ABSTRACT
BACKGROUND: Astragalus grows mainly in drought areas. Cycloastragenol (CAG) is a tetracyclic triterpenoid allelochemical extracted from traditional Chinese medicine Astragalus root. Phospholipase C (PLC) and Gα-submit of the heterotrimeric G-protein (GPA1) are involved in many biotic or abiotic stresses. Nitric oxide (NO) is a crucial gas signal molecule in plants. RESULTS: In this study, using the seedlings of Arabidopsis thaliana (A. thaliana), the results showed that low concentrations of CAG induced stomatal closure, and high concentrations inhibited stomatal closure. 30 µmol·L-1 CAG significantly increased the relative expression levels of PLC1 and GPA1 and the activities of PLC and GTP hydrolysis. The stomatal aperture of plc1, gpa1, and plc1/gpa1 was higher than that of WT under CAG treatment. CAG increased the fluorescence intensity of NO in guard cells. Exogenous application of c-PTIO to WT significantly induced stomatal aperture under CAG treatment. CAG significantly increased the relative expression levels of NIA1 and NOA1. Mutants of noa1, nia1, and nia2 showed that NO production was mainly from NOA1 and NIA1 by CAG treatment. The fluorescence intensity of NO in guard cells of plc1, gpa1, and plc1/gpa1 was lower than WT, indicating that PLC1 and GPA1 were involved in the NO production in guard cells. There was no significant difference in the gene expression of PLC1 in WT, nia1, and noa1 under CAG treatment. The gene expression levels of NIA1 and NOA1 in plc1, gpa1, and plc1/gpa1 were significantly lower than WT, indicating that PLC1 and GPA1 were positively regulating NO production by regulating the expression of NIA1 and NOA1 under CAG treatment. CONCLUSIONS: These results suggested that the NO accumulation was essential to induce stomatal closure under CAG treatment, and GPA1 and PLC1 acted upstream of NO.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Nitric Oxide/metabolism , Signal Transduction , Plant Stomata/physiology , GTP-Binding Protein alpha Subunits/genetics , GTP-Binding Protein alpha Subunits/metabolismABSTRACT
We investigated phenotypes of the double mutants of the calcium (Ca2+) signaling genes plc-1, splA2, and cpe-1 encoding for a phospholipase C1 (PLC-1), a secretory phospholipase A2 (sPLA2), and a Ca2+/H+ exchanger (CPE-1), respectively, to understand the cell functions regulated by their genetic interactions. Mutants lacking plc-1 and either splA2 or cpe-1 exhibited numerous defects including reduced colonial growth, stunted aerial hyphae, premature conidiation on plates with delayed germination, inappropriate conidiation in submerged culture, and lesser mycelial pigmentation. Moreover, the ∆plc-1; ∆splA2 and ∆plc-1; ∆cpe-1 double mutants were female-sterile when crossed with wild type as the male parent. In addition, ∆plc-1, ∆splA2, and ∆cpe-1 single mutants displayed higher carotenoid accumulation and an increased level of intracellular reactive oxygen species (ROS). Therefore, the pleiotropic phenotype of the double mutants of plc-1, splA2, and cpe-1 suggested that the genetic interaction of these genes plays a critical role for normal vegetative and sexual development in N. crassa.
Subject(s)
Calcium Signaling , Fungal Proteins/genetics , Fungal Proteins/metabolism , Hyphae , Mutation , Neurospora crassa/physiology , Phenotype , Antioxidants/metabolism , Biomass , Gene Expression Regulation, Fungal , Pheromones/metabolism , Reactive Oxygen Species/metabolismABSTRACT
Objective To investigate signal transduction mechanism of phospholipase C?1 (PLC?1) in colorectal cancer cells. Methods Gel electrophoresis mobility shift assay (EMSA), immunocytochemistry, zymography and RT-PCR were performed to investigate the function of PLC?1 on nuclear factor-Kappa B (NF-?B), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in LoVo cell. Results Compared with control group, nuclear positive rate of cells treated with epidermal growth factor (EGF) increased significantly (from 26.91%?2.84% to 40.83%?4.36%), while that of cells treated with 2.5mol/L U73122 decreased to 12.20%?1.89%. Meanwhile, pretreatment with 2.5mol/L U73122 before EGF treatment decreased nuclear positive rate of cells from 40.83%?4.36% to 18.21%?1.34%. The results of EMSA further verified that PLC?1 can regulate the activity of NF-?B. RT-PCR results showed that EGF, PLC?1 or NF-?B had no significant effect on the expression of MMP-2, TIMP-2 at mRNA level. Furthermore, zymography indicated that the activity of MMP-2 did not change dramatically by EGF, PLC?1 or NF-?B. Conclusion These data suggested that EGF-PLC?1-NF-?B signaling pathway was operative in LoVo cell, but MMP-2 and TIMP-2 may not be regulated by EGFR-PLC?1-NF-?B signaling pathway.
ABSTRACT
Objective: To investigate the function and mechanism of phospholipase C?1 (PLC?1) in cell-matrix adhesion in colorectal cancer. Methods: Highly metastatic colorectal cancer cell line LoVo and lowly metastatic colorectal cancer cell line SW480 were subjected to cell-matrix adhesion assay. U73122 (a specific inhibitor of PLC) and pyrrolidine dithiocarbamate (PDTC) (an inhibitor of NF-?B) were used to study the effect of PLC?1 and NF-?B on cell-matrix adhesion. Furthermore, Western blot and gel electrophoresis mobility shift assay (EMSA) were performed to detect the mechanism of PLC?1 in colorectal cancer cell adhesion to matrix. Results: Inhibition of PLC?1 or NF-?B resulted in reduction of cell-matrix adhesion in a dose-dependent manner in LoVo cells(P
