Functional Characterization of Tea (Camellia sinensis) MYB4a Transcription Factor Using an Integrative Approach

Abstract

Green tea (Camellia sinensis, Cs) abundantly produces a diverse array of phenylpropanoid compounds benefiting human health. To date, the regulation of the phenylpropanoid biosynthesis in tea remains to be investigated. Here, we report a cDNA isolated from leaf tissues, which encodes a R2R3-MYB transcription factor. Amino acid sequence alignment and phylogenetic analysis indicate that it is a member of the MYB4-subgroup and named as CsMYB4a. Transcriptional and metabolic analyses show that the expression profile of CsMYB4a is negatively correlated to the accumulation of six flavan-3-ols and other phenolic acids. GFP fusion analysis shows CsMYB4a’s localization in the nucleus. Promoters of five tea phenylpropanoid pathway genes are isolated and characterized to contain four types of AC-elements, which are targets of MYB4 members. Interaction of CsMYB4a and five promoters shows that CsMYB4a decreases all five promoters’ activity. To further characterize its function, CsMYB4a is overexpressed in tobacco plants. The resulting transgenic plants show dwarf, shrinking and yellowish leaf, and early senescence phenotypes. A further genome-wide transcriptomic analysis reveals that the expression levels of 20 tobacco genes involved in the shikimate and the phenylpropanoid pathways are significantly downregulated in transgenic tobacco plants. UPLC-MS and HPLC based metabolic profiling reveals significant reduction of total lignin content, rutin, chlorogenic acid, and phenylalanine in CsMYB4a transgenic tobacco plants. Promoter sequence analysis of the 20 tobacco genes characterizes four types of AC-elements. Further CsMYB4a-AC element and CsMYB4a-promoter interaction analyses indicate that the negative regulation of CsMYB4a on the shikimate and phenylpropanoid pathways in tobacco is via reducing promoter activity. Taken together, all data indicate that CsMYB4a negatively regulates the phenylpropanoid and shikimate pathways.Highlight: A tea (Camellia sinensis) MYB4a is characterized to encode a R2R3-MYB transcription factor. It is shown to repressively control the phenylpropanoid and shikimate pathway.

Arabidopsis MYB4 plays dual roles in flavonoid biosynthesis

“…However, it is still unclear how MYB4 represses the expression of early biosynthetic genes like CHS since CHS is not a direct target of MYB4 in our yeast one‐hybrid assay or a direct target controlled by MBW complexes as previously reported (Xu et al , ). This finding is not consistent with a previous report in tea (Li et al , ). Interestingly, MYB4 is involved in the regulation of primary metabolism.…”

“…The binding of MYB4 with AC elements has been reported in many plant species, including maize and switchgrass (Fornale et al , ; Shen et al , ). In addition, MYB4 was also suggested to be a repressor of flavonoid biosynthesis (Fornale et al , ; Li et al , ). However, the detailed molecular mechanisms have not been fully elucidated.…”

“…MYB4 and its homolog MYB7 have also been suggested to repress flavonoid biosynthesis (Jin et al , ; Fornale et al , ). Preliminary results have also suggested that MYB4 from tea ( Camellia sinensis ) could control flavonoid biosynthesis by not only repressing flavonoid biosynthetic genes but also shikimate biosynthetic genes such as 3‐deoxy‐ d ‐arabino‐heptulosonate7‐phosphate synthase ( DAHPS ) and chorismate mutase (CM) (Li et al , ).…”

AtHB2, a class II HD‐ZIP protein, negatively regulates the expression of CsANS, which encodes a key enzyme in Camellia sinensis catechin biosynthesis

“…Their accumulation profiles and gene expression patterns are tissue‐specific as well as development‐dependent (Jiang et al ). The higher promoter activity of CsANS observed in the meristem of the aerial parts of plants is consistent with higher gene expression and greater enzyme activity, as well as more flavonol accumulation, in young leaves (Yang et al , Zhang et al , Li et al , Sun et al , Li et al ). The activity observed in the roots may be responsible for the biosynthesis of proanthocyanidins (Fig.…”

“…ANS catalyzes the penultimate step in anthocyanin biosynthesis, from colorless leucoanthocyanidins to colored anthocyanidins, the first colored compound in the anthocyanin pathway (Nakajima et al , Punyasiri et al ). Based on previous work, the gene expression level of CsANS is consistent with catechin content, and its transcription was induced by light signals (Hong et al , Li et al , ).…”