The regulation of photosynthetic electron transport during photomixotrophic growth in cyanobacteria remains incompletely understood. In this study, we characterized four wild-type strains (WT 1-4) of Synechocystis sp. PCC 6803 and observed distinct strain-specific differences in photosystem II (PSII) function under photomixotrophic conditions. Specifically, WT 1 and WT 2 exhibited near-complete inhibition of electron transfer from QA - to QB following approximately 3 days of glucose supplementation, possibly mediated by binding of the small PSII-associated protein, Psb28-2, and resulting in a metabolic shift toward photoheterotrophy. Observed electron transport blockage was associated with changes in the abundances of various photosynthetic proteins. However, the structural integrity of both Photosystems appeared to be largely preserved. Such stabilization may be driven by a transient downregulation of linear electron transport to prevent overreduction of the electron transport chain under photomixotrophy. In contrast, WT 3 and WT 4 maintained photomixotrophic growth throughout the experiment but exhibited slower growth rates than WT 1 and WT 2. Although glucose uptake was slower in WT 1 and WT 2, both strains accumulated more glycogen than WT 3 and WT 4, suggesting divergent regulation of carbon allocation and storage metabolism. Together, these findings highlight the capacity of cyanobacterial strains to deploy distinct metabolic strategies to optimize photosynthetic function, carbon assimilation, and energy storage under photomixotrophic conditions.

The D2 protein of Photosystem II has five transmembrane helices (A-E). An extended loop, connecting helices D and E, contributes to the binding environments of the primary quinone electron acceptor Q(A), and that of the bicarbonate bound to the non-heme iron between Q(A) and the secondary quinone electron acceptor Q(B). The residues from Ala240 to Thr243 are within a conserved sequence ((240)AEETYSMVTAN(250)) that contributes to the stabilization of both Q(A) and bicarbonate. We have created the A240D, E241A, E242A, E242D and T243A mutants to study the role of these residues. The mutations in the A240D and E241A strains had little impact on PS II performance, except addition of formate altered chlorophyll a fluorescence decay in the E241A mutant following a single-turnover actinic flash. Measurements of variable chlorophyll a fluorescence and thermoluminescence showed the E242A and E242D mutants had impaired acceptor side electron transport consistent with a reduced redox gap between Q(A) and Q(B). In addition, the T243A mutant exhibited a heightened susceptibility to photodamage. These data show that mutations introduced between (241)Glu-Thr(243) of D2 impair PS II activity but are less detrimental than mutations between the corresponding (243)Glu-Thr(245) residues of D1 in the vicinity of the Q(B)-binding site.

Cyanobacteria are important model organisms for studying the process of photosynthesis and the effects of environmental stress factors. This study aimed to identify the inhibitory sites of NaCl in the whole photosynthetic electron transport in Synechocystis sp. PCC 6803 WT cells by using multiple biophysical tools. Exposure of cells to various NaCl concentrations (200 mM to 1 M) revealed the inhibition of Photosystem II (PSII) activity at the water oxidizing complex and between the QA and QB electron acceptors. In contrast to the inhibition of PSII, electron flow through Photosystem I (PSI) was accelerated, indicating enhanced cyclic electron flow. The oxygen-evolving capacity of the cells was inhibited to a larger extent when only CO2 was the final electron acceptor in the Calvin-Benson-Bassham (CBB) cycle than in the presence of the PSII electron acceptor DMBQ, suggesting important NaCl inhibitory site(s) downstream of PSI. Measurements of NADPH kinetics revealed NaCl-induced inhibition of light-induced production of NADPH as well as retardation of NADPH consumption both in the light and in the initial dark period after switching off the light. Chlorophyll fluorescence kinetics, measured in parallel with NADPH fluorescence, showed the enhancement of post-illumination fluorescence rise up to 500 mM NaCl, which was however inhibited at higher NaCl concentrations. Our results show, for the first time, that NaCl inhibits the activity of the CBB cycle at least at two different sites, and confirm earlier results about the NaCl-induced inhibition of the PSII donor and acceptor side and the enhancement of electron flow through PSI.

In Photosystem II (PS II) the D2 and D1 proteins provide binding sites for the primary (QA) and secondary (QB) plastoquinone electron acceptors, respectively. A non-heme iron is located between QA and QB that is coordinated by a bicarbonate ligand and two His residues from D1 (D1-His215 and D1-His272) and two His residues from D2 (D2-His214 and D2-His268). The symmetry of the quinone-Fe-acceptor complex extends to D1-Arg269, which has hydrogen bonds to D1-His272, and D2-Arg265 which has hydrogen bonds to D2-His268. We have examined the role of D2-Arg265 by creating the R265A and R265D mutants in the cyanobacterium Synechocystis sp. PCC 6803. Both mutants exhibited normal photoautotrophic growth, but showed a reduction in oxygen evolution in the presence of the PS II-specific electron acceptor 2,5-dimethyl-1,4-benzoquinone (DMBQ). Chlorophyll a fluorescence induction and decay kinetics were also inhibited in the presence of DMBQ and, in the presence of the native quinone, revealed slowed QA- to QB electron transfer, together with impaired exchange between the QB-binding site and the plastoquinone pool. Addition of formate further inhibited electron transfer, consistent with weakened bicarbonate binding in the mutants, and thermoluminescence measurements revealed a decreased redox gap between QA and QB. Additionally, both mutants displayed heightened sensitivity to high light. These findings demonstrate that D2-Arg265 is important for stability of the acceptor side, bicarbonate-dependent electron transfer, and an optimal QB-binding site. All of our results are also consistent with the architecture of the quinone-Fe-bicarbonate complex supporting photoprotection and regulatory roles that are unique to oxygenic photosynthesis.

Coral health depends on intricate metabolic interactions between the coral host and its symbiotic algae, Symbiodiniaceae. While nutrient exchange is well established, electron-level interactions have remained unexplored. Here, we provide evidence for extracellular electron transport (EET) within coral-algal symbiosis, supported by variable chlorophyll (Chl) fluorescence and ferricyanide reduction measurements. We observed a slow wave in the relaxation of flash-induced Chl fluorescence kinetics under microaerobic conditions in both isolated Fugacium kawagutii (CS156) cells and intact corals, reflecting redox dynamics of the primary quinone electron acceptor (QA) in the photosynthetic electron transport chain. The addition of the extracellular electron acceptor ferricyanide decreased the wave amplitude and QA reduction while being reduced to ferrocyanide, demonstrating EET from the symbiont to extracellular acceptors. Slower Chl fluorescence rise kinetics under continuous illumination in intact corals compared to isolated symbionts indicate that electrons may also flow from symbionts to the host. Under low oxygen conditions, QA was gradually reduced in corals in darkness but not in isolated symbiont cultures, suggesting electron transfer from host to symbiont. Together, these results indicate bidirectional extracellular electron exchange between symbiotic partners, pointing to a previously unrecognized mechanism for redox balancing in coral-algal symbiosis. This pathway likely contributes to metabolic resilience and the maintenance of coral health under fluctuating environmental conditions. ### Competing Interest Statement The authors have declared no competing interest. National Research, Development and Innovation Office (NKFIH), Hungary, FK 146298, PD 146655

The chemical properties of the primary (QA) and secondary (QB) plastoquinone electron acceptors of Photosystem II (PS II) depend on their protein environments. The DE loop of the D2 protein (residues 222-262) contributes to the QA-binding site while the DE loop of the D1 protein (residues 233-266) contributes to the QB-binding environment. The roles of the invariant D2-Met246 and D2-Asn250 residues in the vicinity of the QA-binding site have been investigated in the cyanobacterium Synechocystis sp. PCC 6803 using mutants targeting both residues. The M246F strain was phenotypically similar to control cells; however, the M246A, N250A, and N250H strains had slowed photoautotrophic growth and were sensitive to high light and the addition of formate. In addition, the M246K and N250N strains were unable to assemble PS II. Chlorophyll a fluorescence measurements indicated electron transfer between QA and QB was modified in the M246A, N250A, and N250H strains, and the exchange of plastoquinol between the QB-binding site and the plastoquinone pool in the thylakoid membrane was impaired. Modified electron transfer in these mutants in the presence or absence of formate was restored by the addition of bicarbonate. In addition, thermoluminescence measurements showed a down shift in the redox midpoint potential of the QA/QA- couple in the N250A and N250H strains. These results demonstrate that Met246 and Asn250 play indispensable roles in the quinone-iron-acceptor complex, influencing both QA binding and the binding of the bicarbonate ligand to the non-heme iron that is located between QA and QB.

Singlet oxygen (O-1(2)) is an important reactive oxygen species whose formation by the type-II, light-dependent, photodynamic reaction is inevitable during photosynthetic processes. In the last decades, the recognition that O-1(2) is not only a damaging agent, but can also affect gene expression and participates in signal transduction pathways has received increasing attention. However, contrary to several other taxa, O-1(2)-responsive genes have not been identified in the important cyanobacterial model organism Synechocystis PCC 6803. By using global transcript analysis we have identified a large set of Synechocystis genes, whose transcript levels were either enhanced or repressed in the presence of O-1(2). Characteristic O-1(2) responses were observed in several light-inducible genes of Synechocystis, especially in the hli (or scp) family encoding HLIP/SCP proteins involved in photoprotection. Other important O-1(2)-induced genes include components of the Photosystem II repair machinery (psbA2 and ftsH2, ftsH3), iron homeostasis genes isiA and idiA, the group 2 sigma factor sigD, some components of the transcriptomes induced by salt-, hyperosmotic and cold-stress, as well as several genes of unknown function. The most pronounced O-1(2)-induced upregulation was observed for the hliB and the co-transcribed lilA genes, whose deletion induced enhanced sensitivity against O-1(2)-mediated light damage. A bioreporter Synechocystis strain was created by fusing the hliB promoter to the bacterial luciferase (lux), which showed its utility for continuous monitoring of O-1(2) concentrations inside the cell.

Photosystem II (PS II) is responsible for light-driven water splitting in oxygenic photosynthesis. The Psb27 protein, an assembly factor required for biogenesis of PS II, is found associated with hydrophilic regions of the CP43 core antenna protein in the thylakoid lumen. CP43 and the D1 reaction center protein provide ligands for the Mn4CaO5 oxygen-evolving complex (OEC). Release of Psb27 coincides with conformational changes that enable successful light-driven assembly of the OEC. This stage in biogenesis also requires changes to allow electron transfer between plastoquinone electron acceptors on the opposite side of the membrane. We have introduced charge-swap mutations to target the binding of Psb27 to CP43 during assembly. Here, we show that perturbation of the Psb27-CP43 interaction results in elevated fluorescence, indicating enhanced energy transfer to PS II in fully assembled complexes. In a Psb27:Arg78 to Glu mutant, D1:His252 spontaneously mutated to Gln. D1:His252 is in the DE loop that contributes to quinone binding and protonation. Mutations targeting D1:His252 produced mutants with elevated PS II-specific fluorescence that exceeded that observed in our Psb27 mutants and this was attenuated when the Psb27 charge-swap mutations were introduced into H252Q cells. Perturbation of Psb27 binding to CP43 therefore modified structural changes on the opposite side of the membrane resulting from mutation of D1:His252. The peripheral phycobilisome antenna is lost during thylakoid isolation and thylakoids from our mutants did not display the increased PS II-specific fluorescence. Hence Psb27 binding to CP43 during photoassembly of the OEC can modify phycobilisome-dependent energy transfer into PS II.

A major constraint to global food production is the selection of crops that are better adapted to resource limited environments and soil conditions. In the past decades, the research in this field has focused on the study of the effect of abiotic stress on plants and elucidating signalling pathways that govern the appropriate and coordinated response to abiotic stress. This study identified some tolerant tomato landraces to salt and drought stress from saline zones ofBihor County, in North-Western Romania. Phenotypic characterization of collected landraces provided information regarding drought and salt stress resistance compared with the 'Marmande' cultivar. Root density, leaf area and the reaction under different concentrations of salt and different water content were compared to conclude which landraces are more suitable for future genotypic studies in order to use them in breeding programs. The aim is to obtain genotypes with increased drought and salt stress resistance to improve crop yield and quality in saline environments. This study highlights the potential of local tomato landraces from Bihor County as valuable genetic resources for developing resilient varieties that can thrive in challenging environmental conditions.

In Photosystem II electrons from water splitting pass through a primary quinone electron acceptor (QA) to the secondary plastoquinone (QB). The D2 protein forms the QA-binding site and the D1 protein forms the Q(B)-binding site. A non-heme iron sits between QA and QB resulting in a quinone-Fe-acceptor complex that must be activated before assembly of the oxygen-evolving complex can occur. An extended loop (residues 223-266) between the fourth (helix D) and fifth (helix E) helices of the D1 protein activates forward electron transfer via a conformational change that stabilizes a bidentate bicarbonate ligand to the non-heme iron while simultaneously stabilizing the binding of QB. We show that positioning of D1:Phe265 to provide a hydrogen bond to the distal oxygen of QB is required for forward electron transfer. In addition, mutations targeting D1:Phe265, resulted in a 50 mV decrease in the QB/QB- midpoint potential.

The dinoflagellate algae, Symbiodiniaceae, are significant symbiotic partners of corals due to their photosynthetic capacity. The photosynthetic processes of the microalgae consist of linear electron transport, which provides the energetic balance of ATP and NADPH production for CO2 fixation, and alternative electron transport pathways, including cyclic electron flow, which ensures the elevated ATP requirements under stress conditions. Flash-induced chlorophyll fluorescence relaxation is a non-invasive tool to assess the various electron transport pathways. A special case of fluorescence relaxation, the so-called wave phenomenon, was found to be associated with the activity of NAD(P)H dehydrogenase (NDH) in microalgae. We showed previously that the wave phenomenon existed in Symbiodiniaceae under acute heat stress and microaerobic conditions, however, the electron transport processes related to the wave phenomenon remained unknown. In this work, using various inhibitors, we show that (i) the linear electron transport has a crucial role in the formation of the wave, (ii) the inhibition of the donor side of Photosystem II did not induce the wave, whereas inhibition of the Calvin–Benson cycle accelerated it, (iii) the wave phenomenon was related to the operation of type II NDH (NDH-2). We therefore propose that the wave phenomenon is an important marker of the regulation of electron transport in Symbiodiniaceae.

Summary Singlet oxygen ( 1 O 2 ) is an important reactive oxygen species whose formation by the type-II, light-dependent, photodynamic reaction is inevitable during photosynthetic processes. In the last decades, the recognition that 1 O 2 is not only a damaging agent, but can also affect gene expression and participates in signal transduction pathways has received increasing attention. However, contrary to several other taxa, 1 O 2 -specific genes have not been identified in the important cyanobacterial model organism Synechocystis PCC 6803. By using global transcript analysis we have identified a large set of 1 O 2 -responsive Synechocystis genes, whose transcript levels were either enhanced or repressed in the presence of 1 O 2 . Characteristic 1 O 2 responses were observed in several light-inducible genes of Synechocystis , especially in the hli (or scp ) family encoding HLIP/SCP proteins involved in photoprotection. Other important 1 O 2 -induced genes include components of the Photosystem II repair machinery ( psbA2 and ftsH2 , ftsH3 ), iron homeostasis genes isiA and idiA , the Group-2 sigma factor sig D, some components of the transcriptomes induced by salt-, hyperosmotic and cold-stress, as well as several genes of unknown function. One of the most pronounced 1 O 2 -induced upregulation was observed for the hliB gene, whose deletion provided tolerance against 1 O 2 -mediated light damage. A bioreporter Synechocystis strain was created by fusing the hliB promoter to the bacterial luciferase (lux), which showed its utility for continuous monitoring of 1 O 2 concentrations inside the cell.

Drought stress is one of the most limiting abiotic stresses for plant growth and development. Potato (Solanum tuberosum L.), due to its shallow root system, is considered sensitive to drought. In potato breeding, the wild Solanum species may represent a good resource for disease and abiotic stress resistance genes, but their transfer is limited by sexual incompatibilities. Somatic hybrids (SH) between potato and the wild species Solanum bulbocastanum, sexually incompatible with potato, proved to be late-blight-resistant in laboratory and field assays. The aim of this study was to screen a series of somatic hybrids and derived backcrosses for drought stress tolerance. In vitro stress exposure (with 5% and 15% PEG 6000) allowed the selection of several tolerant genotypes in a short time. The eleven selected genotypes were tested by using a semi-automated plant phenotyping platform at the Biological Research Centre in Szeged, Hungary, where the plants’ biomass accumulation and photosynthesis under long-term drought conditions were monitored. The findings of this study affirm that the somatic hybrids between potato and S. bulbocastanum, along with their backcrosses, constitute valuable pre-breeding material. This is attributed to their possession of both late blight resistance and drought stress tolerance.

Haematococcus lacustris is an important species of green algae because it produces the high-value carotenoid astaxanthin. Astaxanthin production is enhanced by various stress conditions causing the transformation of green vegetative cells to red cells with high amounts of astaxanthin, which plays various photoprotective and antioxidant roles. Although intensive research has been conducted to reveal the regulation of astaxanthin production, the photosynthetic capacity of the various cell forms is unresolved at the single-cell level. In this work, we characterized the photosynthetic and morphological changes of Haematococcus cells, using a combination of microfluidic tools and microscopic chlorophyll fluorescence imaging. We found marked but reversible changes in the variable chlorophyll fluorescence signatures upon the transformation of green cells to red cells, and we propose that the photosynthetic activity as revealed by single-cell chlorophyll fluorescence kinetics serves as a useful phenotypic marker of the different cell forms of Haematococcus.

Flash-induced chlorophyll fluorescence relaxation is a powerful tool to monitor the reoxidation reactions of the reduced primary quinone acceptor, QA- by QB and the plastoquinone (PQ) pool, as well as the charge recombination reactions between the donor and acceptor side components of Photosystem II (PSII). Under certain conditions, when the PQ pool is highly reduced (e.g. in microaerobic conditions), a wave phenomenon appears in the fluorescence relaxation kinetics, which reflects the transient reoxidation and re-reduction of QA- by various electron transfer processes, which in cyanobacteria is mediated by NAD(P)H dehydrogenase (NDH-1). The wave phenomenon was also observed and assigned to the operation of type 2 NAD(P)H dehydrogenase (NDH-2) in the green alga Chlamydomonas reinhardtii under hydrogen-producing conditions, which required a long incubation of algae under sulphur deprivation (Krishna et al. J Exp Bot 70 (21):6321-6336, 2019). However, the conditions that induce the wave remained largely uncharacterized so far in microalgae. In this work, we investigated the wave phenomenon in Chlamydomonas reinhardtii under conditions that lead to a decrease of PSII activity by applying hydroxylamine treatment, which impacts the donor side of PSII in combination with a strongly reducing environment of the PQ pool (microaerobic conditions). A similar wave phenomenon could be induced by photoinhibitory conditions (illumination with strong light in the presence of the protein synthesis inhibitor lincomycin). These results indicate that the fluorescence wave phenomenon is activated in green algae when the PSII activity decreases relative to Photosystem I (PS I) activity and the PQ pool is strongly reduced. Therefore, the fluorescence wave could be used as a sensitive indicator of altered intersystem electron transfer processes, e.g. under stress conditions.

Pathogens are a major threat of plant-based production. Expanding restrictions for the use of classical pesticides is increasing the need of alternative applications to control plant diseases. Nanoparticles have recently received increasing research interest as a potential means to protect plants from adverse conditions including pathogen attack. To assess the beneficial potential of silver nanoparticles to protect plants against the bacterial pathogen Pseudomonas syringae, of which numerous economically relevant pathovars are known, we evaluated the effect of silver nanoparticle pre-treatment in the model pathosystem Arabidopsis thaliana–P. syringae. For this purpose, A. thaliana leaves were treated with different silver nanoparticle concentrations prior to P. syringae infection and visible alterations of the leaf tissue in relation to the individual and combined treatments were scored. While treatment with silver nanoparticles in the concentration range between 0.5 and 10 ppm suppressed P. syringae symptom development, concentrations above 5 ppm caused necroses and chloroses in a dose-dependent manner. This indicates that silver nanoparticles affect plant physiological processes related to cell and tissue integrity that are also associated with the development of infection symptoms caused by P. syringae. Therefore, silver nanoparticle treatments in a suitable concentration range support the maintenance of tissue integrity during pathogen infection in combination with their antimicrobial activity, thus preventing loss of biomass. This makes silver nanoparticles a promising tool for integrative crop protection strategies in commercial production.

Symbiodiniaceae is an important dinoflagellate family which lives in endosymbiosis with reef invertebrates, including coral polyps, making them central to the holobiont. With coral reefs currently under extreme threat from climate change, there is a pressing need to improve our understanding on the stress tolerance and stress avoidance mechanisms of Symbiodinium spp. Reactive oxygen species (ROS) such as singlet oxygen are central players in mediating various stress responses; however, the detection of ROS using specific dyes is still far from definitive in intact Symbiodinium cells due to the hindrance of uptake of certain fluorescent dyes because of the presence of the cell wall. Protoplast technology provides a promising platform for studying oxidative stress with the main advantage of removed cell wall, however the preparation of viable protoplasts remains a significant challenge. Previous studies have successfully applied cellulose-based protoplast preparation in Symbiodiniaceae; however, the protoplast formation and regeneration process was found to be suboptimal. Here, we present a microfluidics-based platform which allowed protoplast isolation from individually trapped Symbiodinium cells, by using a precisely adjusted flow of cell wall digestion enzymes (cellulase and macerozyme). Trapped single cells exhibited characteristic changes in their morphology, cessation of cell division and a slight decrease in photosynthetic activity during protoplast formation. Following digestion and transfer to regeneration medium, protoplasts remained photosynthetically active, regrew cell walls, regained motility, and entered exponential growth. Elevated flow rates in the microfluidic chambers resulted in somewhat faster protoplast formation; however, cell wall digestion at higher flow rates partially compromised photosynthetic activity. Physiologically competent protoplasts prepared from trapped cells in microfluidic chambers allowed for the first time the visualization of the intracellular localization of singlet oxygen (using Singlet Oxygen Sensor Green dye) in Symbiodiniaceae, potentially opening new avenues for studying oxidative stress.

Symbiodiniaceae live in endosymbiosis with corals. In the last few decades, mass bleaching events have occurred in the coral reefs, causing damage in the ecosystem and the associated species. Global temperature increase is affecting the algae, disturbing the whole symbiosis and leads to coral bleaching. However, the heat tolerance is strongly determined by the species (formerly genetic clades) harbored by the coral host. We assessed three different strains of Symbiodiniaceae family, i.e., Fugacium kawagutii (CS156), Symbiodinium tridacnidorum (2465), and Symbiodinium microadriaticum (2467), which display different heat tolerance under heat stress conditions. Flash-induced chlorophyll fluorescence relaxation is a useful tool to monitor various components of the photosynthetic electron transport chain and the redox reactions of plastoquinone pool. We observed the appearance of a wave phenomenon in the fluorescence relaxation by heating the strains in combination with microaerobic conditions. The characteristics of this fluorescence wave were found to be strain-specific and possibly related to the transient oxidation and re-reduction of the plastoquinone pool. The appearance of the wave phenomenon appears to be related to cyclic electron flow as well because it is accompanied with enhanced post-illumination chlorophyll fluorescence rise. These results will potentially reveal further details of the role of cyclic electron transport in Symbiodiniaceae and its relevance in heat stress tolerance.

The Pannonian Plain, as the most productive region of Southeast Europe, has a long tradition of agronomic production as well as agronomic research and plant breeding. Many research institutions from the agri-food sector of this region have a significant impact on agriculture. Their well-developed and fruitful breeding programmes resulted in productive crop varieties highly adapted to the specific regional environmental conditions. Rapid climatic changes that occurred during the last decades led to even more investigations of complex interactions between plants and their environments and the creation of climate-smart and resilient crops. Plant phenotyping is an essential part of botanical, biological, agronomic, physiological, biochemical, genetic, and other omics approaches. Phenotyping tools and applied methods differ among these disciplines, but all of them are used to evaluate and measure complex traits related to growth, yield, quality, and adaptation to different environmental stresses (biotic and abiotic). During almost a century-long period of plant breeding in the Pannonian region, plant phenotyping methods have changed, from simple measurements in the field to modern plant phenotyping and high-throughput non-invasive and digital technologies. In this review, we present a short historical background and the most recent developments in the field of plant phenotyping, as well as the results accomplished so far in Croatia, Hungary, and Serbia. Current status and perspectives for further simultaneous regional development and modernization of plant phenotyping are also discussed.

Two plastoquinone electron acceptors, QA and QB, are present in Photosystem II (PS II) with their binding sites formed by the D2 and D1 proteins, respectively. A hexacoordinate non-heme iron is bound between QA and QB by D2 and D1, each providing two histidine ligands, and a bicarbonate that is stabilized via hydrogen bonds with D2-Tyr244 and D1-Tyr246. Both tyrosines and bicarbonate are conserved in oxygenic photosynthetic organisms but absent from the corresponding quinone-iron electron acceptor complex of anoxygenic photosynthetic bacteria. We investigated the role of D2-Tyr244 by introducing mutations in the cyanobacterium Synechocystis sp. PCC 6803. Alanine, histidine, and phenylalanine substitutions were introduced creating the Y244A, Y244H, and Y244F mutants. Electron transfer between QA and QB was impaired, the back-reaction with the S2 state of the oxygen-evolving complex was modified, and PS II assembly was disrupted, with the Y244A strain being more affected than the Y244F and Y244H mutants. The strains were also highly susceptible to photodamage in the presence of PS II-specific electron acceptors. Thermoluminescence and chlorophyll a fluorescence decay measurements indicated that the redox potential of the QA/QA- couple became more positive in the Y244F and Y244H mutants, consistent with bicarbonate binding being impacted. The replacement of Tyr244 by alanine also led to an insertion of two amino acid repeats from Gln239 to Ala249 within the DE loop of D2, resulting in an inactive PS II complex that lacked PS II-specific variable fluorescence. The 66 bp insertion giving rise to the inserted amino acids therefore created an obligate photoheterotrophic mutant.