Light-dependent volume changes and reactions in chloroplasts. I. Action of alkenylsuccinic acids and phenylmercuric acetate and possible relation to mechanisms of stomatal control.

Chloroplasts are known to change their volume in vitro by 3 mechanisms. Light and osmotic pressure appear to be the most significant factors for controlling chloroplast volume. The simplest type of volume change shown by isolated chloroplasts has been described by Nishida (8), who found by absorbancy, gravimetric, and volumetric techniques that chloroplasts change th.eir volume -in response to exposure to solutions of different tonicity. Hence, chloroplasts in vitro are osmotically sensitive structures like mitochondria and cells. The 2 other mechanisms for bringing about volume changes in chloroplasts require the action of light. One mechanism seems closely geared to the energy transfer reactions that are coupled to electron flow. Packer (12) has shown that suspensions of spinach chloroplasts exhibit light-induced. increases of scattered light that are rapid and reversible, occurring in a time interval of 20 to 100 seconds. Substances that interact with the energy transfer pathway. such as ammoniuml chloride and ADP, inhibit light-scattering increases (2), while ATP under conclitions favoring its hydrolysis promotes light-scattering increases (14). These observations are consistent with the view that volume chan.ges are under the control of energy-linked intermedi.ates (2,14,15). Itoh et al. (3) have shown that this aceion of light brings about a low-amplitude shrinkage that results in a 50 to 60 % decrease in volume of whole chloroplasts, as measured by the volume distribution of chloroplasts in the Coulter counter. Chloroplasts isolated from Etglena (1) have also been reported to manifest light-dependent volume ch.anges. Another action of light on chloroplast volume results in high-amplitude swelling (16, 17). Swelling is brought about slowly in the dark but can be accelerated by light, especially if a cofactor such as phenazine methosulfate has been added to the chloroplasts. Light-dependent, high-amplitude swelling of chloroplasts requires 10 to 90 minutes for completion and has not been found to be reversible either in darkness or by the addition of ATP. Moreover, ammonium ch.loride and ADP do not affect the time course of this process. High-amplitude chloroplast swelling is powerfully inhibited by inorganic phosphate, one of the general requirements for low-amplitude, light-dependent shrinkage. Under conditions for low-amplitude chloroplast shrinking in the light, an energy-dependent translocation of certain ions, such as calcium, phosphate, and sodium, occurs by a lightand energy-dependent mechanism (9). Since the osmotic and turgid properties of plant cells have been reported to be under the influence of light, it seemed possible that the light-dependent movements of water and ions manifested by chloroplasts in vitro might be involved in such processes within ithe cell. In particular, it is known,that light induces the opening of stomata and that this process is accompanied by the increased turgor of guard cells (5, 24). Since guard ce.lls -or their chloroplasts cannot be readily isolated, an indirect approach was undertaken to test the action of certain compounds, such as the alkenylsuccinic acids (22) and phenyl.mercuric acetate (19), that have been found to be effective agents for the control of stomatal aperture. This approach was suggested by the classical investigation of Zelitch (21-24) that has established the importance of photosynthetic reactions in mechanisms of stomatal control by use of such inhibitors. It has been found that these substances inhibit not only reactions of electron transport and photophosphorylation, but also alter the action of light on chloroplast volume.