Photoactivation of the Water-Oxidation System in Isolated Intact Chloroplasts Prepared from Wheat Leaves Grown under Intermittent Flash Illumination

Abstract

Photoactivation of the latent oxygen-evolving system in intact chloroplasts isolated from wheat ( Triticum aestivum L.) leaves grown under intermittent flash illumination was investigated, and the following results were obtained: (a) The water-oxidation activity generated on ifluminating the isolated intact chloroplasts was as high as that generated in intact leaves, indicating that all the machinery necessary for the activity generation is assembled within intact chioroplasts. (b) The generation of wateroxidation activity was accompanied by enhancement of the activity of diphenylcarbazide-oxidation, and both processes share the same photochemical reaction but with respective rate-limiting dark reactions of different efficiencies. (c) A23187, an ionophore for divalent cations, strongly inhibited the generation of water-oxidation activity but did not affect the activity once generated, which suggested that Mn atoms in the chloroplasts are susceptible to the ionophore before photoactivation but turn immune after photoactivation. (d) The generation of water-oxidation activity was not affected by the inhibitors of ATP formation and CO2 fixation, but was inhibited by nitrite, methylviologen and phenylmercuric acetate which suppress or inhibit the reduction of ferredoxin in intact chloroplasts. It was inferred that some factor(s) probably present in stroma to be reduced by PSI photoreaction is involved in the process of photoactivation.There are many reports on the reactivation of 02 evolution in isolated chloroplasts after various inactivation treatments which preferentially block the 02-evolving system. Yamashita and Tomita (21,22) found that Tris-inactivated oxygen evolution is restored by simply washing the chloroplasts with reduced DCIP2 solution, in some cases, followed by illumination in the presence of Mn2 , Ca2' and DTT. In these reactivation experiments with isolated chloroplasts, the coexistence of reducing agent has been shown to be the common requirement.In reactivation experiments with whole algal cells, on the other hand, Cheniae and Martin (4) have demonstrated a good reactivation of 02 evolution in NH20H-treated cells of Anacystis nidu-

Studies on the Photoactivation of the Water-Oxidizing Enzyme

“…Such contrasting requirements for the light-dependent recovery of Vo2 in the various systems, our inability to obtain significant yields of the photoactivation as reported by Yamashita and Tomita (57,58), and the generally low yields of photoactivation in isolated chloroplasts (39,40) compared to algal cells (9-1 1) prompted these studies in attempts to gain additional insights into the specifics of the photoactivation process itself and to identify factors that have limited our observation ofthe photoactivation process in in vitro systems over the years. Here, we report on studies made with NH2OH-extracted wheat leaf segments and Chlamydomonas.…”

“…Moreover, in algae, photoactivation is independent of protein synthesis requirements. Similar minimum require-ments have been noted for the photoactivation process in intact chloroplasts prepared from wheat leaves grown under intermittent flash illumination (39,40). However, these studies yielded evidence suggesting a requirement of some stromal factor reduced by PSI photoreactions and a requirement of Ca2" in addition to Mn2" (39,40).…”

“…Additionally, light requirements for the appearance of V02 have been noted for Mn-deficient higher plants made , spruce needles greened in darkness (38), some cold sensitive higher plants following cold stress regimes (31, 34), Trisacetone extracted spinach chloroplasts (57), and intact chloroplasts from wheat leaves greened under widely spaced flashes (39,40 …”

“…This process has been defined (42) as a multiquantum process driven by PSII quantum events leading to Mn ligation into the inactive apo-S-State complex with the conversion of the inactive complex into an active water oxidizing S-State complex.This multiquantum process has been observed with Mn-deficient algae made Mn-sufficient (9), NH20H extracted algae (10), dark grown Chlorella capable of synthesis in darkness of the pigments of photosynthesis, PSI and PSII trapping centers and electron transport components (1 1), wheat leaves greened under widely spaced flash regimes leading to the synthesis of the pigments of photosynthesis, PSI and PSII trapping centers and electron transport components but not the active water oxidizing S-State complex (22), and Tris-extracted spinach chloroplasts reactivated by DCIPH23 washings (56).Additionally, light requirements for the appearance of V02 have been noted for Mn-deficient higher plants made , spruce needles greened in darkness (38), some cold sensitive higher plants following cold stress regimes (31, 34), Trisacetone extracted spinach chloroplasts (57), and intact chloroplasts from wheat leaves greened under widely spaced flashes (39,40 …”

“…Additionally, light requirements for the appearance of V02 have been noted for Mn-deficient higher plants made Mn-sufficient (20), spruce needles greened in darkness (38), some cold sensitive higher plants following cold stress regimes (31,34), Trisacetone extracted spinach chloroplasts (57), and intact chloroplasts from wheat leaves greened under widely spaced flashes (39,40). It is not at all clear that the same biochemical process(es) is involved in all these instances where light requirements have been demonstrated for the appearance of V02 capacity.…”