Studies On The Molecular Cloning And Characterization Of Phospholipase D In Chorispora Bungean

Chorispora bungeana Fisch. and C.A. Mey (Chorispora bungeana) is a rare alpine subnival plant species that is highly tolerant of stress environment. Phospholipase D (PLD) is a key enzyme involved in membrane phospholipid catabolism during plant growth, development and stress responses. We have isolated and partially characterized a full-length cDNA encoding PLD from the calluses of Chorispora bungeana, with the aim of furthering our understanding of the role of PLD at the molecular level. Although PLD has been studied in many plants, no examinations of its metabolism, regulation and molecular properties during environmental stresses have been undertaken. In this study, we investigated the response of PLD to environmental stresses and the role of PLD in metabolism pathway of Chorispora bungeana. The main results are as follows:(1) Through PCR and RACE techniques, a whole sequence of PLD cDNA was isolated from Chorispora bungeana (GenBank accession No. HM756247). The cloned full-length cDNA of Chorispora bungeana (CbPLD) was2939bp. The cDNA contained a2712bp ORF encoding a protein of903amino acids with a calculated molecular weight of about100.5kDa and with a PI of7.12. A5'untranslated region (UTR) of70bp was found upstream of the first ATG codon, and a3'untranslated region (UTR) of39bp was found downstream from the stop codon. Secondary structure analysis showed that CbPLD had C2domain,"IYIENQYF" region and two HKD motifs belonging to PLD family as their activity region. The putative CbPLD peptide contained26.14%alpha helix,19.38%extended strand,5.54%beta turn, and48.95%random coil. The alpha helix and random coil constituted interlaced domination of the main part of the secondary structure.A comparison of the predicted protein sequences of the CbPLD with PLDs of other plants shows that CbPLD is highly identical to PLD from Arabidopsis thaliana PLD beta (85%). (2) CbPLD expression patterns under stresses were analyzed by semi-quantitative RT-PCR. The results indicated that CbPLD is ubiquitously expressed in Chorispora bungeana, with almost no tissue specificity even though the expression levels showed slight variations between tissue types. The accumulation pattern of the CbPLD mRNA in response to low temperature was studied. Following exposure to-4℃, the transcript levels of CbPLD progressively increased, almost reaching the peak at6days of treatment, and then declined. After exposure to4℃, the transcript level of CbPLD increased rapidly. The level of the CbPLD transcript almost reached the peak at1day and remained at a high level, then decreased at12days. Immunoblots of PLD protein showed a molecular mass around100kDa. In parallel with the RT-PCR and immunoblots analyses, there were no distinguishable differences. The increases of PLD protein content are corresponding to the different levels of CbPLD mRNA. It seems to be a clear correlation between increase in CbPLD transcription levels and protein contents at different days of low temperature treatments. These results suggested that the major cause of the increases of PLD protein content during the low temperature stresses in Chorispora bungeana callus could be due to the up-regulation of CbPLD gene.The transcripts of CbPLD accumulated highly when Chorispora bungeana was treated with abscisic acid (ABA), salinity, hydrogen peroxide (H2O2) and heat. These results indicate that the CbPLD may play an important role in response to stresses in Chorispora bungeana.(3) The influence of freezing treatment on CbPLD activities was studied in Chorispora bungeana. The results showed that:During the freezing treatment (-4℃), PLD activities in both microsomal and mitochondrial membranes showed significant increases at day3, and remained at a high level at day6, then declined to a moderate level. The RT-PCR analyses showed that PLD activity partially corresponded to the PLD gene transcript level. Freezing injury, as measured by electrolyte leakage and malondialdehyde content, peaked at day6and then gradually decreased to a low level. Alleviation of freezing injury was related to a decreased content of membrane-associated Ca2+and in parallel with the changes of PLD activities. The influence of freezing treatment on PLD catalytic mechanism showed that freezing treatment resulted in increases in the Km and Vmax for microsomal and mitochondrial PLD, respectively. Both the optimal pH and calcium ion concentration of the mitochondrial and microsomal PLD were changed under freezing treatment. The findings indicated that PLD is involved in membrane deterioration and the signaling pathway in response to freezing stress, and the specific mechanism of cold resistance of Chorispora bungeana is linked with PLD.(4) The metabolism regulations of CbPLD in response to freezing stress were analyzed. The results showed that CbPLD have mediated the regulation pathway of membrane stability, osmotic adjustment and regulated CAT activity in anti-oxidative system. It also involved in the pathway of ABA signaling.