Identification of key amino acids for the evolution of promoter target specificity of anthocyanin and proanthocyanidin regulating MYB factors.

A complex of R2R3-MYB and bHLH transcription factors, stabilized by WD40 repeat proteins, regulates gene transcription for plant cell pigmentation and epidermal cell morphology. It is the MYB component of this complex which specifies promoter target activation. The Arabidopsis MYB TT2 regulates proanthocyanidin (PA) biosynthesis by activating the expression of ANR (anthocyanidin reductase), the gene product of which catalyzes the first committed step of this pathway. Conversely the closely related MYB PAP4 (AtMYB114) regulates the anthocyanin pathway and specifically activates UFGT (UDP-glucose:flavonoid-3-O-glucosyltransferase), encoding the first enzyme of the anthocyanin pathway. Both at the level of structural and regulatory genes, evolution of PA biosynthesis proceeded anthocyanin biosynthesis and we have identified key residues in these MYB transcription factors for the evolution of target promoter specificity. Using chimeric and point mutated variants of TT2 and PAP4 we found that exchange of a single amino acid, Gly/Arg(39) in the R2 domain combined with an exchange of a four amino acid motif in the R3 domain, could swap the pathway selection of TT2 and PAP4, thereby converting in planta specificity of the PA towards the anthocyanin pathway and vice versa. The general importance of these amino acids for target specificity was also shown for the grapevine transcription factors VvMYBPA2 and VvMYBA2 which regulate PAs and anthocyanins, respectively. These results provide an insight into the evolution of the different flavonoid regulators from a common ancestral gene.

G protein-coupled receptor-type G proteins are required for light-dependent seedling growth and fertility in Arabidopsis.

G protein-coupled receptor-type G proteins (GTGs) are highly conserved membrane proteins in plants, animals, and fungi that have eight to nine predicted transmembrane domains. They have been classified as G protein-coupled receptor-type G proteins that function as abscisic acid (ABA) receptors in Arabidopsis thaliana. We cloned Arabidopsis GTG1 and GTG2 and isolated new T-DNA insertion alleles of GTG1 and GTG2 in both Wassilewskija and Columbia backgrounds. These gtg1 gtg2 double mutants show defects in fertility, hypocotyl and root growth, and responses to light and sugars. Histological studies of shoot tissue reveal cellular distortions that are particularly evident in the epidermal layer. Stable expression of GTG1(pro):GTG1-GFP (for green fluorescent protein) in Arabidopsis and transient expression in tobacco (Nicotiana tabacum) indicate that GTG1 is localized primarily to Golgi bodies and to the endoplasmic reticulum. Microarray analysis comparing gene expression profiles in the wild type and double mutant revealed differences in expression of genes important for cell wall function, hormone response, and amino acid metabolism. The double mutants isolated here respond normally to ABA in seed germination assays, root growth inhibition, and gene expression analysis. These results are inconsistent with their proposed role as ABA receptors but demonstrate that GTGs are fundamentally important for plant growth and development.

A truncated MYB transcription factor from Antirrhinum majus regulates epidermal cell outgrowth.

Plant MYB genes can be divided into subgroups on the basis of additional conserved regions of sequence. In some cases, genes within a subgroup share similarities in function, as well as sequence. The functions of three proteins in subgroup 9 have been described, with AmMYBMX regulating the differentiation of conical-papillate petal epidermal cells, PhMYB1 involved in extending the growth of these cells, and AmMYBML1 involved in differentiation of several petal epidermal cell types. Here, the isolation of a gene encoding a new member of MYB subgroup 9, AmMYBML3 (Antirrhinum majus MYB MIXTA-LIKE 3) is described, which contains the defining regions of conserved sequence but is lacking the majority of the C-terminus, including the amphipathic alpha-helix presumed necessary for transcriptional activation. AmMYBML3 is expressed in all aerial organs, but its expression is restricted to outgrowing epidermal cells, including trichomes, stigmatic papillae, and petal conical-papillate cells. Ectopic expression of AmMYBML3 in tobacco results in the formation of conical-papillate cells in the usually flat carpel epidermis. These data suggest that this protein is capable of altering epidermal development, thus resulting in cellular outgrowth, despite the missing C-terminus, and may act in conjunction with other transcriptional activators to enhance cellular outgrowth from the epidermis of all aerial organs.

The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development.

Proanthocyanidins (PAs; or condensed tannins) can protect plants against herbivores, contribute to the taste of many fruits, and act as dietary antioxidants beneficial for human health. We have previously shown that in grapevine (Vitis vinifera) PA synthesis involves both leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR). Here we report the characterization of a grapevine MYB transcription factor VvMYBPA1, which controls expression of PA pathway genes including both LAR and ANR. Expression of VvMYBPA1 in grape berries correlated with PA accumulation during early berry development and in seeds. In a transient assay, VvMYBPA1 activated the promoters of LAR and ANR, as well as the promoters of several of the general flavonoid pathway genes. VvMYBPA1 did not activate the promoter of VvUFGT, which encodes the anthocyanin-specific enzyme UDP-glucose:flavonoid-3-O-glucosyltransferase, suggesting VvMYBPA1 is specific to regulation of PA biosynthesis in grapes. The Arabidopsis (Arabidopsis thaliana) MYB transcription factor TRANSPARENT TESTA2 (TT2) regulates PA synthesis in the seed coat of Arabidopsis. By complementing the PA-deficient seed phenotype of the Arabidopsis tt2 mutant with VvMYBPA1, we confirmed the function of VvMYBPA1 as a transcriptional regulator of PA synthesis. In contrast to ectopic expression of TT2 in Arabidopsis, constitutive expression of VvMYBPA1 resulted in accumulation of PAs in cotyledons, vegetative meristems, leaf hairs, and roots in some of the transgenic seedlings. To our knowledge, this is the first report of a MYB factor that controls genes of the PA pathway in fruit, including both LAR and ANR, and this single MYB factor can induce ectopic PA accumulation in Arabidopsis.

Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples.

Anthocyanins are secondary metabolites found in higher plants that contribute to the colors of flowers and fruits. In apples (Malus domestica Borkh.), several steps of the anthocyanin pathway are coordinately regulated, suggesting control by common transcription factors. A gene encoding an R2R3 MYB transcription factor was isolated from apple (cv Cripps' Pink) and designated MdMYB1. Analysis of the deduced amino acid sequence suggests that this gene encodes an ortholog of anthocyanin regulators in other plants. The expression of MdMYB1 in both Arabidopsis (Arabidopsis thaliana) plants and cultured grape cells induced the ectopic synthesis of anthocyanin. In the grape (Vitis vinifera) cells MdMYB1 stimulated transcription from the promoters of two apple genes encoding anthocyanin biosynthetic enzymes. In ripening apple fruit the transcription of MdMYB1 was correlated with anthocyanin synthesis in red skin sectors of fruit. When dark-grown fruit were exposed to sunlight, MdMYB1 transcript levels increased over several days, correlating with anthocyanin synthesis in the skin. MdMYB1 gene transcripts were more abundant in red skin apple cultivars compared to non-red skin cultivars. Several polymorphisms were identified in the promoter of MdMYB1. A derived cleaved amplified polymorphic sequence marker designed to one of these polymorphisms segregated with the inheritance of skin color in progeny from a cross of an unnamed red skin selection (a sibling of Cripps' Pink) and the non-red skin cultivar Golden Delicious. We conclude that MdMYB1 coordinately regulates genes in the anthocyanin pathway and the expression level of this regulator is the genetic basis for apple skin color.

Development of three different cell types is associated with the activity of a specific MYB transcription factor in the ventral petal of Antirrhinum majus flowers.

Petal tissue comprises several different cell types, which have specialised functions in pollination in different flowering plant species. In Antirrhinum majus, the MIXTA protein directs the formation of conical epidermal cells in petals. Transgenic experiments have indicated that MIXTA activity can also initiate trichome development, dependent on the developmental timing of its expression. MIXTA is normally expressed late in petal development and functions only in conical cell differentiation. However, an R2R3 MYB transcription factor very similar to MIXTA (AmMYBML1), which induces both trichome and conical cell formation in transgenic plants, is expressed very early during the development of the ventral petal. Its cellular expression pattern suggests that it fulfils three functions: trichome production in the corolla tube, conical cell development in the petal hinge epidermis and reinforcement of the hinge through differential mesophyll cell expansion. The DIVARICATA (DIV) gene is required for ventral petal identity. In div mutants, the ventral petal assumes the identity of lateral petals lacking these three specialised cell types, and expression of AmMYBML1 is significantly reduced compared with wild type, supporting the proposed role of AmMYBML1 in petal cell specification. We suggest that AmMYBML1 is regulated by DIV in association with the B-function proteins DEFICIENS and GLOBOSA, and, consequently, controls specification of particular cells within the ventral petal which adapt the corolla to specialised functions in pollination.