On the regulation of localized and delocalized proton gradient energy coupling (ATP formation) in chloroplast bioenergetics
Abstract
The characteristics of dual energy coupling phenomena as well as the role of Ca++ ions in thylakoid membranes isolated from pea (Pisum sativa) or spinach (Spinacea oleracea) was investigated using violaxanthin deepoxidase (VDE). The samples with a delocalized coupling energy gradient were prepared using different treatments, including: (1) High-salt-storage (100 mM KCI); (2) Carrying out the reactions under “basal” (-ADP) conditions; (3) Dilute uncoupler-induced delocalization; (4) Osmotic-shock-induced delocalization and (5) EGTA-induced delocalization. The reaction of VDE was observed right after the light was given in all the thylakoids with delocalized, but not thylakoids with the localized, coupling mode. Because VDE is an intrinsic enzyme requiring lumenal acidity near or below pH 6.0. the above findings provided direct evidence that the lumenal pH of thylakoids with a delocalized coupling gradient was at least lower than 6.2 and the lumenal pH of thylakoids with localized coupling mode was higher than 6.2, when the external pH of both samples was 8.0. When 1 mM of Ca2+ was included in the osmotic shock medium, the osmotic shock process failed to convert the localized coupling gradient to a delocalized coupling mode, as revealed by a low VDE activity observed in the “osmotic-shock + Ca 2+” case. The addition of Ca2+ to the samples pretreated with EGTA also prevented the switchover of thylakoids from a localized to a delocalized coupling mode. These data confirmed that Ca2+ ion is a key controlling factor in the localized to delocalized coupling mode switching, and that the activation of violaxanthin deepoxidase occurs under the same conditions when the thylakoids are delocalized. This study supports the idea that a possible physiological role of Ca2+-controlled delocalization is to trigger violaxanthin deepoxidase for photoprotection.
Degree
Ph.D.
Advisors
Dilley, Purdue University.
Subject Area
Biophysics|Botany
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