Elucidation Of Individual Functions Of Arabidopsis R2R3MYB Transcription Factors Regulating Aliphatic Glucosinolate Biosynthesis

Aliphatic glucosinolates (AGSLs) are nitrogen-and sulfur-containing plant secondary metabolites which are derived from methionine. Arabidopsis transcription factors, MYB28, MYB29and MYB76, positively regulate aliphatic glucosinolate biosynthesis. The interaction among these MYBs is complex, and their individual functions have not been fully elucidated. In a functional multiple-knockout by myb28myb29, the expression of all three MYBs was repressed and no AGSL was detected, providing a ideal transgenic background for elucidating individual MYB function. In previous studies, overexpression and complementary of MYBs was generally driven by constitutive strong promoters. However, own promoter of a gene consists an important part of transcriptional regulation. In study of transcription factors, control of MYBs driven by their own promoters can reflect more realities.Using wild-type Arabidopsis DNA as template, three MYB genes containing their own promoters were cloned and transformed to myb28myb29, and individual transgenic lines of three MYB genes driven by their own promoters were. We analyzed the AGSL contents and expression levels of AGSL biosynthetic genes.MYB28expression activated most of the biosynthetic genes and rescued accumulation of short-chain AGSLs. MYB29also activated the same pathway, but induction of genes involved in side-chain elongation was weaker than that by MYB28, resulting in a minor rescue of short-chain AGSLs. Neither MYB28nor MYB29recovered accumulation of long-chain AGSLs, probably due to the inability to induce MAM3expression. MYB76could not solely activate AGSL biosynthesis, and MYB76expression was induced by MYB28and MYB29. A sulfur deficiency experiment suggested that the expression of MYB28was induced by sulfur deficiency, and expression of MYB29and MYB76responds more sensitively to sulfur stress than MYB28. Furthermore, MYB29and/or MYB76inhibited the induction of MYB28expression.We illustrate the unique functions of individual MYB transcription factors driven by their own promoters.