Study On The Mechanism Of PP1c Gene In Response To Formaldehyde Stress In Plants

When plants are used to purify the formaldehyde pollution in the air, formaldehyde is primarily absorbed by plants through stomata. Studies have shown that under very low concentrations of formaldehyde in the environment can cause a significant decrease of stomatal conductance, therefore, stomatal conductance is an important limitative factor which affects the purification efficiency of formaldehyde of plants. The mechanism of stomatal switch has been the key topics of plant physiology research, studies have shown that protein phosphatase 1 (PP1) functions downstream of phototropins and upstream of the H+-ATPase in the blue light signaling pathway of guard cells, catalyzes the phosphorylation of plasma membrane H+-ATPase and promotes stomatal opening. PP1 is composed of regulatory subunit and catalytic subunit (PP1c), four genes encoding PPlc have been cloned from Vicia faba, VfPP1c-1, VfPP1c-2, VfPP1c-3 and VfPP1c-4, respectively, VfPP1c-1, VfPP1c-3 and VfPP1c-4 are mainly expressed in guard cells. Antisense expression of VfPP1c-1 gene significantly suppressed the blue light signal transduction pathway in guard cells in Vicia faba, thus VfPP1c-1 gene encoding PP1c plays an important role in the Blue light induced stomatal opening. Previous research in this paper showed that under gaseous formaldehyde stress, the transcription level of VfPP1c-1 gene was significantly inhibited. To further understand the mechanism of VfPP1c-1 gene in response to formaldehyde stress in plants. V. faba and transgenic tobacco were used as experimental materials in this study, which focus on the physiological and molecular level to study the role of PP1c in plants under formaldehyde stress. The main following results were obtained:1. With 0,5,10,20 and 40 ppm gaseous formaldehyde processing potted Vicia faba plants for 1h, the expression VfPPlc-1 gene in the leaves of V. faba was inhibited broad bean. Potted V. faba plants were treated with low concentrations of gaseous formaldehyde releasing from Furniture cabinet for 24-72 h, the results showed that with the extension of treatment time, the expression of VfPP1c-1 gene was inhibited gradually, after treatment for 48 h and 72 h, and the expression of VfPP1c-1 gene in the leaves of V. faba in the cabinet was significantly lower than that of outside the cabinet. This shows that the low and high concentrations of gaseous formaldehyde stress inhibit the expression of VfPP1c-1 gene in V. faba, and VfPP1c-1 gene is involved in response to formaldehyde stress in V. faba.2. PA (phosphatidic acid) inhibits the activity of PP1c,1-butanol (1-BuOH) by inhibiting the hydrolysis of phospholipids to produce PA catalyzed by phospholipase D promotes the phosphorylation of plasma membrane H+-ATPase which increases the activity of plasma membrane H+-ATPase; NO is an important member in abscisic acid (ABA)-induced stomatal closure signal transduction pathways, NO can promote the generation of PA, sodium nitroprusside (SNP) is a NO donor, thus applying exogenous NO can inhibit the activity of PP1c. Tau (Tautomycin) is a specific inhibitor of PP1c. The lower epidermis of potted V.faba treated withactivator and inhibitor of PPlc, to study whether the changes of PPlc activity affect the stomatal conductance and stomatal aperture in the leaves of V. faba under gaseous formaldehyde stress. The results show that under 20ppm gaseous formaldehyde stress, exogenous applying 1-BuOH for 3h can alleviate the inhibition of stomatal conductance and stomatal aperture stressed by gaseous formaldehyde in the leaves of V. faba, however, exogenous applying 300 μ.M SNP and 10 μ M Tau processed for 3h and 5h respectively, which increased the inhibition of stomatal conductance stomatal aperture stressed by gaseous formaldehyde in the leaves of V. faba. The measurements of the plasma membrane H+ -ATPase activity and H+pump activity indicate that under formaldehyde stress 1-BuOH enhance the plasma membrane H+-ATP ase activity and H+ pump activity in the leaves of V. faba, while SNP and Tau inhibit the plasma membrane H+ -ATPase activity and H+ pump activity; Immunoprecipitation analysis show that under formaldehyde Stress,1-BuOH can enhance the phosphorylation of H+ -ATPase and 14-3-3 binding to it in the leaves of V.faba, thereby activating the plasma membrane H+ -ATPase, while the roles of SNP and Tau are opposite. These results demonstrate that the inhibition of PP1c expression, under gaseous formaldehyde stress, resulting in decreas in the phosphorylation levels of plasma membrane H+-ATPase, thus affecting it combine with 14-3-3 protein, reducing the activity of plasma membrane H+ -ATPase and H+ pump, so reducing the stomatal conductance and stomatal aperture.3. Tobacco is an important model plant used in research in the field of botany, this paper using the changes of VfPP1c-1 gene expression transgenic tobacco to examine the role of VfPP1c-1 gene in plants in response to gaseous formaldehyde stress. The results showed that the overexpression of VfPP1c-1 gene in tobacco produces a large amount of PP1c protein, under 20ppm gaseous formaldehyde stress, which enhance the phosphorylation of plasma membrane H+-ATPase and its binding to 14-3-3 protein and increase the plasma membrane H+ -ATPase activity and H+ pump activity, thereby increasing the stomatal conductance and stomatal aperture of transgenic tobacco, and ultimately enhance the absorption capacity of gaseous formaldehyde of transgenic tobacco. In addition, the use of RNAi interference technology to obtain transgenic tobacco with NPPl gene suppressive expression, the analysis of suppression NPP1 gene in response to formaldehyde stress on wild type tobacco, these results indicate that the inhibition of the expression of NPPl gene also inhibited the expression of other homologous genes, and the phosphorylation of plasma membrane H+-ATPase and the binding of 14-3-3 protein, thus reducing the activity of the plasma membrane H+-ATPase and H+ pump activity, resulting in the falling of stomatal conductance and stomatal aperture, and ultimately reduce the formaldehyde absorption capacity and resistance in transgene tobacco. These findings verify the role of VfPP1c-1 gene in response to gaseous formaldehyde in plants, and provide a new strategy and genetic resources for using genetic engineering and molecular biology methods to improve the absorptive capacity of formaldehyde in plant.