RESUMO
Plants fend off potentially damaging ultraviolet (UV)-B radiation by synthesizing and accumulating UV-B-absorbing flavonols that function as sunscreens. Regulation of this biosynthetic pathway is largely transcriptional and controlled by a network of transcription factors, among which the PRODUCTION OF FLAVONOL GLYCOSIDES (PFG) family of R2R3-MYB transcription factors was recently identified with a pivotal function. Here, we describe the response of Arabidopsis seedlings to narrow-band UV-B radiation at the level of phenylpropanoid pathway genes using whole-genome transcriptional profiling and identify the corresponding flavonol glycosides accumulating under UV-B. We further show that the bZIP transcriptional regulator ELONGATED HYPOCOTYL5 (HY5) is required for the transcriptional activation of the PFG1/MYB12 and PFG3/MYB111 genes under UV-B and visible light. A synthetic protein composed of HY5 with the VP16 activation domain is sufficient to activate PFG1/MYB12 expression in planta. However, even though myb11 myb12 myb111 triple mutants have strongly reduced CHS levels in darkness as well as in constant light, neither light- nor UV-B-inducibility seems impaired. Notwithstanding this, absence of the three PFG family transcription factors results in reduced UV-B tolerance, whereas PFG1/MYB12 overexpression leads to an increased tolerance. Thus, our data suggest that HY5-dependent regulation of PFG gene expression contributes to the establishment of UV-B tolerance.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Raios Ultravioleta , Aciltransferases/metabolismo , Antocianinas/análise , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Clorofila/análise , Clonagem Molecular , Flavonoides/análise , Regulação da Expressão Gênica de Plantas , Mutação , Proteínas Nucleares/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , RNA de Plantas/metabolismo , Ativação TranscricionalRESUMO
In Arabidopsis thaliana, most mutants impaired in flavonoid accumulation were identified through screens for altered seed pigmentation. Mutations in more than 20 loci have been described that can result in a transparent testa (tt) or tannin deficient seed (tds) phenotype. For some of these mutants it is still unclear if they represent additional loci or if they are allelic to known mutations. In this study, we found that tt17 is allelic to tt11 and tds4 and identified a point mutation in tt17 that affects the gene encoding Leucoanthocyanidin Dioxygenase (LDOX). The mutation results in replacement of a cysteine close to the active site of the enzyme by the hydrophobic amino acid tyrosine. Effects of this mutation on protein structure and activity are discussed in the context of LDOX sequences from various genotypes. Regulation of the LDOX promoter was analyzed and found to be directly controlled by different MYB-BHLH-TTG1 transcription factor complexes containing the BHLH factors EGL3 and TT8. Experiments with single and double loss-of-function mutants identified EGL3 and TT8 as necessary regulators of anthocyanin accumulation in developing A. thaliana seedlings.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/enzimologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica de Plantas , Oxigenases/genética , Alelos , Sequência de Aminoácidos , Proteínas de Arabidopsis/metabolismo , Modelos Moleculares , MutaçãoRESUMO
Flavonol synthase (FLS) (EC-number 1.14.11.23), the enzyme that catalyses the conversion of flavonols into dihydroflavonols, is part of the flavonoid biosynthesis pathway. In Arabidopsis thaliana, this activity is thought to be encoded by several loci. In addition to the FLAVONOL SYNTHASE1 (FLS1) locus that has been confirmed by enzyme activity assays, loci displaying similarity of the deduced amino acid sequences to FLS1 have been identified. We studied the putative A. thaliana FLS gene family using a combination of genetic and metabolite analysis approaches. Although several of the FLS gene family members are expressed, only FLS1 appeared to influence flavonoid biosynthesis. Seedlings of an A. thaliana fls1 null mutant (fls1-2) show enhanced anthocyanin levels, drastic reduction in flavonol glycoside content and concomitant accumulation of glycosylated forms of dihydroflavonols, the substrate of the FLS reaction. By using a leucoanthocyanidin dioxygenase (ldox) fls1-2 double mutant, we present evidence that the remaining flavonol glycosides found in the fls1-2 mutant are synthesized in planta by the FLS-like side activity of the LDOX enzyme.