Phenotypic changes of tea plant leaves
Tea shoots of ‘Longjing 43’ at 3, 6, and 9 days after shading treatments (S0, S1, and S2) were harvested and investigated (Fig. 2). Under shading treatments, the color of leaves seems more green, and the leaves are softer, especially in the S2 treatment. Leaf area increased during leaf development, and leaf areas of S0 are obvious larger than that in shading treatments, S1 and S2 (Fig. 3). The leaf areas at 3, 6, and 9 days of S0 are 1.2-, 1.5-, and 1.5-folds of S1, respectively, and 1.5-, 2.2-, and 2.5-folds of S2.
Lignin content levels in tea plant leaves under shading treatments
Tea plant leaves with shading treatments were collected and analyzed for lignin content (Fig. 4). With the growth and development of tea plant, lignin accumulation of the leaves showed an upward trend in S0 and S1, while, the change was not significant in S2. The lignin contents were obvious higher in S0 than that in S1 and S2 at 9 days. The content of lignin decreased with increasing shading (S0 > S1 > S2). The results showed that shading might have a negative influence on lignin accumulation in tea plant leaves.
Anatomical structure analysis of tea plant leaves under shading treatments
To further understand the effect of shading on lignin levels in tea plant, the anatomical structure of tea plant leaves was investigated. The transverse sections of leaves were obtained and stained with safranin-O and fast green to highlight the basic anatomical structure of tea plant leaves (Fig. 5). The lignin in the tea plant leaves was mainly distributed in the secondary walls of the xylem region. Compared with control, the lignification of the xylem of leaves under 80% shading treatment appeared to be declining at 9d. The transverse sections were placed under UV excitation to determine the effect of shading treatments on tea plant leaves (Fig. 6). Lignin was also found in the xylem region of tea plant leaves, which was consistent with the observation in Fig. 5.
Analysis of the promoter regions of the genes involved in the lignin pathway in tea plant
The sequences of CsPAL, CsC4H, Cs4CL, CsHCT, CsC3′H, CsCCoAOMT, CsF5H, CsCOMT, CsCCR, CsCAD, CsPER, and CsLAC, 2000 bp upstream of the transcription start site were analyzed to understand the regulatory mechanisms that control the expression of these genes.
As shown in Table 2 and Supplementary Table 1, each gene involved in the lignin pathway contain several light-responsive elements. For example, Box 4 elements appear at the promoters of all lignin pathway genes. G-box, GT1-motif, and TCT-motif are also light-responsive elements, and present in most lignin pathway genes. Those results suggested that the genes involved in the lignin pathway might be regulated by light.
Other cis-regulatory elements, such as the TGA-element, CGTCA-motif, ABRE, ERE, STRE, W box, ARE, are present in most of genes involved in the lignin pathway (Supplementary Table 1). These elements are related to the signal pathways of methyl jasmonic acid (MeJA), ethylene (Eth), abscisic acid (ABA), anaerobic, and other biotic and abiotic stresses. The results indicated that expression of those genes in lignin pathway may also involved in the response to hormones, biotic and abiotic stresses.
Interaction network of the proteins involved in the lignin pathway in tea plant
To better understand the interactions among the structural genes involved in the lignin pathway in tea plant, an interaction network was predicted using STRING software basis on the orthologs in Arabidopsis (Fig. 7). Most of the proteins could interacted with more than eight proteins, and OPCL1 (Cs4CL) interacted with all of the other members. LAC3 (CsLAC) and RCI3 (CsPER) are the necessary enzymes for lignin polymerization, which interacted with the least proteins. LAC3 (CsLAC) only interacted with CYP84A1 (CsF5H), CYP98A3 (CsC3'H), RCI3 (CsPER), and CCoAOMT1 (CsCCoAOMT); RCI3 (CsPER) interacted with CYP84A1 (CsF5H), OMT1 (CsCOMT), ATCAD4 (CsCAD), LAC3 (CsLAC) and CCoAOMT1 (CsCCoAOMT).
Expression patterns of the genes involved in the lignin pathway under shading treatments
To further investigate the effect of shading treatments on lignin pathway, the expression profiles of the 12 lignin biosynthetic related genes were selected for RT-qPCR. CsPAL, CsC4H, and Cs4CL, participated in the general phenylpropanoid pathway. CsHCT, CsC3′H, CsCCoAOMT, CsF5H, CsCOMT, CsCCR, CsCAD, CsPER, and CsLAC, were involved in the specific lignin pathway.
General phenylpropanoid pathway The expression levels of CsPAL, CsC4H, and Cs4CL were gradually increased in leaves during tea plant growth under S0 and S1 treatments (Fig. 8). The trends of Cs4CL was decreased with increasing shading (S0 > S1 > S2). The expression profiles of CsPAL and CsC4H were obvious decreased in S2 compared with S0 (control), while they were increased at 9 d in S1.
Specific lignin pathway With the growth and development of the leaves in tea plant, the expression profiles of all specific lignin related genes showed an upward trend in S0 and S1, whereas CsCCoAOMT, CsCOMT, and CsLAC were reduced significantly in S2 treatment (Fig. 9). Most genes showed slightly higher expression levels in S0 compared with S1 treated group. The expression profiles of CsHCT, CsF5H, CsCCR, CsPER, and CsLAC were obvious decreased in S2 compared with that in S0. CsC3′H, and CsCAD, showed a similar trend, which is, declining in 3 d and 6 d, while increasing in 9 d with increasing shading.
Correlation analysis of lignin content and expression levels of related genes
The correlation coefficients were calculated by correlating lignin content with expression levels of genes in the lignin pathway by Pearson analysis (Fig. 10 and Table 3). The correlations were calculated from the 40% shading treatment (S0&S1), 80% shading treatment (S0&S2), and the whole shading treatments (S0&S1&S2). The expression levels of all lignin related genes were positively correlated with the lignin content in S0&S1, S0&S2, and S0&S1&S2.
Among the genes, the expression profiles of CsPAL, Cs4CL, CsF5H, and CsLAC exhibited significant positively correlation with lignin content in S0&S1, S0&S2, and S0&S1&S2. CsHCT expression was significant correlated with lignin content in S0&S2 and S0&S1&S2; while, CsCOMT was sgnificant correlated with lignin content in S0&S1 and S0&S1&S2. In addition, the gene expression profiles of CsCCoAOMT showed significant positive correlation with the lignin content in S0&S1, whereas Cs4CL was sgnificant correlated in S0&S2, and CsCCR and CsPER were sgnificant correlated in S0&S1&S2.