Submitted to: Biochemical Society Transactions
Publication Type: Proceedings
Publication Acceptance Date: July 23, 2000
Publication Date: N/A
Interpretive Summary: Optimal eating quality of ripe tomato fruit is of short duration, after which the fruit become over-ripe or "senescent." Breakdown of various structural components of the cells that make up the fruit tissues causes the undesirable textural changes associated with fruit senescence. Understanding the action and control of specific enzymes involved in the breakdown of cell structural components will help scientists devise strategies to develop new tomato lines with improved fruit quality and shelf-life. We have identified one such enzyme, PLD, which degrades phospholipids, the primary building blocks of cell membranes. In this study, we characterized the gene that codes for tomato fruit PLD. This will enable us to genetically alter the level of PLD and thus determine whether the enzyme plays an important role in tomato ripening and senescence. It is expected that reduction of the level of PLD may also maintain quality and extend storage-life of tomato slices, since this enzyme becomes much more active when the fruit tissues are cut.
Phospholipase D (PLD) initiates phospholipid (PL) catabolism in plant cells and is also involved in signal transduction and retailoring of membrane PL. Total PL declines and phosphatidic acid rises in pericarp tissue during tomato fruit ripening, suggesting that increased PLD activity alters membrane structure. To assess the role of PLD in tomato ripening, we have begun a molecular genetic approach. Using a castor bean PLD-alpha cDNA as a probe, a PLD-alpha cDNA (LEPLD2) was isolated from our tomato fruit library. It has an open reading frame of 2421 nucleotides, encoding a polypeptide of 807 amino acids with a molecular mass of 92 kDa. The deduced LEPLD2 PLD-alpha has over 75 percent sequence identity with PLD-alphas from castor bean, tobacco and tomato. LEPLD2 transcript, detected by RNA gel blot analysis, was very low in roots, low in stems, moderate in leaves, high in flowers, and increased in fruit during development and ripening. Expression of LEPLD2 in E. coli yielded active enzyme, and a FLAG/PLD-alpha fusion protein produced by transformed E. coli migrated close to the calculated 94 kDa on SDS- PAGE.