The Regulation Of Stomatal Movement By Vacuole Fusion In Plant Guard Cells

Stomata are pores distributed on the epidermis of leaves, young stems and other organs for higher terrestrial plants. Stomata are composed of a pair of highly specialized guard cell, from which a pore formed between the two guard cells. The main function of the stomata is to allow the entry of sufficeint carbon dioxide (CO2) for photosynthesis and the movement of water vapor out of the leaf through transpiration. Transpiration is the major way to lost water in plant, and approximately90%of water taken up by the plant is lost through the process of transpiration. Therefore, the regulation of stomatal aperture is important for the efficient use of water as well as for plant response to drought stress.Studies have shown that the number and size of vacuoles in guard cells change with stomatal movement. These results indicate that vacuole fusion and fission may play a critical role in controlling stomatal movement. However, the molecular mechanism regarding the regulation of stomatal movement by vacuole fusion remains largely unknown.Our goal is to understand the molecular mechanisms governing stomatal movement by vacule fusion in plants. We first analyzed the relationship between stomatal movement and vacuole fusion in Zygophyllum xanthonylon, a well known desert plant in Northwest China. Then, we investigated the role of AtVAM-X gene in yeast vacuole fusion using a yeast heterologous expression system. Finally, we examined the stomatal movement of the Arabidopsis mutant vam-x using the molecular genetics techniques. This study will pave the way for the understanding of the mechanisms underlying plant drought tolerance as well as for the development of new crop plants that are tolerant to drought stress.The major results are listed below:1. We found that the guard cells of Zygophyllum xanthonylon had a great number of small vacuoles when stomata were closing. The small vacuoles fused with each other to produce larger vacuoles during stomatal opening. There results suggest that vacuole fusion/fission may control stomatal movement in Zygophyllum xanthonylon.2. Under the treatments of150mM NaCl or150mM KC1, the stomatal aperture of Zygophyllum xanthonylon was increased by58.3%and42.0%, respectively, while it was increased29.6%and37.1%in Arabidopsis, respectively, indicating that the stomatal aperature of Zygophyllum xanthonylon is more sensitive than Arabidopsis to the treatment of high concentration NaCl or KC1. However, a similar change in stomatal aperture was observed in Zygophyllum xanthonylon and Arabidopsis under the treatment of CaCl2, PEG6000or H2O2. These results suggest that NaCl and KC1play an important role in regulating stomatal movement in Zygophyllum xanthonylon.3. ScNhxl p is a Na+, K+/H+antiporter localized at the tonoplast of the yeast Saccharomyces cerevisiae. ScNhxl p plays an important role in regulating yeast vacuole fusion. Deletion of the ScNhxlp gene leads to vacuolar fragmentation. We found that transforming AtVAM-X gene into the BJ3505-Δnhx1mutant strains produced a large central vacuole in yeast, suggesting that AtVAM-X gene is involved in yeast vacuoles fusion.4. In order to verify the function of AtVAM-X gene in stomatal movement, we screened a Arabidopsis vam-x mutant to analyze its stomatal movement. We found that no significant changes were observed in vam-x mutants compared with the wild type plants under the treatments of NaCl, KC1, CaCl2, PEG or H2O2, indicating that stomatal movement is restrained in mutant plants. There results suggest that vacuole fusion plays a critical role in stomatal movement in guard cells.