Action Spectra of O₂ Evolution by Chloroplasts with & without Added Substrate, for Regeneration of O₂ Evolving Ability by Far-red, & for O₂ Uptake

Abstract

Recent studies (3,6,10,12) have shown that photosynthesis requires two photochemical reactions driven by different pigments. Furthermore, it has been suggested (3, 10, 12) that these two light reactions occur in series. Absorption by chlorophyll b and by chloroplhyll a,7, (so-called accessory pigments) are thought to bring about oxidation of water and evolution of O., with a reduction of an acceptor [X, or perhaps plastoquinone (12)]. A second photochemlical step, mediatefl by a long-wavelength form of chlorophyll a, causes reduction of TPN (or an artificial Hill oxidlant) with the electrons coming from reduced X.If some oxidized X were available, O. evolution would be possible without a Hill oxidant and without the functioning of the long-wavelengtlh chlorophyll reaction. (It may be recalled in this regard that a limited O., evolution upon illumination of chloroplasts suspended only in sucrose solutions has been known for over 80 years.) Such a recurring, but small, evolution of 02 by illuminated chloroplasts was also observed in the present study. The assumption that this 02 evolution in the absence of a Hill oxidant is a reflection of the activity of the accessory-pigment reaction can be tested by comparing the action spectra for O. evolution before and after the addition of a Hill oxidant. The action spectrum determined in the absence of an oxidant should have its peaks at wavelengths corresponding to the absorption of chlorophyll b and chlorophyll a,70. In the presence of a Hill oxidant the action spectrum for 02 evolution would be shifted to longer wavelengths and reflect the functioning of both pigment systems. , fig 3) shows a high initial rate (spike) followed by a rapid decrease to a steadystate net 02 evolution after 3 minutes. The subsequent spike after 3 minutes of darkness was smaller than the initial one and smaller also than a later one following 10 minutes of darkness. Figure 3B compares the effect of exposing chloroplasts to the same intensity of 650 myA light under aerobic conditions. Upon illumination, the 02-production spike is seen again. However, the rate of 02 production declines in the light reaching a steadystate net 02 uptake after about three minutes. The chloroplasts show an 02 gulp when the light is turned off. The dark base line, which slowly drifts upwards, is attained again after about three minutes.Chloroplasts retained their ability to evolve 02 70. (upper). Action spectrum for the production of the 02 spike by Swiss chard chloroplasts. The circulating solution used is described in the legend of figure 3. The action spectrum was determined by using equal incident quanta (477 ergs cm-2sec-1 at 650 m,u). At points below 580 mu the light intensities were limited by the output from the monochromator. These points were adjusted upwards appropriately to correspond with other points in the action spectrum. Temp = 19.50, gas phase, N2. 60FIG. 6 (lower). Relative height of the 02 production spike as a function of 650 m,u intensity for chloroplasts used for the action spectrum given i...

A tribute to Lawrence Rogers Blinks (1900–1989): light and algae

“…As a result of these new hypotheses, action spectra were made against a background of monochromatic light. This work showed that at wavelengths of background light, absorbed by biliproteins, the participation of chlorophyll a in the action spectra for red algae could be clearly discerned (Fork 1963a, b), a result anticipated by the work of Blinks (1960a, b, c) who observed similar effects but came to a different conclusion.…”

“…His preferred explanation of these effects was in terms of changes in respiration, but these are also explained by two light reactions (see Hill andBendall 1960 andDuysens 1989), and later became important experimental evidence in favor of the hypothesis of two photosystems. Larkum and Weyrauch (1977) further clarified the photosystem I and II in their experimental work on Griffithsia monilis from Athol Wharf in Sidney Harbor which was built on the pioneering work by Fork (1963a, b). In their results, Larkum and Weyrauch stated.…”

“…In another interaction, David Fork, working with me (Haxo) for his PhD, in his research at La Jolla (Fork 1960) and Blinks (1960a, b, c) quite independently found important new evidence bearing on the problem of ''inactive chlorophyll'' in red algae. As noted elsewhere (Briggs et al 1990), our (Blinks's and my) earlier action spectrum studies of marine algae along with Blinks's new measurements on chromatic transients gave support to the idea of Robert Emerson that accessory pigments and chlorophyll are organized in special ways and that photosynthesis functions with two photosystems.…”

Quantitative Local Photosynthetic Flux Measurements at Isolated Chloroplasts and Thylakoid Membranes Using Scanning Electrochemical Microscopy (SECM)

“…The appearance of an initial oxygen concentration spike has been reported previously, with Fork observing such a effect while investigating oxygen exchange at chloroplasts in solution in the absence of a Hill oxidant, using a macroscale Clark type oxygen electrode. 77 Matsue et al also observed an initial intracellular oxygen spike in algal protoplasts using ultramicroelectrodes with ring radii of approximately 2 µm. 40,41 The oxygen spike was attributed to the consumption of the limited number of, then unknown, electron acceptors by Fork, and to the consumption of the limited number of terminal species (such as NADP) by Matsue.…”

Requirement for Plastoquinone A in the Hill Reaction of Isolated Chloroplasts

“…Chloroplasts lyophilized for 4 hours shiowed good rates of Hill activiity in the absence of uncouplers. PLANT PHYSIOLOGY tion niot coul)led to photol)hosp)hic)rvl.ationi.One of the I)rol)lems enicounltere(l in the mieasureeent of (., evolutioli by the 14ill reactioni is that of light stimuilated C)., uptake reactionls of the chloroplast suspenlsion (9). The rates of 0., uptake with whole chloroplasts were ustually smnall (ca.…”