Abstract

GAs modulate development and growth of the berry. The most important genes of the GA biosynthetic pathway have not been cloned so far in grape and information on their expression under different physiological conditions is lacking. In this context, we first cloned one member of the GA-20 oxidase (GA20-ox) and GA-2 oxidase (GA2-ox) gene families from 'Sultanina', using degenerate primers directed to conserve portions of the gene. As both genes presented intron-like sequences, probes were designed excluding these regions. Southern analyses using these probes revealed only one gene in each case. The same probes were used in Northern blots to study gene expression profiles in different stages of development with and without exogenous application of GA 3. At the same time, we quantified the endogenous levels of the active forms of gibberellins and their precursors by GC-MS. In early stages of berry development, the two metabolic pathways of GAs co-exist (early 13-hydroxilation and no-early 13-hydroxilation) which was demonstrated by the presence of GA1 and GA4 and their precursors. The highest levels of GAs were detected at fruit set, diminishing to basal levels 20 to 30 days after anthesis. The expression of the genes related to the synthesis and degradation of the active forms (GA20-ox and GA2-ox, respectively) was temporally sequential, with a higher expression of GA20-ox prior to fruit set, and induction of GA2-ox after that stage. Unlike GA20-ox, transcripts of GA2-ox increased in response to exogenous addition of GA 3. The expression pattern of genes in response to GA 3 was studied in berries at two pre-veraison stages using microarrays of 5,000 ESTs obtained from different developmental stages. Of these genes, 401 and 177 were at least two-fold induced in one of the two stages compared to the other, respectively. When comparing the GA 3-treated samples, 369 genes were up-regulated and 319 were repressed on berries at 6-mm diameter stage, and 288 were up-regulated and 1,129 repressed in a later stage.