Characterization of microsomal and mitochondrial phospholipase D activities and cloning of a phospholipase D alpha cDNA from strawberry fruits

Abstract

Phospholipase D alpha (PLD, EC 3.1.4.4)) is a key enzyme involved in membrane deterioration that occurs during fruit ripening and senescence. The biochemical and molecular characteristics of PLD was studied in strawberry ( Fragaria ananassa Duch) fruits, which are non-climacteric fruits. PLD activity was primarily associated with the mitochondrial and microsomal fractions and showed increased activity during development. Optimal pH levels of activity were observed at 5.5 and 6.5 for mitochondrial PLD and at 5 and 7 for microsomal PLD. Calcium enhanced microsomal PLD activity at 1–40 μM levels. PLD activity followed Michaelis–Menten kinetics. Lineweaver–Burk analysis gave K m values in the range of 114 and 277 μM using dipalmitoylphosphatidylcholine (DPPC) as substrate for mitochondrial and microsomal PLD, respectively. The V max value for the microsomal PLD was nearly 12-fold higher than that of mitochondrial PLD. A 2874 bp full-length cDNA for PLD alpha was amplified from strawberry fruit mRNA using RT-PCR and 5′- and 3′-RACE encoding an 810 amino acid-polypeptide. The predicted strawberry PLD sequence showed the characteristic C2 domain and the phospholipase domains conferring calcium sensitivity and the enzyme activity, respectively. The strawberry PLD alpha showed a high degree of similarity to other PLD alphas from plants. The implications of PLD regulation during ripening of fruits are discussed.