WD40-REPEAT 5a Functions In Drought Stress Tolerance By Regulating Nitric Oxide Accumulation In Arabidopsis

Drought stress severely affects plant growth and development, causing substantial losses of crop quantity and quality. Previous reports showed that nitric oxide (NO) generation by NO synthase (NOS) in stomatal guard cells plays a vital role in stomatal closure for adaptive plant responses to drought stress. However, no gene or protein encoding plant NOS is identified so far, and the mechanism underlying the regulation of NOS activity in plants is also largely unknown.In this study, we identified WDRSa as an important factor involved in regulation of NOS activity and plant drought stress tolerance by modulating stomatal movement and the expression of drought-stress-responsive genes. The main results are summarized as below:1. By screening the collection of yeast deletion mutants for possible mutants with repressed NO accumulation and cell apoptosis compared with the wild-type yeast by treating the collection with H2O2, we identified TUP1 as a novel regulator of yeast NOS activity. Our results showed that after treatment of 4 mM H2O2 for 30 min, NO accumulation in wild type was significantly increased compared with untreated control. However, NO accumulation was markedly repressed in Atupl mutant. Also, H2O2-induced cell apoptosis was decreased in the mutant compared with that in the wild type. These data suggested that TUP1 functions in H2O2-induced NO accumulation and cell apoptosis by regulating NOS activity in yeast.2. We assayed the NOS activity in the wild type and Atupl mutant subjected to H2O2 treatment with a NOS assay kit. Our results showed that H2O2 treatment induced NOS activity in the wild-type yeast. However, H2O2-induced NOS activity in Atupl mutant was largely repressed, indicating that TUP1 functions in H2O2-induced NO accumulation and cell apoptosis by regulating NOS activity.3. Arabidopsis WD40-REPEAT 5a (WDR5a), a homolog of yeast TUP1, shares 33% identity with TUP1 protein in the overlapping part. Our complementation assay showed that expression of Arabidopsis WDR5? in ?tup1 mutant increased NO accumulation and NOS activity, indicating the conserved role of TUP1/WDR5a in regulating NOS activity and NO accumulation in yeast and Arabidopsis.4. We identified wdr5a mutants with reduced expression of WDRSa, and found that ABA/H2O2-induced NO accumulation in guard cells and stomatal closure were dramatically repressed in the mutants, indicating the important role of WDR5a in ABA/H2O2-induced NO accumulation and stomatal movement. We also assayed the NOS activity, and our data showed that H2O2-induced NOS activity in the mutants was lower than that in the wild type. Further, we assessed no difference of the L-arginine content in the wild type and wdr5a mutants. These results showed that reduced NO accumulation in wdr5a mutants was not due to the insufficiency of NOS substrate L-arginine, but due to reduced NOS activity.5. We examined the water loss of the detached leaves of both wild-type and mutant plants. Our results showed that the detached leaves of the mutant lost water faster than those of wild-type plants, suggesting that drought-induced stomatal closure was repressed in the mutants. The temperature assessment with infrared images showed that wdr5a mutant had lower leaf surface temperature than the wild type subjected to drought stress, indicating the faster water transpiration of the mutant leaves than that of the wild type. Further, we assayed the stomatal closure in the wild type and wdr5a mutants subjected to drought stress. Our results showed that both stomatal aperture and ratio in the mutants were larger than those in the wild type, and mutants displayed a more were sensitive phenotype with severe wilting and chlorosis of rossette leaves than the wild type. These results demonstrated that WDR5a functions in plant tolerance to drought stress by regulating stomatal closure.6. It was reported that NO regulates stomatal movement as well as the expression of stress-responsive genes. Our results showed that drought-induced genes such as KIN1, KIN2, RD22, RD29A and RD29B were significantly induced in the wild type by drought stress, whereas the enhanced expression of the genes was suppressed in the mutants, suggesting that WDR5a also modulates the expression of drought-stress-responsive genes by regulating NO accumulation in plants.