In all higher plants studied to date, the anthocyanin pigment pathway is regulated by a suite of transcription factors that include Myb, bHLH and WD-repeat proteins. However, in Arabidopsis thaliana, the Myb regulators remain to be conclusively identified, and little is known about anthocyanin pathway regulation by TTG1-dependent transcriptional complexes. Previous overexpression of the PAP1 Myb suggested that genes from the entire phenylpropanoid pathway are targets of regulation by Myb/bHLH/WD-repeat complexes in Arabidopsis, in contrast to other plants. Here we demonstrate that overexpression of Myb113 or Myb114 results in substantial increases in pigment production similar to those previously seen as a result of over-expression of PAP1, and pigment production in these overexpressors remains TTG1- and bHLH-dependent. Also, plants harboring an RNAi construct targeting PAP1 and three Myb candidates (PAP2, Myb113 and Myb114) showed downregulated Myb gene expression and obvious anthocyanin deficiencies. Correlated with these anthocyanin deficiencies is downregulation of the same late anthocyanin structural genes that are downregulated in ttg1 and bHLH anthocyanin mutants. Expression studies using GL3:GR and TTG1:GR fusions revealed direct regulation of the late biosynthetic genes only. Functional diversification between GL3 and EGL3 with regard to activation of gene targets was revealed by GL3:GR studies in single and double bHLH mutant seedlings. Expression profiles for Myb and bHLH regulators are also presented in the context of pigment production in young seedlings.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-313X.2007.03373.x

Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings.

Pigments are present in all living matter and provide attractive colors and play basic roles in the development of organisms. Human beings, like most animals, come in contact with their surroundings through color, and things can or cannot be acceptable based on their color characteristics. This review presents the basic information about pigments focusing attention on the natural ones; it emphasizes the principal plant pigments: carotenoids, anthocyanins, and betalains. Special considerations are given to their salient characteristics; to their biosynthesis, taking into account the biochemical and molecular biology information generated in their elucidation; and to the processing and stability properties of these compounds as food colorants.

Natural Pigments: Carotenoids, Anthocyanins, and Betalains — Characteristics, Biosynthesis, Processing, and Stability

Seed Dormancy and Germination

Flavonoids are secondary metabolites that accumulate in most plant seeds and are involved in physiological functions such as dormancy or viability. This review presents a current view of the genetic and biochemical control of flavonoid metabolism during seed development. It focuses mainly on proanthocyanidin accumulation in Arabidopsis, with comparisons to other related metabolic and regulatory pathways. These intricate networks and their fine-tuned regulation, once they are determined, should contribute to a better understanding of seed coat development and the control of PA and flavonol metabolism. In addition, flavonoids provide an interesting model to study various biological processes and metabolic and regulatory networks.

Genetics and biochemistry of seed flavonoids

The TRANSPARENT TESTA GLABRA1 ( TTG1 ) locus regulates several developmental and biochemical pathways in Arabidopsis, including the formation of hairs on leaves, stems, and roots, and the production of seed mucilage and anthocyanin pigments. The TTG1 locus has been isolated by positional cloning, and its identity was confirmed by complementation of a ttg1 mutant. The locus encodes a protein of 341 amino acid residues with four WD40 repeats. The protein is similar to AN11, a regulator of anthocyanin biosynthesis in petunia, and more distantly related to those of the β subunits of heterotrimeric G proteins, which suggests a role for TTG1 in signal transduction to downstream transcription factors. The 1.5-kb TTG1 transcript is present in all major organs of Arabidopsis. Sequence analysis of six mutant alleles has identified base changes producing truncations or single amino acid changes in the TTG1 protein.

/pdf/the-transparent-testa-glabra1-locus-which-regulates-trichome-3o1m9e2w27.pdf

The TRANSPARENT TESTA GLABRA1 Locus, Which Regulates Trichome Differentiation and Anthocyanin Biosynthesis in Arabidopsis, Encodes a WD40 Repeat Protein

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.

Characterization of Flavonol Synthase and Leucoanthocyanidin Dioxygenase Genes in Arabidopsis (Further Evidence for Differential Regulation of "Early" and "Late" Genes)

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 isomerase in 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.

/pdf/analysis-of-flavanone-3-hydroxylase-in-arabidopsis-seedlings-3jpqk8rgh1.pdf

Analysis of Flavanone 3-Hydroxylase in Arabidopsis Seedlings (Coordinate Regulation with Chalcone Synthase and Chalcone Isomerase)

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.

Disruption of specific flavonoid genes enhances the accumulation of flavonoid enzymes and end-products in Arabidopsis seedlings.

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.

Matthew K. Pelletier

Papers

Characterization of Flavonol Synthase and Leucoanthocyanidin Dioxygenase Genes in Arabidopsis (Further Evidence for Differential Regulation of "Early" and "Late" Genes)

Analysis of Flavanone 3-Hydroxylase in Arabidopsis Seedlings (Coordinate Regulation with Chalcone Synthase and Chalcone Isomerase)

Disruption of specific flavonoid genes enhances the accumulation of flavonoid enzymes and end-products in Arabidopsis seedlings.

Are flavonoids synthesized by a multi-enzyme complex?

Analysis of Flavanone 3-Hydroxylase in