Cloning, characterization and functional expression of a phospholipase Dalpha cDNA from tomato fruit

Phospholipase D (PLD; EC 3.1.4.4) 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 (Lycopersicon esculentum Mill.) 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 2 421 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 11 plant species and LEPLD2 has >/=73% nucleotide sequence identity with PLDalpha cDNAs from castor bean and tobacco, as well as another tomato cDNA. 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 Escherichia coli yielded phosphatidylcholine-hydrolyzing enzyme, and cells transformed with a pFLAG-MAC vector construct produced a FLAG-PLDalpha fusion protein that migrated close to the calculated 94 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.