| Arabidopsis ecotype Columbia (WT) and T-DNA-insertional knockout mutant of PLDal (pldal) were selected as experimental materials, and we selected guard cells as research system, using pharmacological, physiological and cytobiological approaches to study the role and relationship of PLDal, PA, H2O2 and Ca2+ in ABA-mediated stomatal movement.Our results showed that NaCl and ABA can induce stomatal closure in WT, but there is little effect on pldal mutant. EGTA can inhibit or reduce ABA-induced stomatal closure significantly; The production of PLDal-PA (di18:1 PA and di16:0-18:2 PA) and lysophosphatidic acid (Lyso PA) can induce stomatal closure in WT and pldal. EGTA can also inhibit PA of different species-induced stomatal closure in different extent. H2O2 can induce stomatal closure in WT and pldal, and the effects presented concentration dependent. In WT, when [H2O2]≤10-5 mol·L-1, EGTA had effect of inhibting stomatal closure induced by H2O2; While in pldal, at [H2O2]≤10-1 mol·L-1, the effect of H2O2 on stomatal closure was reversible and abolished by EGTA. Exogenous Ca2+ and LaCl3 can also induce stomatal closure in WT and pldal. All these results indicated that PLDal is involved in NaCl and ABA-induced stomatal closure, and it may locate downstream of them. Endogeny Ca2+ in guard cells may be involved in ABA, PA and H2O2 induced stomatal closure; In addition, exogenous Ca can enter into cytoplasm via channels located in plasma membrane, then cause stomatal closure. The mechanism of La3+ inducing stomatal closure may be due to its binding with Ca2+ and K+in channels in membrane system.The calcium sensitive fluorescence indicator Fluo-3 AM was loaded into epiderm in Arabidopsis, confocal laser scanning microscopy was used as main method to investigate the effects of Ca2+, NaCl, ABA, PA and H2O2 on [Ca2+]cyt in guard cells.50 mM NaCl and 10μM ABA can elevate [Ca2+]cyt markedly in WT. While the change in pldal was slower, and level was lower. The result suggests that the absence of PLDal affects Ca2+ responding to ABA in guard cells. [Ca2+]cyt in guard cells of WT and pldal elevated after 50μM PA, 1000μM H2O2 and 0.01,0.1,1,10 mM CaCl2 treatment. However, the change in WT reacted faster than that in pldal, the peak of [Ca2+]cyt in WT was significant higher than pldal. The results above indicate that NaCl and ABA can elevate [Ca2+]cyt in guard cells, then Ca2+ activates PLDal, producing PA, PA elevates [Ca2+]cyt via binding to special targets.Our research showed that PLDα1 can improve Arabidopsis salt tolerance via its production PA, Ca2+ may be partly involved in the process. At the concentration of 200 mmol·L-1 NaCl, under exogenous PA, the survival of WT increased while pldal declined, this result suggests that at higher level of NaCl, the absence of PLDα1 has serious impact on salt tolerance, even exogenous PA was applied. Under EGTA existence, after PA being applied, both WT and pldα1couldn't survive, which indicates that at higher concentration of NaCl, there is an autonomic regulation system controlling Ca2+ homeostasis in plant cells. If this condition is breaked, the salt tolerance will decline, in other words, calcium may be involved in PA-meditated the plant's ability to withstand salt stress. After treatments of 200 mM NaCl and 100,200μM LaCl3 for 4 days, the survival of WT and pldal declined markedly, this depressive effect was La3+ concentration-dependent. Even 50μM PA being applied, there's no effective improvement of salt tolerance. The results indicate that La3+ may inhibit the effect of PA responding to salt stress and regulation of the stress through blocking Ca2+ into the cell. Different pharmacology experiment suggests that calcium may be involved in PA-meditated the plant's ability to withstand salt stress. |
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