Studies On The Physiological, Biochemical And Molecular Characteristics Of Phospholipase D In Strawberry (Fragaria Ananassa Duch.) Fruit

Strawberry (Fragaria ananassa Duch) is one of the highly valuable and important fruit in the world. The whole production of strawberry fruits increases twice during the past six years from 1998 to 2004 in China. After entering into the WTO, the domestic market in China faces the even more severe competition from abroad. Therefore high quality fruits become the key to get into the international market. Strawberry fruits are soft and juicy, but lack of hard shell to protect them. Besides, Strawberry fruit reaches its maturity in short time and then get rotten easily during the process of transportation, storage and marketing, which was accelerated by the catabolic breakdown of cellular structure such as the cell wall and the membrane. Preservation of membrane structure and compartmentalization helps maintain the quality of the fruits, in which the regulation of phospholipase D (PLD) plays an important role. But so far to now, no study of PLD was reported on strawberry. Researches on PLD metabolism, regulation and molecular properties during strawberry fruit development and ripening would be helpful to find new methods and ways to reduce the membrane damage and increase the storage quality and shelf life of strawberry fruits. At the same time, it would also be helpful in finding the potential role of PLD in signal transduction pathway of strawberry fruit, a non-climacteric fruit.The present study examined the roles of PLD in fruit development stages by using strawberry fruits of 'Aromas' and 'Seascape'. The enzymatic characterization, function in vitro and in vivo, sub-cellular localization, and molecular properties were also included in this study. The main results are as follows:(1) It is very important to establish a proper system for detecting PLD activity in strawberry fruits. After optimizing the radiolabeled methods in PLD activity determination, a proper system was established on the preparation of PLD and the detection of its activity in strawberry fruits. This method will help to elucidate the potential roles of PLD in strawberry fruit ripening and senescence process.(2) PLD activity was detected in different development stages of strawberry fruits. In general, PLD activity was higher in 'Seascape' at all stages except the mature white stage. There was very little change in mitochondrial PLD activity during development of 'Aromas' fruits. The microsomal PLD activity increased during fruit development and reached a maximum at the mature white stage, and then declined. PLD activity in the microsomal fraction was the highest at the young immature stage in 'Seascape', after which it declined to nearly half of its original specific activity. Mitochondrial PLD activity also showed a slight decline at the beginning, however it continued toincrease during further development and reached maximal value at the turning orange stage. These results suggest that PLD activity increases during fruit development and ripening and thus PLD may play a role in strawberry fruit ripening.(3) Immunoblots of PLD protein showed similar results with the activity change during strawberry fruit development stages. The results also showed that no matter mitochondrial or microsomal PLD, all had a molecular mass around 92kDa, which was consistent with most reported plant PLDs.(4) The optimum pH for strawberry mitochondrial PLD were 5.5 and 6.5, while for microsomal PLD 5 and 7. Enzyme Kinetic analysis showed that the activities of both mitochondrial and microsomal PLD follow Michaelis-Menten kinetics. Lineweaver-Burk plots showed that the Km constants were 113.89 and 277.01 for mitochondrial and microsomal PLD, respectively. And the maximum velocity for the microsomal PLD was almost 12 times than that of mitochondrial PLD. PLD activity also got activated at around physiological Ca2+ concentration. Proper amounts of calmodulin improved the activation of Ca2+ towards PLD activity. The results show that strawberry fruit mitochondrial PLD and microsomal PLD have different regulatory properties, which may reflect their different functions.