Additional targets of the Arabidopsis autonomous pathway members, FCA and FY.

A central player in the Arabidopsis floral transition is the floral repressor FLC, the MADS-box transcriptional regulator that inhibits the activity of genes required to switch the meristem from vegetative to floral development. One of the many pathways that regulate FLC expression is the autonomous promotion pathway composed of FCA, FY, FLD, FPA, FVE, LD, and FLK. Rather than a hierarchical set of activities the autonomous promotion pathway comprises sub-pathways of genes with different biochemical functions that all share FLC as a target. One sub-pathway involves FCA and FY, which interact to regulate RNA processing of FLC. Several of the identified components (FY, FVE, and FLD) are homologous to yeast and mammalian proteins with rather generic roles in gene regulation. So why do mutations in these genes specifically show a late-flowering phenotype in Arabidopsis? One reason, found during the analysis of fy alleles, is that the mutant alleles identified in flowering screens can be hypomorphic, they still have partial function. A broader role for the autonomous promotion pathway is supported by a microarray analysis which has identified genes mis-regulated in fca mutants, and whose expression is also altered in fy mutants.

A cDNA from grapevine (Vitis vinifera L.), which shows homology to AGAMOUS and SHATTERPROOF, is not only expressed in flowers but also throughout berry development.

An AGAMOUS/SHATTERPROOF homologue (Vvmads1) was isolated from grapevine by differential display between berry and leaf mRNA. The predicted protein sequence of the full-length clone shows a high degree of homology to PLENA (77% identity) and to SHP1 and SHP2 (75% and 74% identity respectively), and is grouped with AGAMOUS/PLENA homologues when the conserved MADS and K domains are compared. Vvmads1 is expressed only in the later stages of flower development and throughout berry development, although expression is reduced after ripening commenced. When Vvmads1 was over-expressed in tobacco, the resulting plants display altered morphologies in the outer two floral whorls. In the most extreme cases, the inner whorls were surrounded by a carpelloid structure created by the modified sepals. Within these sepals were petals which had been split into sections and which were attached at the base of the flower by structures with the appearance of filaments. The results of this study suggest that Vvmads1 has a regulatory role in flower development before fertilisation and a role in fruit development after fertilisation.

Cloning and characterization of Vitis vinifera UDP-glucose:flavonoid 3-O-glucosyltransferase, a homologue of the enzyme encoded by the maize Bronze-1 locus that may primarily serve to glucosylate anthocyanidins in vivo.

We report here the cloning and optimized expression at 16 degrees C and the characterization of a Vitis vinifera UDP-glucose:flavonoid 3-O-glucosyltransferase, an enzyme responsible for a late step in grapevine anthocyanin biosynthesis. The properties of this and other UDP-glucose:flavonoid 3-O-glucosyltransferases, homologues of the product encoded by the maize Bronze-1 locus, are a matter of conjecture. The availability of a purified recombinant enzyme allowed for the unambiguous determination of the characteristics of a flavonoid 3-O-glucosyltransferase. Kinetic analyses showed that kcat for glucosylation of cyanidin, an anthocyanidin substrate, is 48 times higher than for glucosylation of the flavonol quercetin, whereas Km values are similar for both substrates. Activity toward other classes of substrates is absent. Cu2+ ions strongly inhibit the action of this and other glucosyltransferases; however, we suggest that this phenomenon in large part is due to Cu2+-mediated substrate degradation rather than inhibition of the enzyme. Additional lines of complementary biochemical data also indicated that in the case of V. vinifera, the principal, if not only, role of UDP-glucose:flavonoid 3-O-glucosyltransferases is to glucosylate anthocyanidins in red fruit during ripening. Other glucosyltransferases with a much higher relative activity toward quercetin are suggested to glucosylate flavonols in a distinct spatial and temporal pattern. It should be considered whether gene products homologous to Bronze-1 in some cases more accurately should be referred to as UDP-glucose:anthocyanidin 3-O-glucosyltransferases.

Treatment of Grape Berries, a Nonclimacteric Fruit with a Synthetic Auxin, Retards Ripening and Alters the Expression of Developmentally Regulated Genes.

Treatment of grape (Vitis vinifera L.) berries with the synthetic auxin-like compound benzothiazole-2-oxyacetic acid (BTOA) caused a delay in the onset of ripening of approximately 2 weeks. This was manifested as a retardation of the increases in berry weight, color, deformability, and hexose concentration. BTOA treatment also delayed by 2 weeks the increase in abscisic acid level that normally accompanies ripening and altered the expression of a number of developmentally regulated genes. A putative vacuolar invertase, which is normally expressed from berry set until ripening and turned off after ripening commences, remained expressed throughout development in BTOA-treated grape berries. This elevated expression resulted in increased levels of invertase activity. In contrast, the up-regulation of four other genes normally switched on at the time of ripening was delayed in BTOA-treated fruit. These included chalcone synthase and UDP-glucose-flavonoid 3-O-glucosyl transferase, both of which are involved in anthocyanin synthesis, a chitinase, and a ripening-related gene of an unknown function. These observations support the view that auxins (perhaps in conjunction with abscisic acid) may have a role in the control of grape berry ripening by affecting the expression of genes involved in the ripening process.

Expression of anthocyanin biosynthesis pathway genes in red and white grapes.

The expression of seven genes from the anthocyanin biosynthesis pathway was determined in different tissues of Shiraz grapevines. All of the tissues contained proanthocyanidins, but only the berry skin accumulated anthocyanins. In most tissues, all of the flavonoid genes except UDP glucose-flavonoid 3-o-glucosyl transferase (UFGT) were expressed, but UFGT expression was only detected in berry skin. Similar patterns of expression were observed in the skin of other red grapes. In white grapes, UFGT expression was not detected. White grape cultivars appear to lack anthocyanins because they lack UFGT, although they also had decreased expression of other flavonoid pathway genes.

Analysis of the Expression of Anthocyanin Pathway Genes in Developing Vitis vinifera L. cv Shiraz Grape Berries and the Implications for Pathway Regulation.

Anthocyanin synthesis in Vitis vinifera L. cv Shiraz grape berries began 10 weeks postflowering and continued throughout berry ripening. Expression of seven genes of the anthocyanin biosynthetic pathway (phenylalanine ammonia lyase [PAL], chalcone synthase [CHS], chalcone isomerase [CHI], flavanone-3-hydroxylase [F3H], dihydroflavonol 4-reductase [DFR], leucoanthocyanidin dioxygen-ase [LDOX], and UDP glucose-flavonoid 3-o-glucosyl transferase [UFGT]) was determined. In flowers and grape berry skins, expression of all of the genes, except UFGT, was detected up to 4 weeks postflowering, followed by a reduction in this expression 6 to 8 weeks postflowering. Expression of CHS, CHI, F3H, DFR, LDOX, and UFGT then increased 10 weeks postflowering, coinciding with the onset of anthocyanin synthesis. In grape berry flesh, no PAL or UFGT expression was detected at any stage of development, but CHS, CHI, F3H, DFR, and LDOX were expressed up to 4 weeks postflowering. These results indicate that the onset of anthocyanin synthesis in ripening grape berry skins coincides with a coordinated increase in expression of a number of genes in the anthocyanin biosynthetic pathway, suggesting the involvement of regulatory genes. UFGT is regulated independently of the other genes, suggesting that in grapes the major control point in this pathway is later than that observed in maize, petunia, and snapdragon.

An apple polyphenol oxidase cDNA is up-regulated in wounded tissues.

A full-length cDNA clone encoding apple (Malus domesticus) polyphenol oxidase (PPO) was isolated from a fruit peel cDNA library. Southern analysis indicated that apple PPO is encoded by a divergent multigene family. By northern analysis, PPO mRNA was only detected in a fruit sample taken one week after full bloom. PPO mRNA accumulated in wounded tissues, and also in peel tissue showing the symptoms of superficial scald, a post-harvest disorder. The induction of PPO mRNA provides the first evidence for transcriptional control of PPO expression after wounding or the manifestation of a physiological disorder.