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United States Department of Agriculture

Agricultural Research Service

Title: Cloning, Characterization, and Functional Expression of a Phospholipase Da from Tomato Fruit

Authors
item Whitaker, Bruce
item Smith, David
item Green, Karen

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 7, 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.

Technical Abstract: Phospholipase D (PLD) initiates phospholipid (PL) catabolism in plant cells and is also involved in signal transduction and retailoring of membrane PL. Phosphatidic acid (PA), the product of PLD hydrolysis of PL, increases in pericarp tissue during ripening of tomato fruit, suggesting that increased PLD activity may be involved in loss of membrane function associated with ripening. However, a recent report showed a decline in soluble PLD activity in both normal and nonripening mutant fruit over the span that encompasses full ripening. To directly assess the role of PLD in tomato ripening, we have initiated a molecular genetic approach. Using a PLDalpha cDNA from castor bean as a probe, a PLDalpha cDNA (LEPLD2) was isolated from a tomato fruit library. It has an open reading frame of 2421 nucleotides, predicted to encode a polypeptide of 807 amino acids, with a molecular mass of 91.9 kDa. These values are close to those of PLDalphas from eleven plant species and LEPLD2 has 73 to 74 percent nucleotide sequence identity with PLDalpha cDNAs from castor bean, tobacco and tomato. LEPLD2 transcript was detected in all tissues of the tomato plant by RNA gel blot analysis. Levels were 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 phosphatidylcholine-hydrolyzing enzyme, and cells transformed with a pFLAG-MAC vector construct produced a FLAG-PLD fusion protein that migrated close to the calculated 94 kDa on SDS-PAGE.

Last Modified: 4/21/2014
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