Alpha Carbonic Anhydrase 5 Mediates Stimulation of ATP Synthesis by Bicarbonate in Isolated Arabidopsis Thylakoids.

We studied bicarbonate-induced stimulation of photophosphorylation in thylakoids isolated from leaves of Arabidopsis thaliana plants. This stimulation was not observed in thylakoids of wild-type in the presence of mafenide, a soluble carbonic anhydrase inhibitor, and was absent in thylakoids of two mutant lines lacking the gene encoding alpha carbonic anhydrase 5 (αCA5). Using mass spectrometry, we revealed the presence of αCA5 in stromal thylakoid membranes of wild-type plants. A possible mechanism of the photophosphorylation stimulation by bicarbonate that involves αCA5 is proposed.

Quantification of superoxide radical production in thylakoid membrane using cyclic hydroxylamines.

Applicability of two lipophilic cyclic hydroxylamines (CHAs), CM-H and TMT-H, and two hydrophilic CHAs, CAT1-H and DCP-H, for detection of superoxide anion radical (O2(∙-)) produced by the thylakoid photosynthetic electron transfer chain (PETC) of higher plants under illumination has been studied. ESR spectrometry was applied for detection of the nitroxide radical originating due to CHAs oxidation by O2(∙-). CHAs and corresponding nitroxide radicals were shown to be involved in side reactions with PETC which could cause miscalculation of O2(∙-) production rate. Lipophilic CM-H was oxidized by PETC components, reducing the oxidized donor of Photosystem I, P700(+), while at the same concentration another lipophilic CHA, TMT-H, did not reduce P700(+). The nitroxide radical was able to accept electrons from components of the photosynthetic chain. Electrostatic interaction of stable cation CAT1-H with the membrane surface was suggested. Water-soluble superoxide dismutase (SOD) was added in order to suppress the reaction of CHA with O2(∙-) outside the membrane. SOD almost completely inhibited light-induced accumulation of DCP(∙), nitroxide radical derivative of hydrophilic DCP-H, in contrast to TMT(∙) accumulation. Based on the results showing that change in the thylakoid lumen pH and volume had minor effect on TMT(∙) accumulation, the reaction of TMT-H with O2(∙-) in the lumen was excluded. Addition of TMT-H to thylakoid suspension in the presence of SOD resulted in the increase in light-induced O2 uptake rate, that argued in favor of TMT-H ability to detect O2(∙-) produced within the membrane core. Thus, hydrophilic DCP-H and lipophilic TMT-H were shown to be usable for detection of O2(∙-) produced outside and within thylakoid membranes.

Effects of heterocyclic and tertiary permeant amines on the electron transfer in thylakoid membranes.

The effect of low concentrations (up to 50 muM) of lipophilic permeant amines on the electron transfer in thylakoid membranes of pea chloroplasts has been investigated. In the presence of heterocyclic amines (9-aminoacridine and neutral red), the electron transfer, initiated from H(2)O to PS I acceptors, has been shown to be inhibited to a level amounting to less than 50% of control, this taking place for both the basal (at alkaline pH) and the gramicidin-uncoupled transport (at pH 6.5-8.5). Under the same conditions, tertiary amines (dibucaine, tetracaine) cause only a 10-15% inhibition of transport. With all the amines, the rate of electron transport from H(2)O to DCBQ, PS II acceptor is decreased to 80-90% of control at pH above 8.0, but this effect is completely removed when pH is lowered to 7.7-6.5. In the region of PS I, all the amines accelerate the basal transport, but do not influence the uncoupled electron transfer. A conclusion has been drawn that, parallel with uncoupling, heterocyclic and tertiary amines also cause an inhibition of PS II, appearing at alkaline pH values. Additionally, heterocyclic amines seem to brake electron flow at the level of plastoquinone reduction.