Studies on the System Regulating Proton Movement across the Chloroplast Envelope

Abstract

Studies were undertaken to further characterize the spinach (Spinacea oleracea) chloroplast envelope system, which facilitates H+ movement into and out of the stroma, and, hence, modulates photosynthetic activity by regulating stromal pH. It was demonstrated that high envelope-bound Mg2+ causes stromal acidification and photosynthetic inhibition. High envelope-bound Mg2+ was also found to necessitate the activity of a digitoxinand oligomycin-sensitive ATPase for the maintenance of high stromal pH and photosynthesis in the illuminated chloroplast. In chloroplasts that had high envelope Mg2+ and inhibited envelope ATPase activity, 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide was found to raise stromal pH and stimulate photosynthesis. 2-(Diethylamino)-N-(2,6-dimethylphenyl)acetamide is an amine anesthetic that is known to act as a monovalent cation channel blocker in mammalian systems. We postulate that the system regulating cation and Ho fluxes across the plastid envelope includes a monovalent cation channel in the envelope, some degree of (envelope-bound Mg2 modulated) H+ flux linked to monovalent cation antiport, and ATPase-dependent H+ efflux.Upon illumination, the pH of the spinach chloroplast stroma rises by approximately 0.4 to 0.5 pH unit (11). This stromal alkalinization is a regulatory mechanism that facilitates optimal photosynthesis in the light (24), primarily due to the high pH optima ofthe photosynthetic carbon reduction cycle enzymes fructose 1,6-bisphosphatase and sedoheptulose 1,7-bisphosphatase (1, 8). For quite some time, the rise in stromal pH in the light has been primarily attributed to H+ pumping into the thylakoid lumen (1 1, 24). However, optimal photosynthetic capacity, both ofisolated chloroplasts ( 11) and in situ in illuminated leaves (17) (7), Huber and co-workers (13, 15) concluded that a K+/H+ antiporter facilitated this exchange. This hypothesized antiport mechanism was thought to result in either stromal acidification or alkalinization, depending on the extrachloroplastic monovalent cation concentration. However, in later studies, Demmig and Gimmler (6, 7) speculated that H+ pumping into the thylakoid during illumination is not entirely chargebalanced, and the resultant membrane potential developed between the stroma and external solution (inside negative) drives K+ and/or H+ movement into the stroma. K+ efflux was thought to result in H+ influx due to changes in this membrane potential. They concluded, in contrast to Huber, that K+/H+ exchange across the envelope was not facilitated by specific intrinsic envelope proteins acting as "antiporters."The studies of 15)

Surface Charge-Mediated Effects of Mg2+ on K+ Flux across the Chloroplast Envelope Are Associated with Regulation of Stromal pH and Photosynthesis

“…One clear effect of Mg2" binding to the envelope is an inhibition of K+ uptake. However, the data reported here support the results of previous studies (2,11,14,18) in that Mg2e inhibition of photosynthesis was demonstrated to be caused directly by stromal acidification. The data suggest that Mg2+-induced stromal acidification is not due to the action of a Mg2+-activated K+/H+ antiporter or to changes in the ionic strength of the bulk solution external to the chloroplast but possibly due to the metal ion's effects on the current through monovalent cation channels in the envelope.…”

“…This hypothesis seemed plausible in light of our recent work in which we postulated the presence of K+ channels in chloroplast envelopes (18).…”

A probable Na + (K + )/H + exchanger on the chloroplast envelope functions in pH homeostasis and chloroplast development in Arabidopsis thaliana

“…Earlier biochemical and physiological data suggested that an unknown Na ϩ (K ϩ )͞H ϩ antiporter is localized in the chloroplast envelope, partially regulating stromal acidification and alkalization (7,8,30). The stroma pH level is an important factor for high photosynthetic activity.…”

“…Electroneutral monovalent cation͞proton antiport across the chloroplast envelope was shown to have a strong effect on the chloroplast stroma pH and could thereby either help or inhibit photosynthesis (6,7). Subsequent work (8,9) indicated the existence of a complex regulatory system, including a monovalent cation͞proton antiporter, monovalent cation channels, and ATPase-dependent H ϩ efflux, across the chloroplast envelope. However, none of the hypothesized chloroplast envelope transporters have been identified as yet.…”

K+-conducting ion channel of the chloroplast inner envelope: functional reconstitution into liposomes.

“…Inhibitor studies with intact isolated chloroplasts further suggest that trans-envelope K+ flux may occur through a putative ion channel (2)(3)(4).…”