Functions And Roles Of Phospholipase D In Responses To Chilling Stress And Signal Transduction In Cucumber Fruit

Phospholipases D (PLD EC 3. 1. 4. 4) is a key enzyme that catalyzes the hydrolysis of membrane phospholipids into phosphatidic acid (PA) and a hydrophilic headgroup, which are the backbones of biological membranes. The activities of these enzymes not only have an important impact on the structure and stability of cellular membranes but also play an pivotal role in regulating many critical cellular functions, including cellular signal transdcution, lipid metabolism, vesicle trafficking, cell proliferation, celluar backbone formation and sensescence. In recent years, the role of PLD in transmembrane signal transduction has become a new research topic. The objectives of this study were to evaluate the function of PLD in incidence and development of chilling injury (CI) and the effectiveness of warm treatment treatment to reduce CI and storage physiology, and whether these effects were related to changes in PLD activity and PLDmRNA expression.Warm treatment is an easy and feasible physical treating method. It has precious future for innocuity and non-pollution without deposit of any chemistry on fruits and vegetables. Several previous studies have suggested that warm treatment that increase chilling tolerance is thought to work through the induced synthesis and accumulation of specific heat-shock proteins (hsps), and also for improving storability. Application of warm treatment in this study is to test and verify the role of PLD in incidence and development of CI inversely.Cucumber fruit (Cucumis sativus L) is chilling sensitive and susceptible to CI when fruit are stored at temperatures of less than about 10℃. This paper selected one variety of cucumber "JINYOU-1" as testing material which was sensitive to chilling. Before 2℃ storage, cucumbers were devided into two groups, one was treated in 20℃, 90% RH for 24h (chilling stress group), the other was treated in 37°C, 90% RH for 24h (warm treatment group). In order to investigate the metabolic mechanism of CI and effect of warm treatment on inhibiting CI by regulating PLD in cucumber during low temperature storage, physiological and biochemical changing, PLDmRNA expression, changes of fatty acid in membrane were evaluated after storaged at 2°C for 0, 3, 6, 9, 12, 15 d, respectively. The article also gives a brief introduction on the role of PLD in cucumber cellular CI signal transduction. For analysis, when CI symptom first appeared in warm treatment group, sample fruits were cut into three sections with equal length, namely top (calyx end), middle and bottom (stalk end), respectively. Thus, we can . understand the mechanism of CI induced by the changes of PLD in chilling stress.In this study, a partial PLD gene, encoding a putative phospholipase D, was identified in cucumber fruit. This cloned PLD fragment had 889 bp nucleic acids and the deduced amino acid sequence showed high identity with known PLD genes. Phylogeny analysis of PLD amino acid sequences based on cDNA clones from various plant species revealed that identities in cucumber PLD shared the highest identity (81%) with that of caster bean and belonged to PLDa class.In general, chilling stimulated lipid peroxidation and PLD action in chilling stress group cucumber fruit prior to the development of irreversible injury. Both lipid peroxidation and PLD action should be responsible for creating stress conditions in cucumber fruits.Thus, activation of enzyme hydrolysis of membrane phospholipids may be primary step for initiation of CI. When CI symptom first appeared, there is significant difference between CI index, electrolyte leakage, MDA content, PLD and LOX activity, PA content and PLDmRNA expression in top, middle and bottom, indicating irreversible CI occurred initially in the top area near calyx of fruits, and developed toward the bottom near stalk. We suggest that PLD and LOX are associated with the initiation of CI by involving in membrane deterioration and signaling pathway in response to chilling stress, while the difference in PA content may be direct reason.In the study, the change of respiration rate shared similar trends with ethylene production during storage at 2*C. The correlation analysis indicated that excessive accumulation of PA is main reason forCI occurrence.The relationships between CI and physiologicaland biochemical factors are complicated. Changes of Ca2+ content especially membrane-associated Ca2+ content play a vital role inalleviation of CI by warm treatment treatment. The result showed that warm treatment decreased CI index, maintained low level of PLD and LOX activity, inhibitted PLDmRNA expression, resulted in lower cell membrane permeability and PA content, reduced MDA and proline accumulation and delayed the decrease of soluble protein contents. Above all, chilling stress decrease content of membrane-associated Ca2+, which increased PLD mRNA expression firstly, leading to high level of PLD activity;at same time, relative higher levels of cytosolic Ca2+ concentrations could increase to lethal levels, thus activating autocatalytic membrane lipid degradation and LOX activity. As a result, PLD, cooperating with LOX make PA content increased. That may be the main reason for initiation of CI.