The Role Of MKP1 In The Stomatal Closure Of Arabidopsis Thaliana Induced By UV-B And Its Relationship With MPK6, H ₂ 2 And NO

Mitogen-activated protein kinase (MAPK, MPK) phosphatases (MKPs) are important regulators of the activities and dynamics of MPKs, and thus play important roles in the plant growth and development as well as plant responses to the environmental factors. Previous studies have shown that UV-B radiation can promote the accumulation of Arabidopsis MKP1 protein, and which makes UV-B-activated MPK3 and MPK6 dephosphorylation and deactivation. Meanwhile, the pharmacological and genetic evidences also suggest that MPK signaling pathways, hydrogen peroxide (H2O2) and nitric oxide (NO) are all involved in the signaling transduction pathways of UV-B-induced stomatal closure. However, the roles of MKP1 and its target proteins MPK3 and MPK6 as well as their interrelationships with the signaling molecules H2O2 and NO in the UV-B-induced stomatal closure are still unclear at present. In this study, by using the leaves of Arabidopsis wild-type (Col-0), the T-DNA insertion homozygous mutants for Atmkpl, Atmpk3 and Atmpk6 (mkpl-1, mkp1-3, mpk3, mpk6-1 and mpk6-4) and the loss of function double mutants for NADPH oxidases{AtrbohD/F) and nitrate reductases (NR)(Nial/Nia2) as experimental materials, and combining the methods of stomatal bioassay, laser scanning confocal microscopy, gene expression analysis and kinase activity examination, the following main problems were explored:(1) the role of MKP1 and its relationship with H2O2 or NO in the UV-B-induced Arabidopsis stomatal closure; (2) the role of MPK3 and MPK6 and their relationships with H2O2 or NO in the UV-B-induced Arabidopsis stomatal closure; (3) the mechanisms of UV-B-induced MKP1 gene expression and how MKP1 negatively regulated the UV-B-induced stomatal closure. The main results and conclusions are as follows:1.0.2-0.7 W·m-2 UV-B radiation could significantly induce stomatal closure of mkpl mutant, while the stomatal closure of wild-type leaves was significantly induced by the dose of UV-B radiation up to 0.5-0.7 W·m-2. Furthermore, mkp1 mutants showed faster and bigger stomatal closure induced by 0.5 W·m-2 UV-B than the wild type. These results show that mkpl mutants are more sensitive to the UV-B-induced stomatal closure than the wild-type.0.5 W·m-2 UV-B greatly induced MKP1 gene expression in Arabidopsis leaves, suggesting that UV-B radiation enhance MKP1 activity. The above results show that UV-B-activated MKP1 negatively regulated the UV-B-induced stomatal closure, which ensure the stomatal aperture at an appropriate level under UV-B radiation.2. The endogenous H2O2 levels in guard cells had no significant difference between mkpl mutant and wild type under either light or 0.5 W·m-2 UV-B radiation, suggesting that the negative role of MKPl in the UV-B-induced stomatal closure is not caused by H2O2.3. Consistent with the faster and bigger stomatal closure response of mkpl mutants to 0.5 W·m-2 UV-B, the NO generation rate and level in guard cells of mkpl mutants were also bigger and higher than that of wild-type under 0.5 W·m-2 UV-B. These results suggest that the UV-B-activated MKP1 negatively regulates the UV-B-induced stomatal closure via inhibiting NO production in guard cells.4. Compared with light controls,0.5 W·m-2 UV-B radiation obviously promoted phosphorylation of MPK3 and MPK6 in leaves of either wild-type or mkpl mutants. However, the UV-B-induced phosphorylation level of MPK3 and MPK6 in leaves of mkpl mutants were always higher than in wild-type, suggesting that the negative role of MKP1 negative in UV-B-induced stomatal closure may be through the dephosphorylati-on and subsequent deactivation of its target protein MPK6 and MPK3.5. UV-B radiation could induce stomatal closure of wild-type and mpk3 mutant, but could not induce stomatal closure of mpk6 mutants, indicating that UV-B-activated MPK6 plays a positive role in the signal transduction pathways of UV-B-induced stomatal closure. UV-B induced H2O2 generation in guard cells of mpk6 mutation, but could not induce NO production in guard cells of mpk6 mutants; Exogenous H2O2 could not induce stomatal closure of mpk6 mutants, also could not rescue the defect of these mutants in the UV-B-induced stomatal closure; However, exogenous NO not only induced stomatal closure of mpk6 mutants, also revered the defect of these mutants in the UV-B-induced and exogenous H2O2-induced stomatal closure. These results indicate that MPK6 mediates UV-B-induced stomatal closure via inducing NO production in guard cells.6. Under UV-B radiation, the stomata of double mutant mkpl-l/mpk6-4 behaved exactly like that of mpk6 single mutants in terms of either H2O2 and NO generation in guard cells or stomatal movement. In other words, UV-B radiation could induce H2O2 generation in guard cells double mutant, but could not induce NO generation in guard cells and stomatal closure of the double mutant, and exogenous NO could induce stomatal closure of the double mutant under either light or UV-B radiation.The results further confirmed that the negative role of MKP1 in the UV-B-induced stomatal closure is dependent on the activity of its target protein MPK6.7. UV-B radiation could not only induce MPK3 and MPK6 activation in leaves of wild-type leaves, but also could induce MPK3 and MPK6 activation in the leaves of NR double mutant Nial/2 and NADPH oxidase double mutant AtrbohD/F, but the activities of UV-B-induced MPK3 and MPK6 in leaves of the both double mutants were higher than that in the leaves of wild-type, which were similar to that in the leaves of mkpl mutants; Meanwhile, gene expression analysis showed that UV-B-induced mkpl gene expression could be inhibited by Nial/2, AtrbohD/F and mpk6 mutation. These results indicate that UV-B induce mkpl gene expression via a positive feedback mechanism of UV-B-induced H2O2 and NO generation and MPK6 activation, which negatively regulated UV-B-activated MPK3 and MPK6.Combined with the results presented herein and previous results that UV-B-induced NO production depends on H2O2 generation in guard cells, we can draw the following signaling transduction networks of UV-B-induced stomatal closure:UV-B radiation activates MPK3 and MPK6 on the one hand, and on the other hand induces H2O2 generation in guard cells, the activated MPK6 and H2O2 together induce NO production in guard cells, which then not only induce stomatal closure, also induce MKP1 gene expression and thus increases its activity, the rise of MKP1 activity promote its target protein MPK3 and MPK6 to dephosphorylate and thus inactivate, which feedback inhibits the UV-B-induced stomatal closure.