CAH3 from Chlamydomonas reinhardtii: Unique Carbonic Anhydrase of the Thylakoid Lumen.

CAH3 is the only carbonic anhydrase (CA) present in the thylakoid lumen of the green algae Chlamydomonas reinhardtii. The monomer of the enzyme has a molecular weight of ~29.5 kDa with high CA activity. Through its dehydration activity, CAH3 can be involved either in the carbon-concentrating mechanism supplying CO2 for RuBisCO in the pyrenoid or in supporting the maximal photosynthetic activity of photosystem II (PSII) by accelerating the removal of protons from the active center of the water-oxidizing complex. Both proposed roles are considered in this review, together with a description of the enzymatic parameters of native and recombinant CAH3, the crystal structure of the protein, and the possible use of lumenal CA as a tool for increasing biomass production in higher plants. The identified involvement of lumenal CAH3 in the function of PSII is still unique among green algae and higher plants and can be used to understand the mechanism(s) of the functional interconnection between PSII and the proposed CA(s) of the thylakoid lumen in other organisms.

Dependence of state transitions on illumination time in arabidopsis and barley plants.

Solar energy absorbed by plants can be redistributed between photosystems in the process termed "state transitions" (ST). ST represents a reversible transition of a part of the PSII light harvesting complex (L-LHCII) between photosystem II (PSII) and photosystem I (PSI) in response to the change in light spectral composition. The present work demonstrates a slower development of the state 1 to state 2 transition, i.e., L-LHCII transition from PSII to PSI, in the leaves of dicotyledonous arabidopsis (Arabidopsis thaliana) than in the leaves of monocotyledonous barley (Hordeum vulgare) plants that was assessed by the measurement of chlorophyll a fluorescence at 77 K and of chlorophyll a fluorescence at room temperature. It is known that the first step of the state 1 to state 2 transition is phosphorylation of Lhcb1 and Lhcb2 proteins; however, we detected no difference in the rate of accumulation of these phosphorylated proteins in the studied plants. Therefore, the parameters, which possibly affect the second step of this transition, i.e., the migration of L-LHCII complexes along the thylakoid membrane, were evaluated. Spin-probe EPR measurements demonstrated that the thylakoid membranes viscosity in arabidopsis was higher compared to that in barley. Moreover, confocal microscopy data evidenced the different size of chloroplasts in the leaves of the studied species being larger in arabidopsis. The obtained results suggest that the observed deference in the development of the state 1 to state 2 transition in arabidopsis and barley is caused by the slower L-LHCII migration rate in arabidopsis than in barley plants rather than by the difference in the Lhcb1 and Lhcb2 phosphorylation.

Action of 2,6-Dichloro-1,4-benzoquinone on the O2-Evolving Activity of Photosystem II in Chlamydomonas reinhardtii Cells with and without Cell Wall: Inhibitory Effect of Its Oxidized Form.

Chlamydomonas reinhardtii is a widely used object in studies on green algae concerning both photosynthesis aspects and possible biotechnological approaches. The measurement of the maximum O2 evolution by photosystem II (PSII) in living algal cells in the presence of artificial acceptors is one of the commonly used methods for determining the photosynthetic apparatus state or its change as compared to a control, parent strain, etc., because PSII is the most sensitive component of the thylakoid membrane. The present study shows the need to use low concentrations of 2,6-dichloro-1,4-benzoquinone (DCBQ) paired with potassium ferricyanide (FeCy) for achieving the maximum O2 evolution rate, while a DCBQ concentration above certain threshold results in strong suppression of O2 evolution. The required DCBQ concentration depends on the presence of the cell wall and should be exactly ~0.1 mM or in the range of 0.2-0.4 mM for cells with and without a cell wall, respectively. The inhibition effect is caused, probably, by a higher content of DCBQ in the oxidized form inside cells; this depends on the presence of the cell wall, which influences the efficiency of DCBQ diffusion into and out of the cell, where it is maintained by FeCy in the oxidized state. The possible mechanism of DCBQ inhibition action is discussed.

Macromolecular conformational changes in photosystem II: interaction between structure and function.

Conformational changes play an important role in the functioning of proteins and their complexes. This is also true for the pigment-protein super-complex of photosystem II (PSII). The data testify about the pH-induced macromolecular conformational changes in the water-oxidizing complex (WOC) on the donor side of PSII, the interaction between the spatial structure of WOC proteins and the distribution of cytochrome b559 redox-forms, and the electron transfer efficiency between QA and QB on the acceptor side of PSII. Changes in the protein environment near QA and QB can be observed after the removal of the bicarbonate ion associated with non-heme Fe or after the addition of herbicides binding to the QB site, which results in the suppression of the electron transfer in this site. The "locking" of the de novo assembled PSII in an inactive state until WOC activation is also accompanied by strong structural perturbations on the PSII acceptor and donor sides with the participation of Psb28 and Psb27 proteins. The triggers for degradation and replacement of damaged PSII proteins are structural changes induced by their oxidative modification and aggregation. Macromolecular changes in the antenna proteins underlie the activation of photoprotective non-photochemical quenching, which are induced by protonation of the lumenal residues of PsbS or/and Lhcsr3, as well as the phosphorylation of antenna proteins. Besides this, many smaller-scale conformational changes may occur in PSII. This review summarizes current knowledge about the possible conformational changes in proteins in the PSII super-complex and describes their proposed influence on PSII function.

Loss of carbonic anhydrase in the thylakoid lumen causes unusual moderate-light-induced rearrangement of the chloroplast in Chlamydomonas reinhardtii as a way of photosystem II photoprotection.

Chlamydomonas reinhardtii cells have a single large cup-shaped chloroplast that can lose lobes under high light to prevent photodamage of the photosynthetic apparatus, including photosystem II (PSII). Here, under moderate light treatment, the development of the unusual morphology of the chloroplast is shown for mutant cia3, which is deficient in carbonic anhydrase (EC 4.2.1.1) CAH3 in the thylakoid lumen, while such light intensity is harmless for wild type (WT) cells for hours. Cia3 cells had more activated PSII photoprotective mechanisms and showed a tendency to shift in the balance of the PSII damage-repair cycle, whereas PSII retained the same photosynthetic efficiency as in the WT. These findings allow speculation about the unique PSII photoprotection strategy by rearranging the chloroplast in the absence of CAH3. CAH3, in turn, is suggested to be an important participant of the C. reinhardtii photosynthetic apparatus operation, functioning in close connection with PSII.

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.

Involvement of Carbonic Anhydrase CAH3 in the Structural and Functional Stabilization of the Water-Oxidizing Complex of Photosystem II from Chlamydomonas reinhardtii.

The involvement of carbonic anhydrases (CA) and CA activity in the functioning of photosystem II (PSII) has been studied for a long time and has been shown in many works. However, so far only for CAH3 from Chlamydomonas reinhardtii there is evidence for its association with the donor side of PSII, where the CA activity of CAH3 can influence the functioning of the water-oxidizing complex (WOC). Our results suggest that CAH3 is also involved in the organization of the native structure of WOC independently of its CA activity. It was shown that in PSII preparations from wild type (WT) the high O2-evolving activity of WOC was observed up to 100 mM NaCl in the medium and practically did not decrease with increasing incubation time with NaCl. At the same time, the WOC function in PSII preparations from CAH3-deficient mutant cia3 is significantly inhibited already at NaCl concentrations above 35 mM, reaching 50% at 100 mM NaCl and increased incubation time. It is suggested that the absence of CAH3 in PSII from cia3 causes disruption of the native structure of WOC, allowing more pronounced conformational changes of its proteins and, consequently, suppression of the WOC active center function, when the ionic strength of the medium is increased. The results of Western blot analysis indicate a more difficult removal of PsbP protein from PSII of cia3 at higher NaCl concentrations, apparently due to the changes in the intermolecular interactions between proteins of WOC in the absence of CAH3. At the same time, the values of the maximum quantum yield of PSII did not practically differ between preparations from WT and cia3, indicating no effect of CAH3 on the photoinduced electron transfer in the reaction center of PSII. The obtained results indicate the involvement of the CAH3 protein in the native organization of the WOC and, as a consequence, in the stabilization of its functional state in PSII from C. reinhardtii.

The Main Structural and Functional Characteristics of Photosystem-II-Enriched Membranes Isolated from Wild Type and cia3 Mutant Chlamydomonas reinhardtii.

Photosystem II (PSII)-enriched membranes retain the original PSII architecture in contrast to PSII cores or PSII supercomplexes, which are usually isolated from Chlamydomonas reinhardtii. Here, we present data that fully characterize the structural and functional properties of PSII complexes in isolated PSII-enriched membranes from C. reinhardtii. The preparations were isolated from wild-type (WT) and CAH3-deficient mutant cia3 as the influence of CAH3 on the PSII function was previously proposed. Based on the equal chlorophyll content, the PSII-enriched membranes from WT and cia3 have the same amount of reaction centers (RCs), cytochrome b559, subunits of the water-oxidizing complex, Mn ions, and carotenes. They differ in the ratio of other carotenoids, the parts of low/intermediate redox forms of cytochrome b559, and the composition of outer light-harvesting complexes. The preparations had 40% more chlorophyll molecules per RC compared to higher plants. Functionally, PSII-enriched membranes from WT and cia3 show the same photosynthetic activity at optimal pH 6.5. However, the preparations from cia3 contained more closed RCs even at pH 6.5 and showed more pronounced suppression of PSII photosynthetic activity at shift pH up to 7.0, established in the lumen of dark-adapted cells. Nevertheless, the PSII photosynthetic capacities remained the same.

The role of carbonic anhydrase α-CA4 in the adaptive reactions of photosynthetic apparatus: the study with α-CA4 knockout plants.

The role of α-carbonic anhydrase 4 (α-CA4) in photosynthetic machinery functioning in thylakoid membranes was studied, using Arabidopsis thaliana wild type plants (WT) and the plants with knockout of At4g20990 gene encoding α-CA4 (αCA4-mut) grown both in low light (LL, 80 µmol quanta m-2 s-1) or in high light (HL, 400 µmol quanta m-2 s-1). It was found that a content of PsbS protein, one of determinants of non-photochemical quenching of chlorophyll fluorescence, increased in mutants by 30% and 100% compared with WT plants in LL and in HL, respectively. Violaxanthin cycle pigments content and violaxanthin deepoxidase activity in HL were also higher in αCA4-mut than in WT plants. The content of PSII core protein, D1, when adapting to HL, decreased in WT plants and remained unchanged in mutants. This indicates, that the decrease in the content of Lhcb1 and Lhcb2 proteins in HL (Rudenko et al. Protoplasma 55(1):69-78, 2018) in WT plants resulted from decrease of both Photosystem II (PSII) complex content and content of these proteins in this complex, whereas in αCA4-mut plants from the latter process only. The absence of α-CA4 did not affect the rate of electron transport through Photosystem I (PSI) in thylakoids of mutant vs. WT, but led to 50-80% increase in the rate of electron transport from H2O to QA, evidencing the location of α-CA4 close to PSII. The latter difference may raise the question about its causal connection with the difference in the D1 protein content change during adapting to increased illumination in the presence and the absence of α-CA4.

Carbonic anhydrase CAH3 supports the activity of photosystem II under increased pH.

The lumenal carbonic anhydrase (CA) CAH3 from green alga Chlamydomonas reinhardtii is the only one CA identified so far in close association with the photosystem II (PSII) multi-subunit protein complex. It was proposed earlier, that CAH3 could facilitate the H+ removal from the active center of the PSII water-oxidizing complex (WOC) under the light, thereby increasing its activity. In the present work, using PSII enriched membranes from the wild type of C. reinhardtii and from the CAH3-deficient mutant cia3, we demonstrate, that the suppression of the photosynthetic activity of PSII by increased pH is more pronounced in preparations from cia3 as compared to the wild type. Experiments with CA inhibitors show that the activity of CAH3 supports the function of PSII and prevents its irreversible inactivation under light upon increased pH. The photosynthetic activity of PSII from cia3 can be restored to the wild type level upon increased pH if an excess of HCO3- is added. These findings testify that the main role of CAH3 in the vicinity of PSII is the acceleration of the HCO3- dehydration reaction. Measurements of the photoinduced electron transfer rate in PSII from water or from an artificial electron donor indicate, that CAH3 has a direct influence on the WOC function. Based on the data obtained in this work we conclude, that in vivo CA-activity of CAH3 may support the photosynthetic activity of PSII at increased pH in the thylakoid lumen and can be observed under the dark to light transition.

Overexpression of Arabidopsis OPR3 in Hexaploid Wheat (Triticum aestivum L.) Alters Plant Development and Freezing Tolerance.

Jasmonates are plant hormones that are involved in the regulation of different aspects of plant life, wherein their functions and molecular mechanisms of action in wheat are still poorly studied. With the aim of gaining more insights into the role of jasmonic acid (JA) in wheat growth, development, and responses to environmental stresses, we have generated transgenic bread wheat plants overexpressing Arabidopsis 12-OXOPHYTODIENOATE REDUCTASE 3 (AtOPR3), one of the key genes of the JA biosynthesis pathway. Analysis of transgenic plants showed that AtOPR3 overexpression affects wheat development, including germination, growth, flowering time, senescence, and alters tolerance to environmental stresses. Transgenic wheat plants with high AtOPR3 expression levels have increased basal levels of JA, and up-regulated expression of ALLENE OXIDE SYNTHASE, a jasmonate biosynthesis pathway gene that is known to be regulated by a positive feedback loop that maintains and boosts JA levels. Transgenic wheat plants with high AtOPR3 expression levels are characterized by delayed germination, slower growth, late flowering and senescence, and improved tolerance to short-term freezing. The work demonstrates that genetic modification of the jasmonate pathway is a suitable tool for the modulation of developmental traits and stress responses in wheat.

Is carbonic anhydrase activity of photosystem II required for its maximum electron transport rate?

It is known, that the multi-subunit complex of photosystem II (PSII) and some of its single proteins exhibit carbonic anhydrase activity. Previously, we have shown that PSII depletion of HCO3-/CO2 as well as the suppression of carbonic anhydrase activity of PSII by a known inhibitor of α­carbonic anhydrases, acetazolamide (AZM), was accompanied by a decrease of electron transport rate on the PSII donor side. It was concluded that carbonic anhydrase activity was required for maximum photosynthetic activity of PSII but it was not excluded that AZM may have two independent mechanisms of action on PSII: specific and nonspecific. To investigate directly the specific influence of carbonic anhydrase inhibition on the photosynthetic activity in PSII we used another known inhibitor of α­carbonic anhydrase, trifluoromethanesulfonamide (TFMSA), which molecular structure and physicochemical properties are quite different from those of AZM. In this work, we show for the first time that TFMSA inhibits PSII carbonic anhydrase activity and decreases rates of both the photo-induced changes of chlorophyll fluorescence yield and the photosynthetic oxygen evolution. The inhibitory effect of TFMSA on PSII photosynthetic activity was revealed only in the medium depleted of HCO3-/CO2. Addition of exogenous HCO3- or PSII electron donors led to disappearance of the TFMSA inhibitory effect on the electron transport in PSII, indicating that TFMSA inhibition site was located on the PSII donor side. These results show the specificity of TFMSA action on carbonic anhydrase and photosynthetic activities of PSII. In this work, we discuss the necessity of carbonic anhydrase activity for the maximum effectiveness of electron transport on the donor side of PSII.