Molecular Basis of the Distinct Metabolic Features in Shoot Tips and Roots of Tea Plants (Camellia sinensis): Characterization of MYB Regulator for Root Theanine Synthesis

Abstract

The physiological and metabolic differences between shoot tips and roots of tea plants are significant, and understanding them is required for improvement of tea quality and plant growth. A high-quality full-length transcriptome sequencing on tea plant roots and shoot tips by PacBio SMRT technology was done to gain a further understanding. Approximately 160699 and 166120 full-length transcripts were recovered in roots and shoots, respectively, including 31232 and 41068 novel isoforms and 16960 and 26029 alternative splicing (AS) isoforms. These supported 21699 full-length reads and 31232 and 41068 novel transcripts from root and shoot, respectively, including 1679 long noncoding RNAs (lncRNAs) and 2772 fusion transcripts, which significantly upgrade the Camellia sinensis genome annotation. The respective 6475 and 6981 transcripts in roots and shoots differ in 3′untranslated regions. Meanwhile, extensive analyses of novel transcripts, ASs, and lncRNAs also revealed a large number of ASs and lincRNAs closely related to the regulation of characteristic secondary metabolites including catechins, theanine, and caffeine. Finally, a root-specific CsMYB6 was characterized to regulate theanine biosynthesis by genetic and molecular analyses. CsMYB6 directly bound to and activate the promoter of theanine synthetase gene (CsTSI). The study lays a foundation for the further investigation of metabolic genomics and regulation in tea plants.

Diverse roles of MYB transcription factors in regulating secondary metabolite biosynthesis, shoot development, and stress responses in tea plants (Camellia sinensis)

“…The C. sinensis genome sequence (Wei et al., 2018) was searched to identify MYB domain‐containing proteins using the hmmsearch package (version 3) and the Basic Local Alignment Search Tool (BLASTP) algorithm with a Hidden Markov Model of MYB Pfam (PF00249) with an E‐value cutoff of 1e−5. All hits were confirmed by SMART analysis (http://smart.embl-heidelberg.de/) on previous full‐length transcriptome data from our lab and other labs (Xu et al., 2017, Zhang et al., 2021).…”

“…AtCPC , encoding a single‐repeat R3‐MYB, is a repressor of anthocyanin biosynthesis in Arabidopsis (Li et al., 2022). Our previous full‐length single‐cell RNA sequencing (RNA‐Seq) data recovered two AtCPC homolog genes, CsCPCa and CsCPCb (Figure 3c, Zhang et al., 2021). Expression pattern analysis revealed that both CsCPCa and CsCPCb were highly expressed in flowers and tender shoots (Figure S8a).…”

“…By using the Pfam program with the MYB DBD model (PF00249) as a query to search against the tea plant genome, we identified 221 typical non‐redundant MYB proteins, which covered 59 1R MYB (MYB‐related), 155 2R MYB (R2R3‐MYB) and seven 3R MYB (R1R2R3‐MYB) proteins (Table S1, Figure S1). Several key MYB TFs that were not assembled in current tea genomes but have been identified and characterized in recent functional characterizations and full‐length transcriptome studies were also included, such as the anthocyanin regulator CsPAP1/MYB75/CsAN1, which is the ortholog of Arabidopsis AtPAP1 (Sun et al., 2016; Wei et al., 2019), and R2R3‐MYB repressors CsMYBL2a and b, as well as R3‐MYB repressors CsCPCa and CsCPCb (Zhang et al., 2021) (Figure 1a, Figure S2). Neighbor‐joining (NJ) phylogenetic trees constructed with MYBs from tea and Arabidopsis plants divided 162 R2R3‐ and R1R2R3‐MYB TFs into 20 subgroups according to clades with at least 50% bootstrap support (Figure 1a, Figure S2).…”

“…Studies have shown that MYB TFs also function as theanine synthesis regulators (Wen et al., 2020, Zhang et al., 2020b). Phylogenetic, co‐expression, and evolutionary analyses enabled us to identify a few MYB TFs that are highly correlated with theanine biosynthesis, of which MYB6 (CsMYB9) is reported as an activator of CsTSI and CsAlaDC (Figure S18, Table S15) (Zhang et al., 2021). Tea plants also accumulate different types of indole derivatives, such as indole, indole‐3‐methyl acetate, indole‐3‐glycerol phosphate, and indole 3‐acetic acid, which contribute to tea flavors (http://pcsb.ahau.edu.cn:8080/TCDB/f).…”

“…Besides MBW complex‐related MYB repressors, R2R3‐MYB repressors such as AtMYB7, AtMYB4, CsMYB7, and CsMYB4 directly suppress metabolic genes or compete with activators for binding to the same cis ‐elements in their promoters (Fornalé et al., 2014; Li et al., 2017; Wang et al., 2019c; Zhao et al., 2021). MYBs have also been reported to regulate terpenoid biosynthesis (Song et al., 2011); and tea plant CsMYB73 and CsMYB6 regulate theanine biosynthesis (Wen et al., 2020, Zhang et al., 2021). However, how other MYB TFs are involved in tea plant growth and secondary metabolism remains largely unknown (Zhao et al., 2020).…”