| Early studies have indicated phospholipid signaling plays a key role in plant growth, development and in responses to environmental stress. Phosphoinositide-specific phospholipase C (PI-PLC) is a critical enzyme in the phospholipid signaling pathway. Signals outside the cell wall are perceived at the cell membrane by receptors linked to a variety of signaling pathways, especially phospholipid signaling pathway. In this pathway, signals activate PI-PLC, which catalyzes the hydrolysis of phosphatidylinositol (4, 5) bisphosphate [PtdIns (4, 5) P2] to form diacylglycerol (DAG) and Inositol (1, 4, 5) -trisphosphate P3 (IP3). In plants, DAG is rapidly phosphorylated by the diacylglycerol kinase (DGK) to phosphatidic acid (PA).In the previous study, we showed that Nicotiana tabacum PLC (NtPLC) is involved in cell death and defense responses. The N. tabacum genome contains at least four NtPLC genes. In this study, to elucidate the expression patterns of NtPLCs and the role of NtPLC4 in disease resistance, expression patterns of all four genes in different organs and in response to various stress were studied by applying Real Time-PCR approach. Based on the expression pattens of NtPLCs, the complete sequence of NtPLC4 gene was cloned, and the sense and the antisense expression vectors of NtPLC4 were constructed. The role of NtPLC4 gene in disease resistance was explored using constitutively expressing sense or antisense NtPLC4 gene tobacco plants. The results are as follows:1. Expression patterns of all four genes in different organs and in response to various stimuli were studied by applying Real Time-PCR approach. The results showed that multiple members of the gene family were differentially expressed in tobacco organs, suggesting putative roles for this enzyme in plant development, including tissue and organ differentiation. NtPLC1 are expressed at relatively low levels in four tissues, while higher expression in stems. NtPLC2 are expressed at higher levels in leaves than in the other organs. NtPLC3 expressed to higher transcript levels in flowers and stem as compared to leaves and roots. NtIPLC4 are expressed at the highest levels in roots and the lowest levels in flowers. This study also indicated that a majority of the NtPLC genes are induced in response to various environmental stimuli, including cold, salt, phytopathologen infection, and the plant hormone abscisic acid. When compared to the non-treated controls, over four-fold and three-fold increases in transcriptive levels were recorded for NtPLC1 and NtPLC3 genes in response to 200 mmol·L (-1) salt. NtPLC1 and NtPLC3 mRNA increased after 24 h treatment with ABA, NtPLC1 and NtPLC3 increased in a dose-dependent manner. NtPLC3 increased five-fold treated with Phytophthora parasitica var. nicotianae at 24 h. NtPLC4 was expressed at highest levels at 30 min after treatment with ParA1 and increased three-fold. Results strongly suggest that transcriptional activation of the NtPLC gene family is important for adapting plants to stress environments. Significantly, NtPLC3 and NtPLC4 markedly induced by stress and pathogens.2. To investigate the function of tobacco NtPLC4, the complete coding region cDNA of NtPLC4 was inserted into the plant expression vector pROKII respectively and promoted by 35S promoter, and then was transferred into the competent cells of Agrobacterium tumefaciens LBA4404 by freeze-thaw method. The construction plasmids were introduced into tobacco (NC89) plants via Agrobacterium tumefaciens-mediated transformation. Polymerase chain reaction (PCR) was performed to analyse NtPLC4 in transgenic plants. Finally, we got 50 sense transgenic plants and 76 antisense transgenic plants.3. No phenotypic differences were noted between NtPLC4 plants and non-transgenic NC89 (wild type)controls during the seedling phase. Transgenic tobacco leaves were inoculated with conidial suspension of Alternaria longipes TBA003, and wild type served as control. The results showed that disease resistance in overexpression of NtPLC4 transgenic plants was singnificantly increased compared to wild type control. The Real Time-PCR results showed that the disease resistance enhanced with overexpression of NtPLC4. |
