The Reversible Disaggregation Of Particles In Guard Cell Vacuoles Mediated By PH And Its Effects On Osmoregulation Of Stomatal Movement

The chemiosmotic hypothesis describes the stomatal opening as a process in which the osmotic materials, mainly potassium, accumulate in guard cells, and as a result of the increase of osmotic pressure and the absorption of water into guard cells the stomata are driven to open. The energy for trans-membrane transport of K+ is the hyperpolarized potential across plasmalemma, which is established by the proton extrusion. However, it is hard to find out direct evidences for the accumulation of foreign K+ in guard cell vacuoles for stomatal opening to satisfy the chemiosmotic hypothesis. Our research group proposed a model for osmotic-regulation in guard cell vacuoles (GCV), which is depended on reversible disaggregation of particles and modulated by pH in GCV. Published evidences for this model show a great increase of the distribution density going with obvious decrease in volume of the particles in GCV of open stomata in comparison with that of the stomata before open (closed). During stomatal opening there is an acidification in GCV of about -0.5 pH unit. The aims of this Mastered work are to show the changes in dimension and distribution density of particles, and the corresponding changes of pH as well in GCV during the stomatal closing, a) The stomatal closure is induced by ABA, and the sap in GCV of open and closed stomata is directly sampled with glass micro-capillary. The content of the sap is negatively stained by uranyl acetate and observed by TEM. Results show consistency with the published observation that particles in GCV of open stomata are increased enormously in distribution density with obviously diminished small volume. However for the closed stomata, induced by ABA, the distribution density of particles in GCV is greatly reduced with the obviously enlarged volume, b) Buffers regulate pH of in vitro GCV sap in the tip of micro-capillaries: certain amount of Buffer (Mes-Tris) is filled from the tip of micro-capillaries beforehand, and the pH of GCV sap that is sampled by the same micro-capillary should be regulated by the buffer. It is proved by observation under TEM that the status of the aggregation of particles in GCV is modulated by pH. The establishment of the in vitro system of GCV sap is by the direct sampling from GCV by glass micro-capillary. This new method is basically different from the traditional way for GCV sap, which begins from the isolation of the protoplast of guard cells, and then vacuoles are isolated from protoplasts. The traditional method is not only complex and laborious, but also very difficult to get rid of the hard contamination from protoplasm. This improved method will be able to significantly reduce the contamination with easier operation, and most importantly it has the advantage to sample the GCV sap at micro level of about 10皀L. This new method will bring significant developments in studying the principles of stomatal movement, and other quick movement in plants, c) Guard cells are incubated with pH dependent fluorescent chemical probe 'BCECF AM' and excited at 488nm, the fluorescent emission ratio method (520nm/640nm) is employed with laser scanning confocal microscopy, about 0.4 pH unit increase in guard cell vacuoles is observed during stomatal closure that is induced by ABA.