Flavonoids: a colorful model for the regulation and evolution of biochemical pathways

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.

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

Flavonoids are secondary metabolites involved in several aspects of plant development and defence. They colour fruits and flowers, favouring seed and pollen dispersal, and contribute to plant adaptation to environmental conditions such as cold or UV stresses, and pathogen attacks. Because they affect the quality of flowers (for horticulture), fruits and vegetables, and their derivatives (colour, aroma, stringency, etc.), flavonoids have a high economic value. Furthermore, these compounds possess pharmaceutical properties extremely attractive for human health. Thanks to easily detectable mutant phenotypes, such as modification of petal pigmentation and seeds exhibiting transparent testa, the enzymes involved in the flavonoid biosynthetic pathway have been characterized in several plant species. Conserved features as well as specific differences have been described. Regulation of structural gene expression appears tightly organized in a spatial and temporal way during plant development, and is orchestrated by a ternary complex involving transcription factors from the R2R3-MYB, basic helix-loop-helix (bHLH), and WD40 classes. This MYB-bHLH-WD40 (MBW) complex regulates the genes that encode enzymes specifically involved in the late steps of the pathway leading to the biosynthesis of anthocyanins and condensed tannins. Although several genes encoding transcription factors from these three families have been identified, many gaps remain in our understanding of the regulation of this biosynthetic pathway, especially about the respective roles of bHLH and WD40 proteins. A better knowledge of the regulatory mechanisms of the flavonoid pathway is likely to favour the development of new biotechnological tools for the generation of value-added plants with optimized flavonoid content.

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Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway

https://www.cell.com/trends/plant-science/pdf/S1360-1385(04)00297-3.pdf

MYB–bHLH–WD40 protein complex and the evolution of cellular diversity

The position-dependent specification of the hair and non-hair cell types in the Arabidopsis root epidermis provides a simple model for the study of cell fate determination in plants. Several putative transcriptional regulators are known to influence this cell fate decision. Indirect evidence from studies with the maize R gene has been used to suggest that a bHLH transcription factor also participates in this process. We show that two Arabidopsis genes encoding bHLH proteins, GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3), act in a partially redundant manner to specify root epidermal cell fates. Plants homozygous for mutations in both genes fail to specify the non-hair cell type, whereas plants overexpressing either gene produce ectopic non-hair cells. We also find that these genes are required for appropriate transcription of the non-hair specification gene GL2 and the hair cell specification gene CPC, showing that GL3 and EGL3 influence both epidermal cell fates. Furthermore, we show that these bHLH proteins require a functional WER MYB protein for their action, and they physically interact with WER and CPC in the yeast two-hybrid assay. These results suggest a model in which GL3 and EGL3 act together with WER in the N cell position to promote the non-hair cell fate, whereas they interact with the incomplete MYB protein CPC in the H position, which blocks the non-hair pathway and leads to the hair cell fate.

/pdf/the-bhlh-genes-glabra3-gl3-and-enhancer-of-glabra3-egl3-chqrgpslhm.pdf

The bHLH genes GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) specify epidermal cell fate in the Arabidopsis root.

The root epidermis of Arabidopsis provides an exceptional model for studying the molecular basis of cell fate and differentiation. To obtain a systems-level view of root epidermal cell differentiation, we used a genome-wide transcriptome approach to define and organize a large set of genes into a transcriptional regulatory network. Using cell fate mutants that produce only one of the two epidermal cell types, together with fluorescence-activated cell-sorting to preferentially analyze the root epidermis transcriptome, we identified 1,582 genes differentially expressed in the root-hair or non-hair cell types, including a set of 208 “core” root epidermal genes. The organization of the core genes into a network was accomplished by using 17 distinct root epidermis mutants and 2 hormone treatments to perturb the system and assess the effects on each gene's transcript accumulation. In addition, temporal gene expression information from a developmental time series dataset and predicted gene associations derived from a Bayesian modeling approach were used to aid the positioning of genes within the network. Further, a detailed functional analysis of likely bHLH regulatory genes within the network, including MYC1, bHLH54, bHLH66, and bHLH82, showed that three distinct subfamilies of bHLH proteins participate in root epidermis development in a stage-specific manner. The integration of genetic, genomic, and computational analyses provides a new view of the composition, architecture, and logic of the root epidermal transcriptional network, and it demonstrates the utility of a comprehensive systems approach for dissecting a complex regulatory network.

/pdf/a-gene-regulatory-network-for-root-epidermis-cell-1fv5qmvqxa.pdf

A gene regulatory network for root epidermis cell differentiation in Arabidopsis.

The specification of the hair and non-hair cells in the Arabidopsis root epidermis provides a useful model for the study of cell fate determination in plants. A network of putative transcriptional regulators, including the related bHLH proteins GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3), is known to influence the patterning of these cell types. Here, we analyze the expression and regulation of GL3 and EGL3 during root epidermis development. Although they are thought to act in both the hair and non-hair cell types, we surprisingly found that GL3 and EGL3 gene expression and RNA accumulation occurs preferentially in the developing hair cells. By analyzing the expression of GL3::GUS and EGL3::GUS reporter fusions in various mutant and overexpression lines, we discovered that the expression of both genes is negatively regulated by WER, GL3 and EGL3 in the developing non-hair cells, and positively regulated by the CPC and TRY proteins in the developing hair cells. Further, the analysis of a GL3-YFP translational fusion, expressed under the GL3 promoter, indicates that the GL3 protein moves from the hair cells to the non-hair cells. These results suggest that GL3/EGL3 accumulation in the N cells is dependent on specification of the hair cell fate, which itself is known to be influenced (via CPC-mediated lateral inhibition) by the non-hair cells. This bi-directional signaling mechanism defines a new regulatory circuit of intercellular communication to specify the epidermal cell types.

The bHLH genes GL3 and EGL3 participate in an intercellular regulatory circuit that controls cell patterning in the Arabidopsis root epidermis.

Root epidermis development in Arabidopsis provides a simple and powerful model for studying cell fate specification. Cellular, molecular, and genetic approaches have been used to define many genes, and their corresponding proteins, that are essential for the position-dependent specification of the two root epidermal cell types. These studies have led to a working model in which a network of transcriptional regulators that is influenced by positional cues establishes differences in gene expression in neighboring cells through a set of positive and negative feedback loops. The continued analysis of this experimental system is likely to provide new insights into mechanisms of transcriptional regulation and cell-cell interactions during development.

Christine Bernhardt

Papers

The bHLH genes GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3) specify epidermal cell fate in the Arabidopsis root.

A gene regulatory network for root epidermis cell differentiation in Arabidopsis.

The bHLH genes GL3 and EGL3 participate in an intercellular regulatory circuit that controls cell patterning in the Arabidopsis root epidermis.

2004 sivb congress symposium proceeding: cell fate specification during development of the arabidopsis root epidermis