopsis plants (p < 0.05) after cold treatments.