The Roles of Microtubules and Protein Phosphatase AtPP2CA in Regulation of Stomatal Movement in Arabidopsis Thaliana
|School||Nanjing Agricultural College|
|Keywords||Microtubule Stomata Light Dark Taxol Oryzalin ABA PP2CA|
Guard cells can sense kinds of environmental stimulus and adjust the stomatal pore to regulate gas and water exchange in and out of plants. Plant hormone ABA is a key regulator of plant growth and development as well as response to stress. PP2Cs from class A, including HAB1, ABI1, ABI2 and PP2CA, are considerd as negative regulators in ABA signaling, especially in seed and vegetative responses to ABA. However, the role of microtubule during stomatal movement has been debated, and PP2CA in that is little to know. To understand more, Arabidopsis wild type (WT, ecotype Columbia), T-DNA-insertional knockout mutant of PP2CA (pp2ca), the transgenic plant GFP::TUA6 with GFP-labled microtubule associated protein and pp2ca mutant with GFP::TUA6 were selected as experimental materials. We used cotyledons of six-day-old Arabidopsis thaliana to study the relationships of microtubules, AtPP2CA and stomatal movement, with pharmacological, physiological and cytobiological approaches.Our results showed that microtubules organization were related to stomatal movement, and participated in stomatal opening partly. On one hand, light induced microtubules polymerization, then microtubules became abundant, which made stomatal opening. Taxol, a microtubule stabilizer, promoted stomatal opening induced by light and inhibited stomatal closure induced by dark or ABA, since microtubles were stabilized, thickened and better organizated. The effect of taxol was concentration-dependented, and there was significant difference in stomotal aperture between the control group and treatment with 20μM taxol. On the other hand, intact microtubules were not needed in stomatal opening induced by light, because that stomata remained opened, though microtubules were depolymerized completely by a microtubule dsrupter named oryzalin.Microtubules were taken in stomatal closure induced by dark or ABA. Microtubules depolymerization was needed in stomatal closure, but not vice versa. ABA or dark could make microtubules depolymerize mostly, and that exhibited a diffused pattern of most microtubules twisted, patched and broken down into diffusive fragments. Finally, stomata closed. But there was no correlation between the degree of stomatal aperture and that of microtubules depolymerization. When added oryzalin into ABA or in the dark, microtubules were depolymerized completely coupling with stomatal closing, however it didn’t promote stomatal closure, similar to ABA or dark treatment. Treated with taxol and ABA sequentially or simultaneously, stomata couldn’t close, because taxol made microtubules re-stabilize. We saw then:microtubules depolymerization was presented in stomatal closure certainly, whether induced by dark or ABA. And microtubules depolymerization neither led to stomatal closure, nor promoted stomatal closure directly. In other words, microtubules depolymerization might be necessary but was not a sufficient condition for stomatal closing.We screened pp2ca homozygote expressing green fluorescent protein by hybridization. The result of qRT-PCR demonstrated no influence on the transcription of AtPP2CA, when GFP overexpressed. And the ABA treatment of Arabidopsis seedlings indicated that overexpression of GFP would not affect PP2CA’s characteristics. So GFP::TUA6pp2ca could be used to research the relationships between PP2CA and micotubules in stomatal movement in Arabidopsis thaliana. We found that stomatal closure in pp2ca mutant was more obvious than WT under ABA treatment. It concluded that PP2CA regulated stomatal movement negatively in ABA signaling.To research the relationships of PP2CA and microtubules in stomatal movement in Arabidopsis thaliana. We treated WT, pp2ca and pldα1 with microtubule specific drugs, that are taxol and oryzalin, and ABA singly or together. In the control group, the state of stomata and microtubule organization of pp2ca was the same to WT and pldα1, suggested that pp2ca didn’t have special phenotype in stomatal aperture and microtubules organization. When treated with ABA alone, ABA couldn’t influence well-organized microtubules and stomatal aperture in pldα1, but had microtubules depolymerize in both WT and pp2ca. Though the same configuration of their microbubules, pp2ca was more sensitive than WT in stomatal aperture. Oryzalin made microtubules depolymerize in three plants without changes in stomatal aperture comparing with the control group. Taxol could inhibit depolymerization, stablize microtubules and promote stomatal opening in all of three. When oryzalin and ABA used together, microtubules depolymerized, and then stomata closed in pldα1. Although pp2ca and WT microtubules depolymerized completely, the stomatal aperture didn’t changed, the same to ABA treatment. When added taxol into ABA treatment, stomatal aperture of WT approached that of the control group, without significant differences. However, the effect wasn’t the same to pp2ca, with significant differences to the control group. All above suggested that both PLDal and PP2CA were key regulators in ABA signaling. But PP2CA regulated ABA signaling negatively not through regulation of microtubules organization probablely.Despite failure in expression of AtPP2CA, changes in microtubules organization of pp2ca was in accord with that of WT under kinds of treatments with or without stomatal aperture changes. Unlike PLDal that was prior to microtubules, PP2CA wasn’t involved in stomatal movement Arabidopsis thaliana that contained microtubules. On the contrary, microtubules and PP2CA might have no direct interaction. It was possible that they roled parallel in stomatal movement, regulated respectively.