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Transcriptional control of phenylpropanoid metabolism

Objective

The phenylpropanoid biosynthetic pathway in plants is among the best characterised biosynthetic pathways with over 20 structural and regulatory genes identified. The levels of key regulatory enzymes in the pathway are established primarily by transcriptional initiation rates, modulated by responses to both environmental and developmental factors. The goal of this project is to understand and manipulate the molecular mechanisms integrating the activity of genes encoding enzymes of the pathway. A united approach aimed at determining the function of transcription factors of the Myc, Myb, bHLH and bZIP classes was taken, taking advantage of genetic and biochemical systems developed in Antirrhinum, Petunia, Arabidopsis, maize, parsley and tobacco.
The mechanisms regulating expression of structural genes representing different parts of the phenylpropanoid pathway were investigated. The core pathway was represented by genes encoding PAL (phenylalanine ammonia-lyase), which catalyse the first committed step in the pathway. Genes encoding enzymes of the flavonoid pathway including those required for flavonol synthesis [chalcone synthase, (CHS), chalcone isomerase (CHI), flavonol synthase (FLS) and flavonol 3 hydroxylase (F3H)], and those committed to the anthocyanin pathway [dihydroflavonol reductase (DFR)], the general pathway [cinnamate 4-hydroxylase (C4H) and coumaryl CoA ligase (4CL)] and the lignin pathway [cinnamyl alcohol dehyrogenase, (CAD)] were studied in a variety of systems. In addition, classes of genes involved in regulating vacuolar pH, which influences pigmentation, were aso included. The influence of light, tissue stress and developmental factors, mediated by the Myc, Myb, bHLH and bZIP class of transcription factors on these classes of genes were studied. The integrated results of this work are categorised according to the classes of structural genes in the phenylpropanoid pathway.

Core pathway
A minimal PAL2 promoter was defined and two classes of promoter element shown to be necessary and sufficient for developmental and environmental regulation. One site binds transcription factors of the bZIP class, and southwestern screening isolated three candidate factors of the TAF family from tobacco. These factors did not activate expression, and were differentially expressed in tobacco plants. The other class of site was present in three copies, and individual sites contributed to tissue-specific and environmentally induced expression patterns. These sites were shown to bind the Myb class of transcription factors. Candidate Myb factors involved in floral expression of PAL (Antirrhinum Myb 305), in wound-induced PAL expression in potato tubers (tomato Myb16) and in xylem specific expression in tobacco and pine (Pinus taeda Myb 143 and 126) were isolated and characterised. Tissue-specific expression of myb genes, coupled to specific interactions between different binding sites on the promoter, conferred the observed expression patterns.

Flavonoid pathway
In Antirrhinum the factor Myb305 was shown to activate transcription of the PAL, CHI and F3H genes. A closely related factor, Myb340, also regulates these genes. Its strong gene activation is modulated by phosphorylation.These two factors appear to modulate flavonol production in carpels, style and developing ovules. Other Myb proteins, encoded by the Rosea and Veinosa genes, control anthocyanin pigmentation in specific regions of flowers. A model is proposed in which the apparently redundant functions of Myb genes control the kinetics of gene expression in distinct spatial and temporal pattterns. In petunia flowers four regulatory loci, AN1, AN2, AN11 and JAF13, which control expression of genes encoding enzymes of the flavonoid pathway, were defined by transposon tagging. The AN1 and JAF13 proteins are bHLH transcription factors, the AN2 protein is a Myb gene, and AN11 is a regulator of previously unknown structure that is highly conserved at the structural and functional level among plants and humans. JAF13 is a homologue of Delila from Antirrhinum, and it is required, together with the Myb AN2, for ectopic expression of a DFR gene. AN2 is expressed specifically in petals, and a related Myb, AN4, is responsible for anthocyanin expression in anthers. Transposon-generated alleles of the bHLH protein AN1 revealed that the DNA binding domain was not required for activation of flavonoid genes. Species-specific differences were noted in control of early and late steps of the flavonoid pathway in maize and petunia that depended upon the promoter elements, not the transcription factors. A detailed structural and functional study of the petunia Ph3 Myb gene was undertaken. The protein is expressed in the epidermal layer of floral tissue and was shown to activate expression of the chsJ gene, indicating a key role in regulation of phenylpropanoid biosynthesis in petunia flowers. Ph3 Myb can bind to two different target sites, and this dual specificity was shown to be due to a single amino acid in the DNA binding domain. In Arabidopsis and parsley the regulation of CHS by light is an important step leading to the production of flavonoid UV protectants. A light-inducible cell culture was used to define the CHS light responsive unit (LRU), necessary and sufficient for light responsiveness and tissue-specificity. This contained an ACGT (ACE) element and a Myb binding site. BZIP transcription factors called CPRF1-4 were isolated from parsley and their mRNAs were shown to be UV inducible. The CPRF1 gene may autoregulate its expression in response to UV. The polyproline motifs of CPRF-1 and -4 were shown to function as activation domains. A Myb protein binding to the LRU was isolated from parsley, although this only activated transcription as a VP16 fusion. This Myb protein defined a new class that contains a single helix-turn-helix DNA binding domain. Knowing the key role of Myb genes in regulating phenylpropanoid gene expression, a systematic sequencing analysis was carried out on the large Arabidopsis Myb family. 77 genes were partially sequenced, and it is estimated that there are over 100 members of this family in Arabidopsis. Screening T-DNA insertion lines led to the isolation of two disruptions in Myb genes. In maize aleurone and pericarp tissue-specific flavonoid synthesis was shown to be controlled by bHLH proteins, and light regulation was controlled by induction of Pl Myb gene expression. Interactions betwen alleles of bHLH proteins lead to partial silencing (REED), correlated with hypermethylation of promoter regions.

Lignin-specific pathway
Antirrhinum Myb308 and Myb330 were shown to be responsible for control of the phenolic and lignin pathways, since over-expression lead to a 30% reduction in lignin content in transgenic tobacco. Overexpression of these genes also lead to accelerated cell death in transgenic tobacco plants. In collaboration, Pinus Myb genes expressed specifically in the developing xylem were isolated and shown to activate the PAL2 and CAD genes via the Myb binding site necessary for xylem-specific expression in tobacco.

MAJOR SCIENTIFIC BREAKTHROUGHS:
We have demonstrated a key role for Myb transcription factors in integrating the expression of sets of phenylpropanoid biosynthetic genes in tissue-specific and environmentally-induced gene expression. The foundations for future genetic analysis of this class of transcription factor have been established in Arabidopsis by the partial characterisation of 77 members of the gene family. Hints at the interactions between bHLH and Myb proteins required for petal specific gene expresison have been obtained, and a novel class of protein, possibly involved in signal transduction, was necessary for expression of phenylpropanoid genes.

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CSC - Cost-sharing contracts

Coordinator

JOHN INNES CENTRE
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Norwich Research Park, Colney
NORWICH
United Kingdom

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Last update: 16 August 1995

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Permalink: https://cordis.europa.eu/project/id/BIO2930101

European Union, 2024