Vacuolar Dynamics Of Guard Cells And The Dynamics Relationship Between Vacuoles And Actin Cytoskeleton During Stomatal Movement

Stomatal movement is important for plants to exchange gas with environment. The process contributes to the optimization of the photosynthesis and transpiration. Studies have showed that the change of vacuolar volume in guard cells is involved in the stomatal movement. However, little is known about the regulatory mechanism for the rapid changes of the vacuolar volume in guard cells during stomatal movement. Actin cytoskeleton of guard cells also is dynamics and involved in various signal pathway in regulating stomatal movement. However, the relationship between the dynamic of vacuoles and that of actin cytoskeleton, and their interacting mechanism during stomatal movement are unclear. Using CLSM and GFP-labeling technology, we studied the dynamics and function of vacuole and the relationship between the dynamic of vacuoles and that of actin cytoskeleton in guard cell during stomatal movement.There are many small spherical vacuoles and tubular vacuoles in guard cells of closed Vicia faba stomata, and the tubular vacuoles link the spherical vacuoles. The small vacuoles are irregular and harbor many complex membrane structures in lumen of vacuoles, such as ingrowths of tonoplast and vesicles. The small vacuoles and complex membrane systems are fused with each other or with the bigger vacuoles to generate the large vacuoles during stomatal opening. Reversely, the large vacuoles in the guard cells split into smaller vacuoles and generate many complex membrane structures in the closing and closed stomata. Vacuole fusion inhibitor, E-64d, can significantly inhibit the stomatal opening of Viciafaba. Furthermore, mutation of the Arabidopsis thaliana SGR3 also leads to retardation of vacuolar fusion and stomatal opening. These evidences indicated that vacuolar fusion is required for stomatal movement. From the disappearance of the complex membranes in vacuolar lumen in the opened stomata, we also can infer that these membranes serve as a reservoir of tonoplast for the changes of vacuolar volume during stomatal movement. From the 3D projection of many optical sections and photobleaching by CLSM, we found that the lumens of different vacuoles in a guard cell of Viciafaba are interconnected and their saps can flow freely. Vacuolar continuum in guard cells might function in stomatal movement via facilitating the diffusion of solutes and water among the different parts of guard cells to maintain the balance of osmotic potential and turgor pressure in the whole guard cell.Using transgenic tobacco (Nicotiana tobacuum) plant overexpressing gfp-mTalin driven by 35s, we observed the F-actin arrangement and distribution in guard cells of stable and moving stomata. The web and radial pattern of the F-actin arrangement in closed and opened stomata were observed. The difference of distribution of actin of guard cells in the closed stomata and the opened was also found. The actin was found in the cortical plasma and cytoplasm in closed stomata, whereas it was mainly located in the cortical plasma in opened ones. We also found the endoplasmic F-actin increased and cortical F-actin decreased accordingly during stomatal movement induced stomatal closing by ABA and dark.Pharmacological experiments showed that cytochalasin B, which inhibit the polymerization of F-actin in guard cells, facilitates not only light and FC-induced stomatal opening, but also the changes of vacuolar number and volume during stomatal opening. In contrast, phalloidin, which polymerize theF-actin, inhibits stomatal opening and vacuolar changes induced light and FC. By dobule labeling method, we found that the vacuoles and F-actin in guard cells of tobacco stomata are colocalization probably. Combining with the pharmacological results, we infered that F-actin depolymerization in guard cells facilitates the vacuolar fusion, thus enhance stomatal opening, and the retardation the vacuolar fusion by inhibiting F-actin depolymerization leads to downregulated stomatal opening.The dynamics and function of vacuoles of guard cells during stomatal movement, and the dynamics relationshi...