Amanda R. Walker

Arabidopsis AUX1 gene: A permease-like regulator of root gravitropism

The plant hormone auxin regulates various developmental processes including root formation, vascular development, and gravitropism. Mutations within the AUX1 gene confer an auxin-resistant root growth phenotype and abolish root gravitropic curvature. Polypeptide sequence similarity to amino acid permeases suggests that AUX1 mediates the transport of an amino acid-like signaling molecule. Indole-3-acetic acid, the major form of auxin in higher plants, is structurally similar to tryptophan and is a likely substrate for the AUX1 gene product. The cloned AUX1 gene can restore the auxin-responsiveness of transgenic aux1 roots. Spatially, AUX1 is expressed in root apical tissues that regulate root gravitropic curvature.

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.

The transcription factor VvMYB5b contributes to the regulation of anthocyanin and proanthocyanidin biosynthesis in developing grape berries.

Among the dramatic changes occurring during grape berry (Vitis vinifera) development, those affecting the flavonoid pathway have provoked a number of investigations in the last 10 years. In addition to producing several compounds involved in the protection of the berry and the dissemination of the seeds, final products of this pathway also play a critical role in berry and wine quality. In this article, we describe the cloning and functional characterization of VvMYB5b, a cDNA isolated from a grape berry (V. vinifera ‘Cabernet Sauvignon’) library. VvMYB5b encodes a protein belonging to the R2R3-MYB family of transcription factors and displays significant similarity with VvMYB5a, another MYB factor recently shown to regulate flavonoid synthesis in grapevine. The ability of VvMYB5a and VvMYB5b to activate the grapevine promoters of several structural genes of the flavonoid pathway was confirmed by transient expression of the corresponding cDNAs in grape cells. Overexpression of VvMYB5b in tobacco (Nicotiana tabacum) leads to an up-regulation of genes encoding enzymes of the flavonoid pathway and results in the accumulation of anthocyanin- and proanthocyanidin-derived compounds. The ability of VvMYB5b to regulate particularly the anthocyanin and the proanthocyanidin pathways is discussed in relation to other recently characterized MYB transcription factors in grapevine. Taken together, data presented in this article give insight into the transcriptional mechanisms associated with the regulation of the flavonoid pathway throughout grape berry development.

The Grapevine R2R3-MYB Transcription Factor VvMYBF1 Regulates Flavonol Synthesis in Developing Grape Berries

Flavonols are important ultraviolet light protectants in many plants and contribute substantially to the quality and health-promoting effects of fruits and derived plant products. To study the regulation of flavonol synthesis in fruit, we isolated and characterized the grapevine (Vitis vinifera ‘Shiraz’) R2R3-MYB transcription factor VvMYBF1. Transient reporter assays established VvMYBF1 to be a specific activator of flavonol synthase1 (VvFLS1) and several other promoters of grapevine and Arabidopsis (Arabidopsis thaliana) genes involved in flavonol synthesis. Expression of VvMYBF1 in the Arabidopsis mutant myb12 resulted in complementation of its flavonol-deficient phenotype and confirmed the function of VvMYBF1 as a transcriptional regulator of flavonol synthesis. Transcript analysis of VvMYBF1 throughout grape berry development revealed its expression during flowering and in skins of ripening berries, which correlates with the accumulation of flavonols and expression of VvFLS1. In addition to its developmental regulation, VvMYBF1 expression was light inducible, implicating VvMYBF1 in the control of VvFLS1 transcription. Sequence analysis of VvMYBF1 and VvFLS1 indicated conserved putative light regulatory units in promoters of both genes from different cultivars. By analysis of the VvMYBF1 amino acid sequence, we identified the previously described SG7 domain and an additional sequence motif conserved in several plant MYB factors. The described motifs have been used to identify MYB transcription factors from other plant species putatively involved in the regulation of flavonol biosynthesis. To our knowledge, this is the first functional characterization of a light-inducible MYB transcription factor controlling flavonol synthesis in fruit.

Two new grape cultivars, bud sports of Cabernet Sauvignon bearing pale-coloured berries, are the result of deletion of two regulatory genes of the berry colour locus.

Bud sports are infrequent changes in phenotype affecting shoots of woody perennials but the molecular basis of these mutations has rarely been identified. In this report, we show that the bronze-coloured berries of the Malian cultivar, a documented bud sport of the wine grape Cabernet Sauvignon (Vitis vinifera L.), lack anthocyanins in the subepidermal cells compared to the red/black berried Cabernet Sauvignon in which both the epidermis and several subepidermal cell layers contain anthocyanin. The Malian phenotype is correlated with an alteration in the genome indicated by a reduction of hybridisation signal using a MYBA probe. In Shalistin, a white-berried bud sport of Malian, the red allele at the berry colour locus appears to have been deleted completely. These data suggest that Malian could be a L1/L2 periclinal chimera, which gave rise to Shalistin by an invasion of epidermal cells (L1) by the mutated subepidermal cells (L2). The red grape Pinot Noir has given rise to a number of pale coloured sports, although the provenance of the extant sports is not known. We show that a clone of Pinot Blanc (white-berried) does not have a deletion of the red allele of the same dimensions as that in Shalistin, though a small deletion is a likely explanation for the altered phenotype. However, the mechanism of deletion of the red allele of the berry colour locus is a possible means by which other red to white clonal mutations of grapevines have occurred.

The R2R3-MYB Transcription Factors MYB14 and MYB15 Regulate Stilbene Biosynthesis in Vitis vinifera

This study reports the identification and functional characterization of two stress-inducible R2R3-MYB–type transcription factors, termed MYB14 and MYB15, which regulate the stilbene biosynthetic pathway in grapevine. Plant stilbenes are phytoalexins that accumulate in a small number of plant species, including grapevine (Vitis vinifera), in response to biotic and abiotic stresses and have been implicated in many beneficial effects on human health. In particular, resveratrol, the basic unit of all other complex stilbenes, has received widespread attention because of its cardio-protective, anticarcinogenic, and antioxidant properties. Although stilbene synthases (STSs), the key enzymes responsible for resveratrol biosynthesis, have been isolated and characterized from several plant species, the transcriptional regulation underlying stilbene biosynthesis is unknown. Here, we report the identification and functional characterization of two R2R3-MYB–type transcription factors (TFs) from grapevine, which regulate the stilbene biosynthetic pathway. These TFs, designated MYB14 and MYB15, strongly coexpress with STS genes, both in leaf tissues under biotic and abiotic stress and in the skin and seed of healthy developing berries during maturation. In transient gene reporter assays, MYB14 and MYB15 were demonstrated to specifically activate the promoters of STS genes, and the ectopic expression of MYB15 in grapevine hairy roots resulted in increased STS expression and in the accumulation of glycosylated stilbenes in planta. These results demonstrate the involvement of MYB14 and MYB15 in the transcriptional regulation of stilbene biosynthesis in grapevine.

Two WD-repeat genes from cotton are functional homologues of the Arabidopsis thaliana TRANSPARENT TESTA GLABRA1 (TTG1) gene

Cotton fibres are single, highly elongated cells derived from the outer epidermis of ovules, and are developmentally similar to the trichomes of Arabidopsisthaliana. To identify genes involved in the molecular control of cotton fibre initiation, we isolated four putative homologues of the Arabidopsis trichome-associated gene TRANSPARENT TESTA GLABRA1 (TTG1). All four WD-repeat genes are derived from the ancestral D diploid genome of tetraploid cotton and are expressed in many tissues throughout the plant, including ovules and growing fibres. Two of the cotton genes were able to restore trichome formation in ttg1 mutant Arabidopsis plants. Both these genes also complemented the anthocyanin defect in a white-flowered Matthiola incana ttg1 mutant. These results demonstrate parallels in differentiation between trichomes in cotton and Arabidopsis, and indicate that these cotton genes may be functional homologues of AtTTG1.

Two basic-helix-loop-helix genes (MYC-146 and GL3) from Arabidopsis can activate anthocyanin biosynthesis in a white-flowered Matthiola incana mutant

Basic helix-loop-helix (bHLH) proteins, similar to mammalian Myc transcription factors, regulate the anthocyanin biosynthetic pathway in both monocots and dicots. Two Arabidopsis bHLH genes, GLABRA3 (GL3) and MYC-146, encode proteins that are similar throughout the predicted amino acid sequence to R and DELILA, which regulate anthocyanin production in maize and snapdragon, respectively. Northern blot analysis indicates that MYC-146 is most highly expressed in flower buds and flowers. Expression of a MYC-146 cDNA from the CaMV 35S promoter was unable to complement the anthocyanin deficiency in a ttg1 mutant of Arabidopsis and resulted in no obvious phenotypic change in Columbia plants. However, transient expression of GL3 and MYC-146 upon microprojectile bombardment of petals of a white-flowered mutant of Matthiola incana was able to complement anthocyanin deficiency. The lack of anthocyanin-deficient Arabidopsis mutants mapping to the locations of GL3 and MYC-146 suggests that the two bHLH proteins may be partially redundant and overlap in function.

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/Arg39 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.

Genetic dissection of a TIR-NB-LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine.

The most economically important diseases of grapevine cultivation worldwide are caused by the fungal pathogen powdery mildew (Erysiphe necator syn. Uncinula necator) and the oomycete pathogen downy mildew (Plasmopara viticola). Currently, grapegrowers rely heavily on the use of agrochemicals to minimize the potentially devastating impact of these pathogens on grape yield and quality. The wild North American grapevine species Muscadinia rotundifolia was recognized as early as 1889 to be resistant to both powdery and downy mildew. We have now mapped resistance to these two mildew pathogens in M. rotundifolia to a single locus on chromosome 12 that contains a family of seven TIR-NB-LRR genes. We further demonstrate that two highly homologous (86% amino acid identity) members of this gene family confer strong resistance to these unrelated pathogens following genetic transformation into susceptible Vitis vinifera winegrape cultivars. These two genes, designated resistance to Uncinula necator (MrRUN1) and resistance to Plasmopara viticola (MrRPV1) are the first resistance genes to be cloned from a grapevine species. Both MrRUN1 and MrRPV1 were found to confer resistance to multiple powdery and downy mildew isolates from France, North America and Australia; however, a single powdery mildew isolate collected from the south-eastern region of North America, to which M. rotundifolia is native, was capable of breaking MrRUN1-mediated resistance. Comparisons of gene organization and coding sequences between M. rotundifolia and the cultivated grapevine V. vinifera at the MrRUN1/MrRPV1 locus revealed a high level of synteny, suggesting that the TIR-NB-LRR genes at this locus share a common ancestor.