Ginga Shimakawa

The Calvin Cycle Inevitably Produces Sugar-Derived Reactive Carbonyl Methylglyoxal During Photosynthesis: A Potential Cause of Plant Diabetes

Sugar-derived reactive carbonyls (RCs), including methylglyoxal (MG), are aggressive by-products of oxidative stress known to impair the functions of multiple proteins. These advanced glycation end-products accumulate in patients with diabetes mellitus and cause major complications, including arteriosclerosis and cardiac insufficiency. In the glycolytic pathway, the equilibration reactions between dihydroxyacetone phosphate and glyceraldehyde 3-phosphate (GAP) have recently been shown to generate MG as a by-product. Because plants produce vast amounts of sugars and support the same reaction in the Calvin cycle, we hypothesized that MG also accumulates in chloroplasts. Incubating isolated chloroplasts with excess 3-phosphoglycerate (3-PGA) as the GAP precursor drove the equilibration reaction toward MG production. The rate of oxygen (O2) evolution was used as an index of 3-PGA-mediated photosynthesis. The 3-PGA- and time-dependent accumulation of MG in chloroplasts was confirmed by HPLC. In addition, MG production increased with an increase in light intensity. We also observed a positive linear relationship between the rates of MG production and O2 evolution (R = 0.88; P < 0.0001). These data provide evidence that MG is produced by the Calvin cycle and that sugar-derived RC production is inevitable during photosynthesis. Furthermore, we found that MG production is enhanced under high-CO2 conditions in illuminated wheat leaves.

FLAVODIIRON2 and FLAVODIIRON4 proteins mediate an oxygen-dependent alternative electron flow in Synechocystis sp. PCC 6803 under CO2-limited conditions.

Two iron-bound flavidoxin-domain proteins mediate an oxygen-dependent alternative electron flow in the cyanobacterium Synechocystis under CO2-limited conditions. This study aims to elucidate the molecular mechanism of an alternative electron flow (AEF) functioning under suppressed (CO2-limited) photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803. Photosynthetic linear electron flow, evaluated as the quantum yield of photosystem II [Y(II)], reaches a maximum shortly after the onset of actinic illumination. Thereafter, Y(II) transiently decreases concomitantly with a decrease in the photosynthetic oxygen evolution rate and then recovers to a rate that is close to the initial maximum. These results show that CO2 limitation suppresses photosynthesis and induces AEF. In contrast to the wild type, Synechocystis sp. PCC 6803 mutants deficient in the genes encoding FLAVODIIRON2 (FLV2) and FLV4 proteins show no recovery of Y(II) after prolonged illumination. However, Synechocystis sp. PCC 6803 mutants deficient in genes encoding proteins functioning in photorespiration show AEF activity similar to the wild type. In contrast to Synechocystis sp. PCC 6803, the cyanobacterium Synechococcus elongatus PCC 7942 has no FLV proteins with high homology to FLV2 and FLV4 in Synechocystis sp. PCC 6803. This lack of FLV2/4 may explain why AEF is not induced under CO2-limited photosynthesis in S. elongatus PCC 7942. As the glutathione S-transferase fusion protein overexpressed in Escherichia coli exhibits NADH-dependent oxygen reduction to water, we suggest that FLV2 and FLV4 mediate oxygen-dependent AEF in Synechocystis sp. PCC 6803 when electron acceptors such as CO2 are not available.

Flavodiiron 2 and 4 Proteins Mediate an O2-dependent Alternative Electron Flow in Synechocystis sp. PCC 6803 under CO2-limited Conditions

Two iron-bound flavidoxin-domain proteins mediate an oxygen-dependent alternative electron flow in the cyanobacterium Synechocystis under CO2-limited conditions. This study aims to elucidate the molecular mechanism of an alternative electron flow (AEF) functioning under suppressed (CO2-limited) photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803. Photosynthetic linear electron flow, evaluated as the quantum yield of photosystem II [Y(II)], reaches a maximum shortly after the onset of actinic illumination. Thereafter, Y(II) transiently decreases concomitantly with a decrease in the photosynthetic oxygen evolution rate and then recovers to a rate that is close to the initial maximum. These results show that CO2 limitation suppresses photosynthesis and induces AEF. In contrast to the wild type, Synechocystis sp. PCC 6803 mutants deficient in the genes encoding FLAVODIIRON2 (FLV2) and FLV4 proteins show no recovery of Y(II) after prolonged illumination. However, Synechocystis sp. PCC 6803 mutants deficient in genes encoding proteins functioning in photorespiration show AEF activity similar to the wild type. In contrast to Synechocystis sp. PCC 6803, the cyanobacterium Synechococcus elongatus PCC 7942 has no FLV proteins with high homology to FLV2 and FLV4 in Synechocystis sp. PCC 6803. This lack of FLV2/4 may explain why AEF is not induced under CO2-limited photosynthesis in S. elongatus PCC 7942. As the glutathione S-transferase fusion protein overexpressed in Escherichia coli exhibits NADH-dependent oxygen reduction to water, we suggest that FLV2 and FLV4 mediate oxygen-dependent AEF in Synechocystis sp. PCC 6803 when electron acceptors such as CO2 are not available.

Functional Analysis of the AKR4C Subfamily of Arabidopsis thaliana: Model Structures, Substrate Specificity, Acrolein Toxicity, and Responses to Light and [CO2]

In Arabidopsis thaliana, the aldo-keto reductase (AKR) family includes four enzymes (The AKR4C subfamily: AKR4C8, AKR4C9, AKR4C10, and AKR4C11). AKR4C8 and AKR4C9 might detoxify sugar-derived reactive carbonyls (RCs). We analyzed AKR4C10 and AKR4C11, and compared the enzymatic functions of the four enzymes. Modeling of protein structures based on the known structure of AKR4C9 found an (α/β)8-barrel motif in all four enzymes. Loop structures (A, B, and C) which determine substrate specificity, differed among the four. Both AKR4C10 and AKR4C11 reduced methylglyoxal. AKR4C10 reduced triose phosphates, dihydroxyacetone phosphate (DHAP), and glyceraldehydes 3-phosphate (GAP), the most efficiently of all the AKR4Cs. Acrolein, a lipid-derived RC, inactivated the four enzymes to different degrees. Expression of the AKR4C genes was induced under high-[CO2] and high light, when photosynthesis was enhanced and photosynthates accumulated in the cells. These results suggest that the AKR4C subfamily contributes to the detoxification of sugar-derived RCs in plants.

O2-dependent large electron flow functioned as an electron sink, replacing the steady-state electron flux in photosynthesis in the cyanobacterium Synechocystis sp. PCC 6803, but not in the cyanobacterium Synechococcus sp. PCC 7942

To determine whether alternative electron flow (AEF) can replace the photosynthetic electron flow in cyanobacteria, we used an open O2-electrode system to monitor O2-exchange over a long period. In air-grown Synechocystis sp. PCC 6803 (S. 6803(WT)), the quantum yield of PSII, Y(II), held even after photosynthesis was suppressed by CO2 shortage. The S. 6803 mutant, deficient in flavodiiron (FLV) proteins 1 and 3, showed the same phenotype as S. 6803(WT). In contrast, Y(II) decreased in Synechococcus sp. PCC 7942 (S. 7942). These results suggest that AEF functioned as the Y(II) in S. 6803 and replaced the photosynthetic electron flux. In contrast, the activity of AEF in S. 7942 was lower. The affinity of AEF for O2 in S. 6803 did not correspond to those of FLVs in bacteria or terminal oxidases in respiration. AEF might be driven by photorespiration. Graphical Abstract Cyanobacterial species dependence of alternative electron flow to replace the electron Flux in Photosynthesis: a, Synechocystis (S. 6803); b, Synechococcus (S. 7942).

Diversity of strategies for escaping reactive oxygen species production within photosystem I among land plants: P700 oxidation system is prerequisite for alleviating photoinhibition in photosystem I

In land plants, photosystem I (PSI) photoinhibition limits carbon fixation and causes growth defects. In addition, recovery from PSI photoinhibition takes much longer than PSII photoinhibition when the PSI core-complex is degraded by oxidative damage. Accordingly, PSI photoinhibition should be avoided in land plants, and land plants should have evolved mechanisms to prevent PSI photoinhibition. However, such protection mechanisms have not yet been identified, and it remains unclear whether all land plants suffer from PSI photoinhibition in the same way. In the present study, we focused on the susceptibility of PSI to photoinhibition and investigated whether mechanisms of preventing PSI photoinhibition varied among land plant species. To assess the susceptibility of PSI to photoinhibition, we used repetitive short-pulse (rSP) illumination, which specifically induces PSI photoinhibition. Subsequently, we found that land plants possess a wide variety of tolerance mechanisms against PSI photoinhibition. In particular, gymnosperms, ferns and mosses/liverworts exhibited higher tolerance to rSP illumination-induced PSI photoinhibition than angiosperms, and detailed analyses indicated that the tolerance of these groups could be partly attributed to flavodiiron proteins, which protected PSI from photoinhibition by oxidizing the PSI reaction center chlorophyll (P700) as an electron acceptor. Furthermore, we demonstrate, for the first time, that gymnosperms, ferns and mosses/liverworts possess a protection mechanism against photoinhibition of PSI that differs from that of angiosperms.

Oxidation of P700 in photosystem I is essential for the growth of cyanobacteria

Oxidation of P700 in photosystem I prevents the photoinhibition of photosystem I to enable cyanobacteria to grow under ambient [CO2]. The photoinhibition of photosystem I (PSI) is lethal to oxygenic phototrophs. Nevertheless, it is unclear how photodamage occurs or how oxygenic phototrophs prevent it. Here, we provide evidence that keeping P700 (the reaction center chlorophyll in PSI) oxidized protects PSI. Previous studies have suggested that PSI photoinhibition does not occur in the two model cyanobacteria, Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942, when photosynthetic CO2 fixation was suppressed under low CO2 partial pressure even in mutants deficient in flavodiiron protein (FLV), which mediates alternative electron flow. The lack of FLV in Synechococcus sp. PCC 7002 (S. 7002), however, is linked directly to reduced growth and PSI photodamage under CO2-limiting conditions. Unlike Synechocystis sp. PCC 6803 and S. elongatus PCC 7942, S. 7002 reduced P700 during CO2-limited illumination in the absence of FLV, resulting in decreases in both PSI and photosynthetic activities. Even at normal air CO2 concentration, the growth of S. 7002 mutant was retarded relative to that of the wild type. Therefore, P700 oxidation is essential for protecting PSI against photoinhibition. Here, we present various strategies to alleviate PSI photoinhibition in cyanobacteria.

Why don't plants have diabetes? Systems for scavenging reactive carbonyls in photosynthetic organisms.

In the present paper, we review the toxicity of sugar- and lipid-derived RCs (reactive carbonyls) and the RC-scavenging systems observed in photosynthetic organisms. Similar to heterotrophs, photosynthetic organisms are exposed to the danger of RCs produced in sugar metabolism during both respiration and photosynthesis. RCs such as methylglyoxal and acrolein have toxic effects on the photosynthetic activity of higher plants and cyanobacteria. These toxic effects are assumed to occur uniquely in photosynthetic organisms, suggesting that RC-scavenging systems are essential for their survival. The aldo-keto reductase and the glyoxalase systems mainly scavenge sugar-derived RCs in higher plants and cyanobacteria. 2-Alkenal reductase and alkenal/alkenone reductase catalyse the reduction of lipid-derived RCs in higher plants. In cyanobacteria, medium-chain dehydrogenases/reductases are the main scavengers of lipid-derived RCs.

Overexpression of flv3 improves photosynthesis in the cyanobacterium Synechocystis sp. PCC6803 by enhancement of alternative electron flow

BackgroundTo ensure reliable sources of energy and raw materials, the utilization of sustainable biomass has considerable advantages over petroleum-based energy sources. Photosynthetic algae have attracted attention as a third-generation feedstock for biofuel production, because algae cultivation does not directly compete with agricultural resources, including the requirement for productive land and fresh water. In particular, cyanobacteria are a promising biomass feedstock because of their high photosynthetic capability.ResultsIn the present study, the expression of the flv3 gene, which encodes a flavodiiron protein involved in alternative electron flow (AEF) associated with NADPH-coupled O2 photoreduction in photosystem I, was enhanced in Synechocystis sp. PCC6803. Overexpression of flv3 improved cell growth with corresponding increases in O2 evolution, intracellular ATP level, and turnover of the Calvin cycle. The combination of in vivo13C-labeling of metabolites and metabolomic analysis confirmed that the photosynthetic carbon flow was enhanced in the flv3-overexpressing strain.ConclusionsOverexpression of flv3 improved cell growth and glycogen production in the recombinant Synechocystis sp. PCC6803. Direct measurement of metabolic turnover provided conclusive evidence that CO2 incorporation is enhanced by the flv3 overexpression. Increase in O2 evolution and ATP accumulation indicates enhancement of the AEF. Overexpression of flv3 improves photosynthesis in the Synechocystis sp. PCC6803 by enhancement of the AEF.

Scavenging Systems for Reactive Carbonyls in the Cyanobacterium Synechocystis sp. PCC 6803

To elucidate the scavenging systems of sugar- and lipid-derived reactive carbonyls (RCs) in the cyanobacterium Synechocystis sp. PCC 6803 (S. 6803), we selected proteins from S. 6803 based on amino-acid (AA) sequence similarities with proteins from Arabidopsis thaliana, and characterized the properties of the GST-fusion proteins expressed. Slr0942 catalyzed the aldo-keto reductase (AKR) reaction scavenging mainly sugar-derived RCs, methylglyoxal (MG). Slr1192 is the medium-chain dehydrogenase/redutase (MDR). It catalyzed the AKR reaction scavenging several lipid-derived RCs, acrolein, propionaldehyde, and crotonaldehyde. Slr0315 is a short-chain dehydrogenase/redutase (SDR), and it catalyzed only the reduction of MG in the AKR reaction. Slr0381 catalyzed the conversion of hemithioacetal to S-lactoylglutahione (SLG) in the glyoxalase (GLX) 1 reaction. Sll1019 catalyzed the conversion of SLG to glutathione and lactate in the GLX2 reaction. GLX1 and GLX2 compose the glyoxalase system, which scavenges MG. These enzymes contribute to scavenging sugar- and lipid-derived RCs as scavenging systems.