Developing a Functional Genomics Approach to Berry Ripening and Defense

For the past year we have focused our work on gene discovery in grape. We are using a gene library from veraison stage fruit as the source of the new genes. We screened more than 700 plasmids to identify candidates for DNA sequencing, and identified 312 that appeared to have inserts large enough to identify the gene once its sequence was obtained. Preparation for sequencing required growing selected E. coli colonies overnight and then extracting plasmid DNA. One round of sequence was obtained using T3/T7 sites on the pBluescript as sequencing primers. After the sequences were acquired they were examined for open reading frames (ORF) and when an ORF was found it was submitted for a BLAST search to determine if there was homology with known genes. We have obtained many important and interesting genes that are expressed during veraison. The results obtained thus far constitute a list of genes that can be organized into related groups. The groups are based on their physiological and biochemical functions. The groups are: l)Protein Synthesis, Processing and Turnover; 2 Abscisic Acid and Water Stress; 3 Oxidative Stress and Redox; 4 Cell Wall and Cell Wall Management; 5 Plant Hormone and Signal Transduction; 6 Transcription Factors; 7 Enzymes of Primary and Secondary Metabolism/Structural Proteins. Several of the genes have very interesting roles and have been shown to be valuable in other systems. For example we found a pectate lyase that has been shown to cause preactivation of defense genes in transgenic potato, providing resistance to the pathogen Erwinia carotovora. However, the role of the pectate lyase in grape berries is unknown. Some of the proteins expressed at veraison seem to be involved with water stress or related to abscisic acid (ABA), the plant hormone commonly associated with responses to water stress. Finding this category was somewhat surprising but is clearly an important area for further study. It has been recognized for some time that ABA has a role in grape ripening, but it may be that its role is associated with water stress experienced by the berry at veraison. We now have several genes related to ABA and water stress with which to address the role of ABA in ripening, and its possible association with water stress. Our results in the past year have pointed out several important features of berry physiology that were unexpected. We have found several genes related to plant hormones such as auxin, ABA and ethylene. It is well known that hormones are important in berry ripening, and we now have clues as to which of these might be important, and tools to study their effect on expression of specific genes. Taken together, the new view of berry ripening that is emerging from our results may perhaps be the most important accomplishment of the 1999 season.