Auxin and Ethylene Induce Flavonol Accumulation through Distinct Transcriptional Networks

Daniel R Lewis,Brenda S.J Winkel,Melissa V Ramirez,Prashanthi Vallabhaneni,Nathan D Miller,Gloria K Muday,Richard F Helm,W Keith Ray

doi:10.1104/pp.111.172502

Abstract

Auxin and ethylene are key regulators of plant growth and development, and thus the transcriptional networks that mediate responses to these hormones have been the subject of intense research. This study dissected the hormonal cross talk regulating the synthesis of flavonols and examined their impact on root growth and development. We analyzed the effects of auxin and an ethylene precursor on roots of wild-type and hormone-insensitive Arabidopsis (Arabidopsis thaliana) mutants at the transcript, protein, and metabolite levels at high spatial and temporal resolution. Indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) differentially increased flavonol pathway transcripts and flavonol accumulation, altering the relative abundance of quercetin and kaempferol. The IAA, but not ACC, response is lost in the transport inhibitor response1 (tir1) auxin receptor mutant, while ACC responses, but not IAA responses, are lost in ethylene insensitive2 (ein2) and ethylene resistant1 (etr1) ethylene signaling mutants. A kinetic analysis identified increases in transcripts encoding the transcriptional regulators MYB12, Transparent Testa Glabra1, and Production of Anthocyanin Pigment after hormone treatments, which preceded increases in transcripts encoding flavonoid biosynthetic enzymes. In addition, myb12 mutants were insensitive to the effects of auxin and ethylene on flavonol metabolism. The equivalent phenotypes for transparent testa4 (tt4), which makes no flavonols, and tt7, which makes kaempferol but not quercetin, showed that quercetin derivatives are the inhibitors of basipetal root auxin transport, gravitropism, and elongation growth. Collectively, these experiments demonstrate that auxin and ethylene regulate flavonol biosynthesis through distinct signaling networks involving TIR1 and EIN2/ETR1, respectively, both of which converge on MYB12. This study also provides new evidence that quercetin is the flavonol that modulates basipetal auxin transport.

Highlights

Auxin and ethylene are key regulators of plant growth and development, and the transcriptional networks that mediate responses to these hormones have been the subject of intense research
We examined the transcript abundance of genes encoding additional enzymes in the flavonoid biosynthetic pathway, including CHALCONE ISOMERASE (CHI) and FLAVANONE 3 HYDROXYLASE (F3H), and Dihydroflavonol 4-Reductase (DFR) with and without Indole-3-acetic acid (IAA) and ACC treatment
CHI showed a similar induction to Chalcone synthase (CHS), while little or no change was observed for F3H in response to either treatment

Summary

Introduction

Auxin and ethylene are key regulators of plant growth and development, and the transcriptional networks that mediate responses to these hormones have been the subject of intense research. The equivalent phenotypes for transparent testa (tt4), which makes no flavonols, and tt, which makes kaempferol but not quercetin, showed that quercetin derivatives are the inhibitors of basipetal root auxin transport, gravitropism, and elongation growth These experiments demonstrate that auxin and ethylene regulate flavonol biosynthesis through distinct signaling networks involving TIR1 and EIN2/ETR1, respectively, both of which converge on MYB12. Transport Inhibitor Response (TIR1) encodes an auxin receptor, an E3-ubiquitin ligase, which functions to degrade transcriptional repressors and thereby induce gene expression (Dharmasiri et al, 2005; Kepinski and Leyser, 2005) These negative transcription factors are encoded by the AUXIN/INDOLE3-ACETIC ACID (AUX/IAA) gene families (Ulmasov et al, 1987), and upon their degradation, Auxin Response Factor (ARF) family proteins are released from complexes and generally act as positive transcription factors enhancing the expression of target genes, which affect growth and developmental responses (Ulmasov et al, 1999; Shin et al, 2007)

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