ABSTRACT
Polyclonal antibodies were developed against the flavonoid biosynthetic enzymes, CHS, CHI, F3H, FLS, and LDOX from Arabidopsis thaliana. These antibodies were used to perform the first detailed analysis of coordinate expression of flavonoid metabolism at the protein level. The pattern of flavonoid enzyme expression over the course of seedling development was consistent with previous studies indicating that chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), and flavonol synthase (FLS) are encoded by 'early' genes while leucoanthocyanidin dioxygenase (LDOX) is encoded by a 'late' gene. This sequential expression may underlie the variations in flavonoid end-products produced during this developmental stage, as determined by HPLC analysis, which includes a shift in the ratio of the flavonols, quercetin and kaempferol. Moreover, immunoblot and HPLC analyses revealed that several transparent testa lines blocked at intermediate steps of the flavonoid pathway actually accumulated higher levels of specific flavonoid enzymes and end-products. These results suggest that specific intermediates may act as inducers of flavonoid metabolism.
Subject(s)
Arabidopsis/enzymology , Arabidopsis/genetics , Flavonoids/biosynthesis , Genes, Plant , Kaempferols , Plant Proteins , Acyltransferases/genetics , Acyltransferases/immunology , Acyltransferases/metabolism , Amino Acid Sequence , Arabidopsis/growth & development , Base Sequence , Escherichia coli/genetics , Gene Expression Regulation, Plant , Glycosides/isolation & purification , Intramolecular Lyases/genetics , Intramolecular Lyases/immunology , Intramolecular Lyases/metabolism , Light , Mixed Function Oxygenases/genetics , Mixed Function Oxygenases/immunology , Mixed Function Oxygenases/metabolism , Molecular Sequence Data , Mutation , Oxidoreductases/genetics , Oxidoreductases/immunology , Oxidoreductases/metabolism , Oxygenases/genetics , Oxygenases/immunology , Oxygenases/metabolism , Quercetin/analogs & derivatives , Quercetin/isolation & purification , Recombinant Proteins/metabolism , Time FactorsABSTRACT
As part of an ongoing investigation into the organization and regulation of the flavonoid biosynthetic pathway, two Arabidopsis thaliana expressed sequence tag (EST) clones (153O10T7 and YAY780) with high homology to leucoanthocyanidin dioxygenase (LDOX) or flavonol synthase (FLS) were identified. EST YAY780 was sequenced and found to encode a protein 49 to 78% identical to all LDOX sequences in the database. EST 153O10T7 was used to isolate a genomic clone encoding a protein with 59 to 61% sequence identity to petunia (Petunia hybrida) and potato (Solanum tuberosum) FLS. DNA blot analysis was used to screen the Arabidopsis genome for sequences related to FLS and LDOX and to determine the positions of the two clones on the RI map. The expression patterns of FLS and LDOX in etiolated seedlings moved to white light and in two putative regulatory mutants (ttg and tt8) were determined by RNA blot analysis. These studies indicate that FLS is an "early" flavonoid gene in Arabidopsis seedlings, whereas LDOX is a "late" gene. Furthermore, FLS is the first flavonoid enzyme identified in Arabidopsis that may be encoded by a gene family.
Subject(s)
Arabidopsis/enzymology , Arabidopsis/genetics , Gene Expression Regulation, Plant , Genes, Plant , Oxidoreductases/genetics , Oxygenases/genetics , Plant Proteins , Amino Acid Sequence , Cloning, Molecular , Darkness , Gene Expression Regulation, Developmental , Gene Expression Regulation, Enzymologic , Information Systems , Light , Molecular Sequence Data , Multigene Family , Oxidoreductases/biosynthesis , Oxidoreductases/chemistry , Oxygenases/biosynthesis , Oxygenases/chemistry , Plants/enzymology , Sequence Homology, Amino Acid , Sequence Tagged Sites , Solanum tuberosum/enzymologyABSTRACT
A genomic clone encoding flavanone 3-hydroxylase (F3H) was isolated from Arabidopsis thaliana. The deduced amino acid sequence is 72 to 94% identical to all previously reported F3H proteins. Low-stringency DNA blot analysis indicated that F3H is encoded by a single gene in Arabidopsis. The F3H locus was mapped to the bottom of chromosome 3 and therefore does not correspond to any of the 13 flavonoid-deficient transparent testa mutants for which a map position is known. Analysis of gene expression in etiolated seedlings exposed to white light and in two putative regulatory mutants, ttg and tt8, demonstrated that the Arabidopsis F3H gene is coordinately expressed with chalcone synthase and chalcone isomerases is seedlings, whereas dihydroflavonol reductase expression is controlled by distinct regulatory mechanisms. The F3H gene may represent a pivotal point in the regulation of flavonoid biosynthesis because its expression is coordinated with different subsets of genes in different plant species.
Subject(s)
Arabidopsis/enzymology , Mixed Function Oxygenases/metabolism , Arabidopsis/genetics , Arabidopsis/growth & development , Base Sequence , Blotting, Southern , Cloning, Molecular , DNA Primers , Gene Expression Regulation, Enzymologic/radiation effects , Gene Expression Regulation, Plant/radiation effects , Genes, Regulator , Light , Mixed Function Oxygenases/genetics , Molecular Sequence Data , MutationABSTRACT
An enormous variety of metabolic processes are characterized by enzyme complexes, which are likely to play important roles in directing the efficient operation and specificity of cellular metabolism. In many cases membranes or cytoskeletal elements provide scaffolding for these highly ordered assemblies of enzymes. Biochemical and immunocytochemical studies indicate that the flavonoid biosynthetic pathway of higher plants involves a complex of sequentially-acting enzymes localized at the cytoplasmic face of the endoplasmic reticulum. This paper describes preliminary efforts to define the organization of this putative flavonoid biosynthetic complex and elucidate its role in controlling the synthesis of different flavonoid end-products in the model plant, Arabidopsis thaliana.
