Control of anthocyanin and non-flavonoid compounds by anthocyanin-regulating MYB and bHLH transcription factors in Nicotiana benthamiana leaves.

Nikolay S. Outchkourov,Carlos A. Carollo,Dirk Bosch,Victoria Gomez-Roldan,Ric C. H. de Vos,Robert D. Hall,Jules Beekwilder

doi:10.3389/fpls.2014.00519

Abstract

Coloration of plant organs such as fruit, leaves and flowers through anthocyanin production is governed by a combination of MYB and bHLH type transcription factors (TFs). In this study we introduced Rosea1 (ROS1, a MYB type) and Delila (DEL, a bHLH type), into Nicotiana benthamiana leaves by agroinfiltration. ROS1 and DEL form a pair of well-characterized TFs from Snapdragon (Antirrhinum majus), which specifically induce anthocyanin accumulation when expressed in tomato fruit. In N. benthamiana, robust induction of a single anthocyanin, delphinidin-3-rutinoside (D3R) was observed after expression of both ROS1 and DEL. Surprisingly in addition to D3R, a range of additional metabolites were also strongly and specifically up-regulated upon expression of ROS1 and DEL. Except for the D3R, these induced compounds were not derived from the flavonoid pathway. Most notable among these are nornicotine conjugates with butanoyl, hexanoyl, and octanoyl hydrophobic moieties, and phenylpropanoid-polyamine conjugates such as caffeoyl putrescine. The defensive properties of the induced molecules were addressed in bioassays using the tobacco specialist lepidopteran insect Manduca sexta. Our study showed that the effect of ROS1 and DEL expression in N. benthamiana leaves extends beyond the flavonoid pathway. Apparently the same transcription factor may regulate different secondary metabolite pathways in different plant species.

Highlights

Higher plants produce a large variety of low-molecular weight secondary compounds, such as phenylpropanoids, terpenoids, and alkaloids (Pichersky and Gang, 2000)
Our study has shown that the effect of ROS1 and DEL expression in N. benthamiana extends beyond anthocyanin production
In this study we report that ectopic expression of two anthocyanin-specific transcription factors (TFs) from A. majus (ROS1 and DEL) in N. benthamiana induces, besides a single anthocyanin delphinidin 3-rutinoside (D3R), a range of non-flavonoid, defensive, anti-herbivore molecules

Summary

Introduction

Higher plants produce a large variety of low-molecular weight secondary compounds, such as phenylpropanoids, terpenoids, and alkaloids (Pichersky and Gang, 2000). The patterns of anthocyanin coloration in different plant organs and tissues of higher plants is under the control of specific transcription factors (TFs) of the MYB and basic helix-loophelix (bHLH) families (Petroni and Tonelli, 2011). MYB TFs that regulate anthocyanin biosynthesis belong to the R2R3-type MYB family (Stracke et al, 2001; Feller et al, 2011) Members of this protein family regulate diverse processes such as the phenylpropanoid pathway, tryptophan biosynthesis, epithelial cell fate identity and plant responses to environmental factors, and they play a role in mediating hormone actions (Stracke et al, 2001; Broun, 2005; Dubos et al, 2010). For the activity of ROS1, a bHLH factor, such as Delila (DEL) from A. majus, is often required (Butelli et al, 2008) in tobacco it has been shown that the expression of ROS1 alone could mediate anthocyanin formation (Orzaez et al, 2009)

Methods

Results

Conclusion