Comparative transcriptome analysis of molecular mechanisms underlying adventitious root developments in Huangshan Bitter tea (Camellia gymnogyna Chang) under red light quality

Abstract

As the formation of adventitious roots (AR) is an important component of in vitro regeneration of tea plants, the propagation and preservation of Huangshan Bitter tea (Camellia gymnogyna Chang) cuttings have been hindered due to its lower rooting rate. As light is a crucial environmental factor that affects AR formation, this study aimed to investigate the special role of red light (RL) in the formation of AR in Huangshan Bitter tea plants, which has not been well understood. Huangshan Bitter tea plants were induced with white light (control, WL) and red light (660 nm, RL) qualities 36 days after induced treatment (DAI) to investigate dynamic AR formation and development, anatomical observation, hormones content change, and weighted gene co-expression network analysis (WGCNA) of the transcriptome. Results showed that RL promoted the rooting rate and root characteristics compared to WL. Anatomical observations demonstrated that root primordium was induced earlier by RL at the 4 DAI. RL positively affected IAA, ZT and GA₃ content and negatively influenced ABA from the 4 to 16 DAI. RNA-seq and analysis of differential expression genes (DEGs) exhibited extensive variation in gene expression profiles between RL and WL. Meanwhile, the results of WGCNA and correlation analysis identified three highly correlated modules and hub genes mainly participated in 'response to hormone', 'cellular glucan metabolic progress', and 'response to auxin'. Furthermore, the proportion of transcription factors (TFs) such as ethylene response factor (ERF), myeloblastosis (MYB), basic helix-loop-helix (bHLH), and WRKYGQK (WRKY) were the top four in DEGs. These results suggested that the AR-promoting potential of red light was due to complex hormone interactions in tea plants by regulating the expression of related genes. This study provided an important reference to shorten breeding cycles and accelerate superiority in tea plant propagation and preservation.

Keywords: Camellia gymnogyna Chang; adventitious root formation; light quality; plant phytohormone signal transduction; transcriptome analysis.

Figure 1

Root formation and development of tea tissue culture seeding between red light (RL) and white light (WL). (A) Dynamic changes of rooting rate over culturing. (B) Root number. (C) Maximum root length (unit: cm). (D) Average root hair length (unit: cm). ** indicates significant differences at p < 0.01 based on independent sample T-test.

Figure 2

Dynamic developments of root morphological and anatomical structure between red light and white light over culturing at 1, 4, 16 and 20 days after induced treatment (DAI). The red arrow was pointed to the tiny adventitious root. The black arrows were pointed to the developing root primordia and adventitious roots. E, epidermis; C, cortex; P, phloem; PI, pith; X, xylem; PR, pith ray; RP, roots primordium; AR, adventitious root; VC, vascular cambium.

Figure 3

Global analysis of the transcriptomes. (A) Heatmap showing the different expression patterns of genes under red light (RL) and white light (WL). (B) Histogram showing the number of DEGs in the comparison group. (C) Venn diagrams showing the intersections among the four comparison groups. RL, red light; WL, white light; and the numbers were days.

Figure 4

Gene expression profile analysis and functional enrichment analysis in red light (RL) and white light (WL). (A) Module profiles of the genes in two light qualities across four AR developmental stages. (B) Venn diagrams showing the number of shared and uniquely expressed genes between two light qualities in profiles 0,19,17 and 4. (C) KEGG pathway enrichment analysis results for the genes clustered in profiles 0,19,17 and 4 between two light qualities. The x-axis represents the pathways, and the y-axis represents the percentage of the number of enriched genes relative to the total number of genes in the pathway.

Figure 5

Weighted gene co-expression network analysis. (A) Module-phytohormone-rooting rate association. Each row corresponds to a module. Columns correspond to IAA, ABA, ZT, GA₃ and rooting rate. The color of each cell at the row-column intersection indicates the correlation coefficient between the module and the hormone. The red-colored cell indicates a positive correlation, and the blue-colored cell means a negative correlation. The values in each cell indicate the correlation coefficient and p-value. (B) A scatterplot of Gene Significance (GS) for four traits vs. Module Membership (MM) in the highest correlation module. (B–E) are IAA, ABA, GA_3, and rooting rate, respectively. (F–H) Eigengene expression profiles and heat map show the FPKM of each gene in the greenyellow, salmon and cyan modules. The y-axis indicates the value of the module eigengene or the gene; the x-axis indicates the sample type and developmental stage. The number of genes in each module is indicated at the top. (I–K) GO term enrichment analysis results of the greenyellow, salmon, and cyan module genes visualized by the ‘TreeMap’ view of REVIGO. Each rectangle is a single cluster representative. The representatives are joined into ‘superclusters’ of loosely related terms, visualized with different colors. The size of the rectangles is adjusted to reflect the p-value. (L–N) Correlation networks of the greenyellow, salmon and cyan modules are visualized by Cytoscape.

Figure 6

Dynamic changes of IAA (A), ABA(B), ZT (E), and GA₃ (F) content at 1, 4, 16 and 20 days after induced treatment (DAI) and the expression of major IAA- (C) and ABA- (D), ZT- (G) and GA₃- (H) related genes under red light (RL) and white light (WL). Lowercase letters above the bars indicate significant differences among the 1, 4, 16 and 20 DAI with p < 0.05. Two‐way analysis of variance (ANOVA) was performed to evaluate the effects of light, induce time, and their interactions (light × induce-time). NS means non‐significant and * indicate significant differences at p < 0.05. IAA, indole-3-acetic acid; ABA, abscisic acid; ZT, zeatin; GA₃, gibberellic acid 3.

Figure 7

Transcription factor expression analysis. (A) Transcription results of transcription factors in DEGs among different comparison groups under red light (RL) and white light (WL). (B) The proportion of various transcription factors in DEGs. (C) WRKY expression pattern in DEGs. (D) ERF expression pattern in DEGs (E) bHLH expression pattern in DEGs. (F) MYB expression pattern in DEGs. RL, red light; WL, white light; and the numbers were days.