Light as a substrate: migration of LHCII antennas in extended Michaelis-Menten model for PSI kinetics.

We extended, for the first time, the Michaelis-Menten (M-M) model to describe the kinetics of photosystem I (PSI) complexes using light as a substrate. Our work is novel as it can be useful for studying the phenomenon of "state transitions" because it quantifies the affinity of light to PSI reaction centers depending on the associated light harvesting complex II (LHCII) antennas. We verified our models by measuring the PSI activity as a function of light intensity using an oxygen electrode for chloroplast from plants grown in low light conditions and treated with far red light. We determined the kinetics constant KM for: PSI-LHCI, PSI-LHCI-LHCII and PSI-PSII megacomplexes and have shown that KM for PSI located in the megacomplexes was smaller in magnitude than PSI-LHCI, thus demonstrating that LHCII antennas are functionally associated with PSI. The parameter [S]1/2used in our models is the equivalent of M-M constant. Far red light increases [S]1/2, which indicates that transition from state 1 to state 2 leads to an energy gain while reaching the PSI reaction centers. We also observed that redistribution of the absorbed excitation energy is realized not only by LHCII migration but also by association of the photosystems in the megacomplexes.

Understanding Maize Response to Nitrogen Limitation in Different Light Conditions for the Improvement of Photosynthesis.

The photosynthetic capacity of leaves is determined by their content of nitrogen (N). Nitrogen involved in photosynthesis is divided between soluble proteins and thylakoid membrane proteins. In C4 plants, the photosynthetic apparatus is partitioned between two cell types: mesophyll cells and bundle sheath. The enzymes involved in the C4 carbon cycle and assimilation of nitrogen are localized in a cell-specific manner. Although intracellular distribution of enzymes of N and carbon assimilation is variable, little is known about the physiological consequences of this distribution caused by light changes. Light intensity and nitrogen concentration influence content of nitrates in leaves and can induce activity of the main enzymes involved in N metabolism, and changes that reduce the photosynthesis rate also reduce photosynthetic N use efficiency. In this review, we wish to highlight and discuss how/whether light intensity can improve photosynthesis in maize during nitrogen limitation. We described the general regulation of changes in the main photosynthetic and nitrogen metabolism enzymes, their quantity and localization, thylakoid protein abundance, intracellular transport of organic acids as well as specific features connected with C4 photosynthesis, and addressed the major open questions related to N metabolism and effects of light on photosynthesis in C4 plants.

Photosynthesis of the Cyanidioschyzon merolae cells in blue, red, and white light.

Photosynthesis and respiration rates, pigment contents, CO2 compensation point, and carbonic anhydrase activity in Cyanidioschizon merolae cultivated in blue, red, and white light were measured. At the same light quality as during the growth, the photosynthesis of cells in blue light was significantly lowered, while under red light only slightly decreased as compared with white control. In white light, the quality of light during growth had no effect on the rate of photosynthesis at low O2 and high CO2 concentration, whereas their atmospheric level caused only slight decrease. Blue light reduced markedly photosynthesis rate of cells grown in white and red light, whereas the effect of red light was not so great. Only cells grown in the blue light showed increased respiration rate following the period of both the darkness and illumination. Cells grown in red light had the greatest amount of chlorophyll a, zeaxanthin, and ß-carotene, while those in blue light had more phycocyanin. The dependence on O2 concentration of the CO2 compensation point and the rate of photosynthesis indicate that this alga possessed photorespiration. Differences in the rate of photosynthesis at different light qualities are discussed in relation to the content of pigments and transferred light energy together with the possible influence of related processes. Our data showed that blue and red light regulate photosynthesis in C. merolae for adjusting its metabolism to unfavorable for photosynthesis light conditions.

Effect of light on the rearrangements of PSI super-and megacomplexes in the non-appressed thylakoid domains of maize mesophyll chloroplasts.

We demonstrated the existence of PSI-LHCI-LHCII-Lhcb4 supercomplexes and PSI-LHCI-PSII-LHCII megacomplexes in the stroma lamellae and grana margins of maize mesophyll chloroplasts; these complexes consist of different LHCII trimers and monomer antenna proteins per PSI photocentre. These complexes are formed in both low (LL) and high (HL) light growth conditions, but with different contents. We attempted to identify the components and structure of these complexes in maize chloroplasts isolated from the leaves of low and high light-grown plants after darkness and transition to far red (FR) light of high intensity. Exposition of plants from high and low light growth condition on FR light induces different rearrangements in the composition of super- and megacomplexes. During FR light exposure, in plants from LL, the PSI-LHCI-LHCII-Lhcb4 supercomplex dissociates into free LHCII-Lhcb4 and PSI-LHCI complexes, and these complexes associate with the PSII monomer. This process occurs differently in plants from HL. Exposition to FR light causes dissociation of both PSI-LHCI-LHCII-Lhcb4 supercomplexes and PSI-PSII megacomplexes. These results suggest a different function of super- and megacomplex organization than the classic state transitions model, which assumes that the movement of LHCII trimers in the thylakoid membraneis considered as a mechanism for balancing light absorption between the two photosystems in light stress. The behavior of the complexes described in this article does not seem to be well explained by this model, i.e., it does not seem likely that the primary purpose of these megacomplexes dynamics is to balance excitation pressure. Rather, as stated in this article, it seems to indicate a role of these complexes for PSI in excitation quenching and for PSII in turnover.

Deletion of psbQ' gene in Cyanidioschyzon merolae reveals the function of extrinsic PsbQ' in PSII.

KEY MESSAGE: We have successfully produced single-cell colonies of C. merolae mutants, lacking the PsbQ' subunit in its PSII complex by application of DTA-aided mutant selection. We have investigated the physiological changes in PSII function and structure and proposed a tentative explanation of the function of PsbQ' subunit in the PSII complex. We have improved the selectivity of the Cyanidioschyzon merolae nuclear transformation method by the introduction of diphtheria toxin genes into the transformation vector as an auxiliary selectable marker. The revised method allowed us to obtained single-cell colonies of C. merolae, lacking the gene of the PsbQ' extrinsic protein. The efficiency of gene replacement was extraordinarily high, allowing for a complete deletion of the gene of interest, without undesirable illegitimate integration events. We have confirmed the absence of PsbQ' protein at genetic and protein level. We have characterized the physiology of mutant cells and isolated PSII protein complex and concluded that PsbQ' is involved in nuclear regulation of PSII activity, by influencing several parameters of PSII function. Among these: oxygen evolving activity, partial dissociation of PsbV, regulation of dimerization, downsizing of phycobilisomes rods and regulation of zeaxanthin abundance. The adaptation of cellular physiology appeared to favorite upregulation of PSII and concurrent downregulation of PSI, resulting in an imbalance of energy distribution, decrease of photosynthesis and inhibition of cell proliferation.

Transformation of the Cyanidioschyzon merolae chloroplast genome: prospects for understanding chloroplast function in extreme environments.

KEY MESSAGE: We have successfully transformed an exthemophilic red alga with the chloramphenicol acetyltransferase gene, rendering this organism insensitive to its toxicity. Our work paves the way to further work with this new modelorganism. Here we report the first successful attempt to achieve a stable, under selectable pressure, chloroplast transformation in Cyanidioschizon merolae-an extremophilic red alga of increasing importance as a new model organism. The following protocol takes advantage of a double homologous recombination phenomenon in the chloroplast, allowing to introduce an exogenous, selectable gene. For that purpose, we decided to use chloramphenicol acetyltransferase (CAT), as chloroplasts are particularly vulnerable to chloramphenicol lethal effects (Zienkiewicz et al. in Protoplasma, 2015, doi: 10.1007/s00709-015-0936-9 ). We adjusted two methods of DNA delivery: the PEG-mediated delivery and the biolistic bombardment based delivery, either of these methods work sufficiently with noticeable preference to the former. Application of a codon-optimized sequence of the cat gene and a single colony selection yielded C. merolae strains, capable of resisting up to 400 µg/mL of chloramphenicol. Our method opens new possibilities in production of site-directed mutants, recombinant proteins and exogenous protein overexpression in C. merolae-a new model organism.

Chloramphenicol acetyltransferase-a new selectable marker in stable nuclear transformation of the red alga Cyanidioschyzon merolae.

In this study, we have shown the applicability of chloramphenicol acetyltransferase as a new and convenient selectable marker for stable nuclear transformation as well as potential chloroplast transformation of Cyanidioschyzon merolae-a new model organism, which offers unique opportunities for studding the mitochondrial and plastid physiology as well as various evolutionary, structural, and functional features of the photosynthetic apparatus.

Differences in photosynthetic responses of NADP-ME type C4 species to high light.

MAIN CONCLUSION: Three species chosen as representatives of NADP-ME C4 subtype exhibit different sensitivity toward photoinhibition, and great photochemical differences were found to exist between the species. These characteristics might be due to the imbalance in the excitation energy between the photosystems present in M and BS cells, and also due to that between species caused by the penetration of light inside the leaves. Such regulation in the distribution of light intensity between M and BS cells shows that co-operation between both the metabolic systems determines effective photosynthesis and reduces the harmful effects of high light on the degradation of PSII through the production of reactive oxygen species (ROS). We have investigated several physiological parameters of NADP-ME-type C4 species (e.g., Zea mays, Echinochloa crus-galli, and Digitaria sanguinalis) grown under moderate light intensity (200 µmol photons m-2 s-1) and, subsequently, exposed to excess light intensity (HL, 1600 µmol photons m-2 s-1). Our main interest was to understand why these species, grown under identical conditions, differ in their responses toward high light, and what is the physiological significance of these differences. Among the investigated species, Echinochloa crus-galli is best adapted to HL treatment. High resistance of the photosynthetic apparatus of E. crus-galli to HL was accompanied by an elevated level of phosphorylation of PSII proteins, and higher values of photochemical quenching, ATP/ADP ratio, activity of PSI and PSII complexes, as well as integrity of the thylakoid membranes. It was also shown that the non-radiative dissipation of energy in the studied plants was not dependent on carotenoid contents and, thus, other photoprotective mechanisms might have been engaged under HL stress conditions. The activity of the enzymes superoxide dismutase and ascorbate peroxidase as well as the content of malondialdehyde and H2O2 suggests that antioxidant defense is not responsible for the differences observed in the tolerance of NADP-ME species toward HL stress. We concluded that the chloroplasts of the examined NADP-ME species showed different sensitivity to short-term high light irradiance, suggesting a role of other factors excluding light factors, thus influencing the response of thylakoid proteins. We also observed that HL affects the mesophyll chloroplasts first hand and, subsequently, the bundle sheath chloroplasts.

The short-term response of Arabidopsis thaliana (C3) and Zea mays (C4) chloroplasts to red and far red light.

MAIN CONCLUSION: Light quality has various effects on photochemistry and protein phosphorylation in Zea mays and Arabidopsis thaliana thylakoids due to different degrees of light penetration across leaves and redox status in chloroplasts. The effect of the spectral quality of light (red, R and far red, FR) on the function of thylakoid proteins in Zea mays and Arabidopsis thaliana was investigated. It was concluded that red light stimulates PSII activity in A. thaliana thylakoids and in maize bundle sheath (BS) thylakoids, but not in mesophyll (M) thylakoids. The light quality did not change PSI activity in M thylakoids of maize. FR used after a white light period increased PSI activity significantly in maize BS and only slightly in A. thaliana thylakoids. As shown by blue native (BN)-PAGE followed by SDS-PAGE, proteins were differently phosphorylated in the thylakoids, indicating their different functions. FR light increased dephosphorylation of LHCII proteins in A. thaliana thylakoids, whereas in maize, dephosphorylation did not occur at all. The rate of phosphorylation was higher in maize BS than in M thylakoids. D1 protein phosphorylation increased in maize and decreased in A. thaliana upon irradiation with both R and growth light (white light, W). Light variations did not change the level of proteins in thylakoids. Our data strongly suggest that response to light quality is a species-dependent phenomenon. We concluded that the maize chloroplasts were differently stimulated, probably due to different degrees of light penetration across the leaf and thereby the redox status in the chloroplasts. These acclimation changes induced by light quality are important in the regulation of chloroplast membrane flexibility and thus its function.

Metabolic responses to lead of metallicolous and nonmetallicolous populations of Armeria maritima.

Metabolic responses to Pb(NO3)2 (Pb) ions of excised leaves of metallicolous (MPs) and nonmetallicolous populations (NMPs) of Armeria maritima, cultivated on normal soil, were examined. Detached leaves were exposure to Pb for 24 h, and metabolic parameters were investigated. Pb decreased the photosynthesis (Pn) rate and photosystem II (PSII) activity, whereas the photochemical efficiency of PSII remained unchanged. In both populations, Pb ions caused increase in O2 uptake of dark-treated leaves; however, respiration after Pn was not affected. Pb increased superoxide dismutase activity in MP leaves and malondialdehyde content in NMP leaves. Other metabolites after Pb treatment were increased (proline or H2O2) or decreased (malate). Ascorbate peroxidase activity and adenosine triphosphate content decreased more in MP than in NMP leaves. Our results indicate that A. maritima is well adapted to heavy metal-contaminated soils, and we discuss potential causes of the stimulation of respiration by Pb ions and possible reasons for the tolerance to oxidative stress of plants growing in a metal-rich habitat.

Light intensity and quality stimulated Deg1-dependent cleavage of PSII components in the chloroplasts of maize.

Recent studies have revealed that photo damages inducing high white light illumination of C3-type plant Arabidopsis thaliana promotes Deg1-mediated degradation of not only photosystem II core proteins D1/D2 but also minor LHCII proteins CP26, CP29 and PSII-associated PsbS protein. Using biochemical and immunological approaches we show that that the substrate pool of the heterologously expressed Deg1 ortholog protease from C4-type plant Zea mays is very similar to that of the A. thaliana in both mesophyll and bundle sheath chloroplasts. The Deg1-mediated degradation of photosystem II components has been observed after high light and red light treatment of maize leaves, while far red light did not induce Deg1-mediated degradation. Moreover, two isoforms of the Deg1 protease have been identified. Their genes are localized in chromosomes 6 and 8. The Pull-Down assay indicated that both proteins were able to bind the same set of chloroplast proteins, nevertheless in vitro digestion of Z. mays thylakoids in the form of inside-out vesicles has raveled that only Deg1 found in chromosome 8 exhibited proteolytic activity. Interestingly, the relative amount of Deg1 proteases in Z. mays bundle sheath chloroplasts (BS) is significantly higher than in mesophyll chloroplasts (M) in spite of lower content of PSII (∼20%) in BS.

[C4 type photosynthesis]. / Fotosynteza typu C4.

C4 photosynthesis includes several anatomical and biochemical modifications that allow plants to concentrate CO2 at the site of Rubisco. The photorespiratory pathway is repressed in C4 plants, since the rates of photosynthesis and biomass production are increased. This is an adaptation to high light intensities, high temperatures and dryness. C4 plants contain two distinct types of photosynthetic cells, mesophyll and bundle sheath. The processes of assimilation and reduction of CO2 are separated spatiality and catayzed by two different enzymes. Only the bundle sheath chloroplasts perform the reactions of the Calvin-Benson cycle with the help of the Rubisco enzyme present exclusively in this cell type. The primary CO2 fixation occurs in mesophyll cells through the action of the phosphoenolpyruvate carboxylase. The light-dependent reactions of the photosynthesis occur exclusively in the latter cell type. These differences in photochemistry lead to distinct redox profiles in both types of cells. C4 plants are divided into three biochemical subtypes on the basis of differences in the mechanisms of decarboxylation of the C4 acids. C4 plants will provide the main source of food for humans and animals in the nearest decade.

Differential phosphorylation of thylakoid proteins in mesophyll and bundle sheath chloroplasts from maize plants grown under low or high light.

In C4 plants, such as maize, the photosynthetic apparatus is partitioned over two cell types called mesophyll (M) and bundle sheath (BS), which have different structure and specialization of the photosynthetic thylakoid membranes. We characterized protein phosphorylation in thylakoids of the two cell types from maize grown under either low or high light. Western blotting with phosphothreonine antibodies and ProQ phosphostaining detected light-dependent changes in the protein phosphorylation patterns. LC-MS/MS with alternating CID and electron transfer dissociation sequencing of peptide ions mapped 15 protein phosphorylation sites. Phosphorylated D2, CP29, CP26, Lhcb2 proteins, and ATPsynthase were found only in M membranes. A previously unknown phosphorylation site was mapped in phosphoenolpyruvate carboxykinase from the BS cells. Phosphorylation stoichiometry was calculated from the ratios of normalized ion currents for phosphorylated to nonphosphorylated peptide pairs from the D1, D2, CP43, and PbsH proteins of photosystem II (PSII). Every PSII in M thylakoids contained on average 1.5 ± 0.1 or 2.3 ± 0.2 phosphoryl groups in plants grown under either low or high light, while in BS membranes the corresponding numbers were 0.25 ± 0.1 or 0.7 ± 0.2, respectively. It is suggested that the phosphorylation level, as well as turnover of PSII depend on the structure of thylakoids.

High light stimulates Deg1-dependent cleavage of the minor LHCII antenna proteins CP26 and CP29 and the PsbS protein in Arabidopsis thaliana.

The chloroplast Deg1 protein performs proteolytic cleavage of the photodamaged D1 protein of the photosystem II (PSII) reaction center, PSII extrinsic subunit PsbO and the soluble electron carrier plastocyanin. Using biochemical, immunological and mass spectrometry approaches we showed that the heterogeneously expressed Deg1 protease from Arabidopsis thaliana can be responsible for the degradation of the monomeric light-harvesting complex antenna subunits of PSII (LHCII), CP26 and CP29, as well as PSII-associated PsbS (CP22/NPQ4) protein. The results may indicate that cytochrome b (6) protein and two previously unknown thylakoid proteins, Ptac16 and an 18.3-kDa protein, may be the substrates for Deg1. The interaction of Deg1 with the PsbS protein and the minor LHCII subunits implies its involvement in the regulation of both excess energy dissipation and state transition adaptation processes.

Phosphorylation of PSII proteins in maize thylakoids in the presence of Pb ions.

Lead is potentially toxic to all organisms including plants. Many physiological studies suggest that plants have developed various mechanisms to contend with heavy metals, however the molecular mechanisms remain unclear. We studied maize plants in which lead was introduced into detached leaves through the transpiration stream. The photochemical efficiency of PSII, measured as an Fv/Fm ratio, in the maize leaves treated with Pb was only 10% lower than in control leaves. The PSII activity was not affected by Pb ions in mesophyll thylakoids, whereas in bundle sheath it was reduced. Protein phosphorylation in mesophyll and bundle sheath thylakoids was analyzed using mass spectrometry and protein blotting before and after lead treatment. Both methods clearly demonstrated increase in phosphorylation of the PSII proteins upon treatment with Pb(2+), however, the extent of D1, D2 and CP43 phosphorylation in the mesophyll chloroplasts was clearly higher than in bundle sheath cells. We found that in the presence of Pb ions there was no detectable dephosphorylation of the strongly phosphorylated D1 and PsbH proteins of PSII complex in darkness or under far red light. These results suggest that Pb(2+) stimulates phosphorylation of PSII core proteins, which can affect stability of the PSII complexes and the rate of D1 protein degradation. Increased phosphorylation of the PSII core proteins induced by Pb ions may be a crucial protection mechanism stabilizing optimal composition of the PSII complexes under metal stress conditions. Our results show that acclimation to Pb ions was achieved in both types of maize chloroplasts in the same way. However, these processes are obviously more complex because of different metabolic status in mesophyll and bundle sheath chloroplasts.

Isolation of cytochrome b6f complex from grana and stroma membranes from spinach chloroplasts.

The cytochrome b6f complex is located in the appressed granal membranes and nonappressed stroma thylakoids. The procedure presents isolation of the complex from both types of thylakoids by washing with NaBr, detergent treatment, ammonium sulfate fractionation, and sucrose gradient centrifugation. Optimal concentration of the detergent is lower for grana than for stroma vesicles. The cytochrome b6f complex from stroma lamellae locates at a higher density in the sucrose gradient than the granal complex. Electrophoretic analyses indicate that both complexes are dimeric and contain four large subunits and at least three small subunits with masses below 4 kDa. Plastocyanin and 15 kDa protein are also identified in the complexes but in variable amounts.

Mechanical isolation of bundle sheath cell strands and thylakoids from leaves of C4 grasses.

Bundle sheath (BS) strand cells and BS thylakoids from C4 plants represent a unique system for various studies using a combination of physiological, biochemical, and molecular approaches. We have developed procedures for mechanical disruption of leaf tissues in order to isolate metabolically active bundle sheath strand cells and thylakoids practically free from cross-contamination coming from mesophyll cells. The procedures are described in detail together with useful practical suggestions. Using mechanical disruption we have shown the supramolecular organization of the dimeric LHCII-PSII in BS thylakoids of maize.

Differential turnover of the photosystem II reaction centre D1 protein in mesophyll and bundle sheath chloroplasts of maize.

Photoinhibition is caused by an imbalance between the rates of the damage and repair cycle of photosystem II D1 protein in thylakoid membranes. The PSII repair processes include (i) disassembly of damaged PSII-LHCII supercomplexes and PSII core dimers into monomers, (ii) migration of the PSII monomers to the stroma regions of thylakoid membranes, (iii) dephosphorylation of the CP43, D1 and D2 subunits, (iv) degradation of damaged D1 protein, and (v) co-translational insertion of the newly synthesized D1 polypeptide and reassembly of functional PSII complex. Here, we studied the D1 turnover cycle in maize mesophyll and bundle sheath chloroplasts using a protein synthesis inhibitor, lincomycin. In both types of maize chloroplasts, PSII was found as the PSII-LHCII supercomplex, dimer and monomer. The PSII core and the LHCII proteins were phosphorylated in both types of chloroplasts in a light-dependent manner. The rate constants for photoinhibition measured for lincomycin-treated leaves were comparable to those reported for C3 plants, suggesting that the kinetics of the PSII photodamage is similar in C3 and C4 species. During the photoinhibitory treatment the D1 protein was dephosphorylated in both types of chloroplasts but it was rapidly degraded only in the bundle sheath chloroplasts. In mesophyll chloroplasts, PSII monomers accumulated and little degradation of D1 protein was observed. We postulate that the low content of the Deg1 enzyme observed in mesophyll chloroplasts isolated from moderate light grown maize may retard the D1 repair processes in this type of plastids.

Structural organization of photosynthetic apparatus in agranal chloroplasts of maize.

We investigated the organization of photosystem II (PSII) in agranal bundle sheath thylakoids from a C(4) plant maize. Using blue native/SDS-PAGE and single particle analysis, we show for the first time that PSII in the bundle sheath (BS) chloroplasts exists in a dimeric form and forms light-harvesting complex II (LHCII).PSII supercomplexes. We also demonstrate that a similar set of photosynthetic membrane complexes exists in mesophyll and agranal BS chloroplasts, including intact LHCI.PSI supercomplexes, PSI monomers, PSII core dimers, PSII monomers devoid of CP43, LHCII trimers, LHCII monomers, ATP synthase, and cytochrome b(6)f complex. Fluorescence functional measurements clearly indicate that BS chloroplasts contain PSII complexes that are capable of performing charge separation and are efficiently sensitized by the associated LHCII. We identified a fraction of LHCII present within BS thylakoids that is weakly energetically coupled to the PSII reaction center; however, the majority of BS LHCII is shown to be tightly connected to PSII. Overall, we demonstrate that organization of the photosynthetic apparatus in BS agranal chloroplasts of a model C(4) plant is clearly distinct from that of the stroma lamellae of the C(3) plants. In particular, supramolecular organization of the dimeric LHCII.PSII in the BS thylakoids strongly suggests that PSII in the BS agranal membranes may donate electrons to PSI. We propose that the residual PSII activity may supply electrons to poise cyclic electron flow around PSI and prevent PSI overoxidation, which is essential for the CO(2) fixation in BS cells, and hence, may optimize ATP production within this compartment.

High light induced accumulation of two isoforms of the CF1 alpha-subunit in mesophyll and bundle sheath chloroplasts of C4 plants.

The effect of light irradiance on the amount of ATP synthase alpha-subunit in mesophyll (M) and bundle sheath (BS) chloroplasts of C(4) species such as maize (Zea mays L., type NADP-ME), millet (Panicum miliaceum, type NAD-ME) and guinea grass (Panicum maximum, type PEP-CK) was investigated in plants grown under high, moderate and low light intensities equal to 800, 350 and 50 micromol photons m(-2) s(-1), respectively. The results demonstrate that alpha-subunit of ATP synthase in both M and BS chloroplasts is altered by light intensity, but differently in the investigated species. Moreover, we identified two isoforms of the CF(1) alpha-subunit, called alpha and alpha. The CF(1) alpha-subunit was the major isoform and was present in all light conditions, whereas alpha was the minor isoform in low light. A strong increase in the level of the alpha-subunit in maize mesophyll and bundle sheath thylakoids was observed after 50 h of high light treatment. The alpha and alpha-subunits from investigated C(4) species displayed apparent molecular masses of 64 and 67 kDa, respectively, on SDS/PAGE. The presence of the alpha-subunit of ATPase was confirmed in isolated CF(1) complex, where it was recognized by antisera to the alpha-subunit. The N-terminal sequence of alpha-subunit is nearly identical to that of alpha. Our results indicate that both isoforms coexist in M and BS chloroplasts during plant growth at all irradiances. We suggest the existence in M and BS chloroplasts of C(4) plants of a mechanism(s) regulating the ATPase composition in response to light irradiance. Accumulation of the alpha isoform may have a protective role under high light stress against over protonation of the thylakoid lumen and photooxidative damage of PSII.