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Regulation of photosynthetic electron flow in isolated chloroplasts by bicarbonate, formate and herbicides

This thesis describes some efforts that were made to gain a better understanding of the processes involved in the regulation of photosynthetic electron flow by bicarbonate, formate and herbicides in chloroplasts. In the past decade a large amount of research has been devoted to get insight into the mechanism of herbicide action on electron flow at the acceptor side of photosystem II. This thesis will deal mainly with studies on the regulation of electron flow at the acceptor side of photosystem II by bicarbonate and formate. Some details of the mechanism of this regulation as well as its integration in the overall process of photosynthesis were investigated.In Chapter 2 experiments are described that were aimed to provide a more quantitative description of the mutual interaction between herbicides, bicarbonate and their binding environment at the acceptor side of photosystem II. This interaction was studied by measuring the effects of bicarbonate and herbicides on electron flow in CO 2 -depleted chloroplasts. The kinetics of the reactivation of the inhibited Hill reaction in CO 2 -depleted chloroplasts by dark-incubation with bicarbonate suggest that the binding of bicarbonate involves a reaction with (pseudo) first order kinetics. It is shown that in the presence of the herbicides i -dinoseb and DCMU the reactivation of the Hill reaction in CO 2 -depleted chloroplasts by bicarbonate is retarded. It is shown that any competitive inhibitor of the binding of bicarbonate can be expected to effectively retard the binding of bicarbonate (a theoretical treatment is given in Appendix 1). Although i -dinoseb appeared to be an apparently competitive inhibitor of the stimulation of the Hill reaction by bicarbonate under equilibrium conditions, i -dinoseb and bicarbonate probably do not compete for a common binding site. This was inferred from the kinetics of the binding of i -dinoseb. These kinetics were shown to be to fast to be explained by simple competition. Therefore it is concluded that the binding of i -dinoseb affects the binding of bicarbonate in an allosteric way.In Chapter 3 a new procedure for CO 2 -depletion of chloroplasts is presented. This method yields CO 2 -depleted chloroplasts in which the Hill reaction can be almost completely reactivated by bicarbonate at pH 6.5. It is further shown that formate is a competitive inhibitor of the reactivation of the Hill reaction by bicarbonate. The true reactivation constant K r was calculated to be 78 μM bicarbonate at pH 6.5. Under the same conditions the inhibitor constant K i was calculated to be 2 mM formate. Experiments performed at bicarbonate and formate concentrations lower than K r resp. K i show that under these conditions electron flow proceeds at high rates. These observations suggest that, contrary to hitherto presented concepts, binding of bicarbonate to the regulatory site at the acceptor side of PSII is not a requirement for electron flow at the acceptor side of PS II. The results presented in Chapter 3 further suggest that formate is a potent inhibitor of electron flow at the acceptor side of PSII. The inhibitory action of formate is counteracted in a competitive way by bicarbonate.It is proposed in Chapter 3 that photorespiration may affect photosynthetic electron flow. Under certain conditions formate is produced during photorespiration. As photorespiratory formate production is dependent on the oxygen concentration, a negative feedback loop from photosynthetically produced oxygen to formate may exist under these conditions. This negative feedback loop can be powerful mechanism for the regulation of electron flow in vivo .In Chapter 4 the effects of formate and bicarbonate on the Hill reaction are further studied and characterized, especially during prolonged illumination. Formate is shown to stimulate the Hill reaction in non-depleted chloroplasts. It is therefore proposed that formate acts as an uncoupling agent. In the presence of an uncoupler formate was found not to stimulate electron flow. Instead only inhibiton of electron flow by formate was observed. The extent of this inhibition was not constant in time but appeared to increase during prolonged illumination The inhibition of electron flow by formate could be partially prevented by including bicarbonate in the reaction medium, but in the light bicarbonate appeared to be a less efficient counteracting agent than in the dark. It is estimated that the inhibitor constant K i of formate may be more than a magnitude lower in the light than in the dark, implying that formate affects steady state electron flow at sub mM concentrations.In Chapter 5 an alternative method is presented which can effectively be used to study the effects of formate and bicarbonate on electron flow in intact chloroplasts. This method is based on measurement of the magnitude of the flash-induced transmembrane potential using the P 515 response as a linear indicator of the transmembrane potential difference. In CO 2 -depleted chloroplasts the overall rate-limiting step of electron flow was found to posess a half-time of about 200-250 ms in the absence of bicarbonate. This half-time was much smaller in the presence of bicarbonate. Moreover gramicidin insensitive absorbance changes in the 500-550 nm region were found to be affected by CO 2 -depletion and readdition of bicarbonate. These absorbance changes are speculated to be associated, with protolytic reactions at the acceptor side of PSII.Chapter 6 describes effects of formate and bicarbonate on electron flow in isolated intact chloroplasts. Formate was found to inhibit PGA dependent oxygen evolution, but this inhibition could not be correlated with inhibition of electron flow at the acceptor side of PSII. Inhibition of electron flow was observed when isolated intact chloroplasts were incubated with formate at low pH. This inhibition of electron flow was abolished by addition of bicarbonate. The treatment with formate at low pH however appeared to be detrimental to carbon metabolism; PGA and CO 2 -dependent oxygen evolution were found to be irreversibly inhibited.A summarizing discussion is given in Chapter 7, in which the physiological significance of regulation of electron flow by formate and bicarbonate is discussed in relation to carbon metabolism.