Integrative analysis of endogenous phytohormones and transcriptomics reveals mechanism of light-induced callus formation in tea (Camellia sinensis) cuttings

Cutting is the mainly asexual propagation method for tea plant, and moreover, callus formation is a key point for the survival rate of tea cuttings. As reported, light is crucial for callus formation of tea cuttings via influencing phytohormone contents and plant hormone signal transduction. However, the molecular mechanism of light-induced callus formation is not clear. In this study, callus growth was analyzed in short tea cuttings exposed to light conditions with/without shading. The results showed the callus formation proportion exposed to light was twice that of shading treatment on the 21st day. To discover the regulatory process of light-induced callus formation, transcriptome and phytohormone analyses of stems were performed at the 7th and 14th day. KEGG results displayed that “starch and sucrose metabolism” and “early auxin hormone signaling” were enriched by up-regulated genes in S7d_VS_L7d, while “motor proteins” and “photosynthesis-antenna proteins” were enriched by up-regulated genes in S14d_VS_L14d. Hormonal analysis displayed that the tZ, IAA and ABA were higher with light treatment in the L14d, and the tZ and IAA content increased from 7d to 14d, while ABA content decreased. Integrated transcriptomics results, the key genes of tZ and IAA biosynthesis were also up-regulated as the tZ and IAA content, and the ABA cleavage genes-abscisic acid 8’-hydroxylase (ABA8ox) were highly expressed at 14d under light treatment. Moreover, the CKs signal transduction were also promoted by light, corresponding with the expression level of genes related to cell cycle and division in cuttings under this condition. Weighted gene co-expression network analysis (WGCNA) of DEGs in turquoise module and protein-protein interaction (PPI) analysis of DEGs in L14d_vs_S14d revealed that CsMYB82 is the hub gene during light-induced callus formation, and the interaction of CsMYB82 and CsGL3 was verified via yeast two-hybrid (Y2H) assay and bimolecular fluorescence complementation (BiFC) assay. Structural analyses illustrated that CsMYB82 can interact CsGL3 via hydrogen bonds and salt bridges. Through the phytohormones and transcriptome analysis, carbohydrate metabolism and phytohormones metabolism were closely correlated with light treatments. And the change of phytohormones might result in the interactions of CsMYB82 and CsGL3, leading to the cell fate transition and highly expression of genes related with cell cycle and motor proteins. This study illustrates light is an important factor for callus formation according to a complex and orderly process, and also provides an understanding of the asexual reproduction mechanism of tea plants.