Characterization of Genes Involved in the Grapevine’s Defense Response
We collected berry samples at several stages in berry development. The deformability, brix, berry number and the berry weight was recorded to document the developmental stage. Soluble proteins were extracted from the fruit at each stage and separated by gel electrophoresis. The corresponding western blots are being used to evaluate the relative abundance of two malic enzyme isoenzymes during ripening. We made three plasmid constructs using cDNA clones from two grape malic enzymes (VvMEl and VvME2) that were used for expression of the respective proteins in E. coli cultures. One construct was a full length cDNA for VvMEl, the second was an unprocessed full length cDNA for VvME2, and the third was a VvME2 that had been truncated 50 amino acids from the start methionine to give a version of VvME2 that is thought to be present in grape tissue after import of the polypeptide into plastids. The constructs were successfully transformed into an E. coli strain and results show that malic enzyme activity in crude extracts of the bacterial cells induced with IPTG increases over the uninduced cells. Thus, we have successfully introduced the grape malic enzyme cDNAs into E. coli and obtained expression of the active enzyme protein. In the current year we used a grape berry cDNA library to identify 10 genes that are expressed specifically at the beginning of ripening, and half of them correspond to proteins with known functions. One of the first genes identified is nearly identical to an enzyme that catalyzes rotation of peptide proline bonds (peptidyl-prolyl cis-trans isomerase). This enzyme aids polypeptide folding for proper conformation after synthesis of proteins in the cell. The second gene is very similar to a putative surface protein from Arabidopsis. Of the 158 amino acids in the open reading frame 113 were identical, and if conservative substitutions are considered 126 of the 158 matched. The third gene is nearly identical to ribosomal protein L10 (Large subunit, protein 10) from Arabidopsis. In an 86 amino acid segment, 72 were identical to the protein from Arabidopsis, and 20 other matches were found with ribosomal proteins from various organisms such as humans, pigs, yeast and rats. The fourth gene is very interesting and turned out to be a cap-binding protein. These are proteins that specifically recognize the 7-methyl guanosine group at the 5′ end of eukaryotic rnRNAs. The one we have identified from grape most closely matches a cap-binding protein from wheat called eIF4E (eukaryotic initiation factor 4E). The precise role of this protein in plants is not known, although work in Arabidopsis is currently underway in other laboratories and should soon reveal how eIF4E influences mRNA translation in plant cells. Another gene we identified is a malate/oxoglutarate transporter nearly identical to one reported from potato. There was also homology with a malate/oxoglutarate transporter from rice and Panicum miliaeum. These transporters are known to be located in the membranes of vacuoles, plastids and mitochondria where they regulate movement of malate and oxoglutarate in and out of these organelles. Thus, of the 10 genes we isolated, we were able to assign function to 5. The remaining 5 have no clear homology to proteins in the databases, but more sophisticated search strategies are expected to identify functional domains of these proteins which may suggest the function they have in vivo.