RESUMO
Improving photosynthesis, the fundamental process by which plants convert light energy into chemical energy, is a key area of research with great potential for enhancing sustainable agricultural productivity and addressing global food security challenges. This perspective delves into the latest advancements and approaches aimed at optimizing photosynthetic efficiency. Our discussion encompasses the entire process, beginning with light harvesting and its regulation and progressing through the bottleneck of electron transfer. We then delve into the carbon reactions of photosynthesis, focusing on strategies targeting the enzymes of the Calvin-Benson-Bassham (CBB) cycle. Additionally, we explore methods to increase CO2 concentration near the Rubisco, the enzyme responsible for the first step of CBB cycle, drawing inspiration from various photosynthetic organisms, and conclude this section by examining ways to enhance CO2 delivery into leaves. Moving beyond individual processes, we discuss two approaches to identifying key targets for photosynthesis improvement: systems modeling and the study of natural variation. Finally, we revisit some of the strategies mentioned above to provide a holistic view of the improvements, analyzing their impact on nitrogen use efficiency and on canopy photosynthesis.
RESUMO
The superior productivity of C4 plants is achieved via a metabolic C4 cycle which acts as a CO2 pump across mesophyll and bundle sheath (BS) cells and requires an additional input of energy in the form of ATP. The importance of chloroplast NADH dehydrogenase-like complex (NDH) operating cyclic electron flow (CEF) around Photosystem I (PSI) for C4 photosynthesis has been shown in reverse genetics studies but the contribution of CEF and NDH to cell-level electron fluxes remained unknown. We have created gene-edited Setaria viridis with null ndhO alleles lacking functional NDH and developed methods for quantification of electron flow through NDH in BS and mesophyll cells. We show that CEF accounts for 84% of electrons reducing PSI in BS cells and most of those electrons are delivered through NDH while the contribution of the complex to electron transport in mesophyll cells is minimal. A decreased leaf CO2 assimilation rate and growth of plants lacking NDH cannot be rescued by supplying additional CO2. Our results indicate that NDH-mediated CEF is the primary electron transport route in BS chloroplasts highlighting the essential role of NDH in generating ATP required for CO2 fixation by the C3 cycle in BS cells.
Assuntos
Cloroplastos , NADH Desidrogenase , Complexo de Proteína do Fotossistema I , Transporte de Elétrons , Cloroplastos/metabolismo , NADH Desidrogenase/metabolismo , NADH Desidrogenase/genética , Complexo de Proteína do Fotossistema I/metabolismo , Setaria (Planta)/metabolismo , Setaria (Planta)/genética , Dióxido de Carbono/metabolismo , Células do Mesofilo/metabolismo , Fotossíntese , Feixe Vascular de Plantas/metabolismo , Folhas de Planta/metabolismoRESUMO
Sedoheptulose-1,7-bisphosphatase (SBPase) is one of the rate-limiting enzymes of the Calvin cycle, and increasing the abundance of SBPase in C3 plants provides higher photosynthetic rates and stimulates biomass and yield. C4 plants usually have higher photosynthetic rates because they operate a biochemical CO2-concentrating mechanism between mesophyll and bundle sheath cells. In the C4 system, SBPase and other enzymes of the Calvin cycle are localized to the bundle sheath cells. Here we tested what effect increasing abundance of SBPase would have on C4 photosynthesis. Using green foxtail millet (Setaria viridis), a model C4 plant of NADP-ME subtype, we created transgenic plants with 1.5 to 3.2 times higher SBPase content compared to wild-type plants. Transcripts of the transgene were found predominantly in the bundle sheaths suggesting the correct cellular localization of the protein. The abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit was not affected in transgenic plants overexpressing SBPase, and neither was leaf chlorophyll content or photosynthetic electron transport parameters. We found no association between SBPase content in S. viridis and saturating rates of CO2 assimilation. Moreover, a detailed analysis of CO2 assimilation rates at different CO2 partial pressures, irradiances, and leaf temperatures showed no improvement of photosynthesis in plants overexpressing SBPase. We discuss the potential implications of these results for understanding the role of SBPase in regulation of C4 photosynthesis.
Assuntos
Setaria (Planta) , Setaria (Planta)/genética , Setaria (Planta)/metabolismo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Dióxido de Carbono/metabolismo , Fotossíntese/genética , Plantas Geneticamente Modificadas/metabolismoRESUMO
When C4 leaves are exposed to low light, the CO2 concentration in the bundle sheath (BS) cells decreases, causing an increase in photorespiration relative to assimilation, and a consequent reduction in biochemical efficiency. These effects can be mitigated by complex acclimation syndromes, which are of primary importance for crop productivity but are not well studied. We unveil an acclimation strategy involving the coordination of electron transport processes. First, we characterize the anatomy, gas exchange and electron transport of C4 Setaria viridis grown under low light. Through a purposely developed biochemical model, we resolve the photon fluxes and reaction rates to explain how the concerted acclimation strategies sustain photosynthetic efficiency. Our results show that a smaller BS in low-light-grown plants limited leakiness (the ratio of CO2 leak rate out of the BS over the rate of supply via C4 acid decarboxylation) but sacrificed light harvesting and ATP production. To counter ATP shortage and maintain high assimilation rates, plants facilitated light penetration through the mesophyll and upregulated cyclic electron flow in the BS. This shade tolerance mechanism, based on the optimization of light reactions, is possibly more efficient than the known mechanisms involving the rearrangement of carbon metabolism, and could potentially lead to innovative strategies for crop improvement.
Assuntos
Setaria (Planta) , Aclimatação , Trifosfato de Adenosina/metabolismo , Dióxido de Carbono/metabolismo , Elétrons , Fotossíntese , Folhas de Planta/metabolismo , Setaria (Planta)/metabolismo , Zea mays/metabolismoRESUMO
Photosynthetic efficiency and sink demand are tightly correlated with rates of phloem loading, where maintaining low cytosolic sugar concentrations is paramount to prevent the downregulation of photosynthesis. Sugars Will Eventually be Exported Transporters (SWEETs) are thought to have a pivotal role in the apoplastic phloem loading of C4 grasses. SWEETs have not been well studied in C4 species, and their investigation is complicated by photosynthesis taking place across two cell types and, therefore, photoassimilate export can occur from either one. SWEET13 homologues in C4 grasses have been proposed to facilitate apoplastic phloem loading. Here, we provide evidence for this hypothesis using the C4 grass Setaria viridis. Expression analyses on the leaf gradient of C4 species Setaria and Sorghum bicolor show abundant transcript levels for SWEET13 homologues. Carbohydrate profiling along the Setaria leaf shows total sugar content to be significantly higher in the mature leaf tip compared with the younger tissue at the base. We present the first known immunolocalization results for SvSWEET13a and SvSWEET13b using novel isoform-specific antisera. These results show localization to the bundle sheath and phloem parenchyma cells of both minor and major veins. We further present the first transport kinetics study of C4 monocot SWEETs by using a Xenopus laevis oocyte heterologous expression system. We demonstrate that SvSWEET13a and SvSWEET13b are high-capacity transporters of glucose and sucrose, with a higher apparent Vmax for sucrose, compared with glucose, typical of clade III SWEETs. Collectively, these results provide evidence for an apoplastic phloem loading pathway in Setaria and possibly other C4 species.
Assuntos
Transporte Biológico/genética , Glucose/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Floema/metabolismo , Setaria (Planta)/genética , Setaria (Planta)/metabolismo , Sacarose/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Fotossíntese , Transcrição GênicaRESUMO
Sorghum is one of the most important crops providing food and feed in many of the world's harsher environments. Sorghum utilizes the C4 pathway of photosynthesis in which a biochemical carbon-concentrating mechanism results in high CO2 assimilation rates. Overexpressing the Rieske FeS subunit of the Cytochrome b6 f complex was previously shown to increase the rate of photosynthetic electron transport and stimulate CO2 assimilation in the model C4 plant Setaria viridis. To test whether productivity of C4 crops could be improved by Rieske overexpression, we created transgenic Sorghum bicolor Tx430 plants with increased Rieske content. The transgenic plants showed no marked changes in abundances of other photosynthetic proteins or chlorophyll content. The steady-state rates of electron transport and CO2 assimilation did not differ between the plants with increased Rieske abundance and control plants, suggesting that Cytochrome b6 f is not the only factor limiting electron transport in sorghum at high light and high CO2 . However, faster responses of non-photochemical quenching as well as an elevated quantum yield of Photosystem II and an increased CO2 assimilation rate were observed from the plants overexpressing Rieske during the photosynthetic induction, a process of activation of photosynthesis upon the dark-light transition. As a consequence, sorghum with increased Rieske content produced more biomass and grain when grown in glasshouse conditions. Our results indicate that increasing Rieske content has potential to boost productivity of sorghum and other C4 crops by improving the efficiency of light utilization and conversion to biomass through the faster induction of photosynthesis.
Assuntos
Sorghum , Sorghum/genética , Sorghum/metabolismo , Biomassa , Dióxido de Carbono/metabolismo , Folhas de Planta/metabolismo , Fotossíntese/genética , Grão Comestível/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Produtos AgrícolasRESUMO
The model heterocyst-forming filamentous cyanobacterium Anabaena sp. PCC 7120 (Anabaena) is a typical example of a multicellular organism capable of simultaneously performing oxygenic photosynthesis in vegetative cells and O2 -sensitive N2 -fixation inside heterocysts. The flavodiiron proteins have been shown to participate in photoprotection of photosynthesis by driving excess electrons to O2 (a Mehler-like reaction). Here, we performed a phenotypic and biophysical characterization of Anabaena mutants impaired in vegetative-specific Flv1A and Flv3A in order to address their physiological relevance in the bioenergetic processes occurring in diazotrophic Anabaena under variable CO2 conditions. We demonstrate that both Flv1A and Flv3A are required for proper induction of the Mehler-like reaction upon a sudden increase in light intensity, which is likely important for the activation of carbon-concentrating mechanisms and CO2 fixation. Under ambient CO2 diazotrophic conditions, Flv3A is responsible for moderate O2 photoreduction, independently of Flv1A, but only in the presence of Flv2 and Flv4. Strikingly, the lack of Flv3A resulted in strong downregulation of the heterocyst-specific uptake hydrogenase, which led to enhanced H2 photoproduction under both oxic and micro-oxic conditions. These results reveal a novel regulatory network between the Mehler-like reaction and the diazotrophic metabolism, which is of great interest for future biotechnological applications.
Assuntos
Anabaena , Dióxido de Carbono , Dióxido de Carbono/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Anabaena/genética , Anabaena/metabolismo , Oxigênio/metabolismo , Fotossíntese/fisiologiaRESUMO
Photosynthetic manipulation provides new opportunities for enhancing crop yield. However, understanding and quantifying the importance of individual and multiple manipulations on the seasonal biomass growth and yield performance of target crops across variable production environments is limited. Using a state-of-the-art cross-scale model in the APSIM platform we predicted the impact of altering photosynthesis on the enzyme-limited (Ac ) and electron transport-limited (Aj ) rates, seasonal dynamics in canopy photosynthesis, biomass growth, and yield formation via large multiyear-by-location crop growth simulations. A broad list of promising strategies to improve photosynthesis for C3 wheat and C4 sorghum were simulated. In the top decile of seasonal outcomes, yield gains were predicted to be modest, ranging between 0% and 8%, depending on the manipulation and crop type. We report how photosynthetic enhancement can affect the timing and severity of water and nitrogen stress on the growing crop, resulting in nonintuitive seasonal crop dynamics and yield outcomes. We predicted that strategies enhancing Ac alone generate more consistent but smaller yield gains across all water and nitrogen environments, Aj enhancement alone generates larger gains but is undesirable in more marginal environments. Large increases in both Ac and Aj generate the highest gains across all environments. Yield outcomes of the tested manipulation strategies were predicted and compared for realistic Australian wheat and sorghum production. This study uniquely unpacks complex cross-scale interactions between photosynthesis and seasonal crop dynamics and improves understanding and quantification of the potential impact of photosynthesis traits (or lack of it) for crop improvement research.
Assuntos
Nitrogênio , Água , AustráliaRESUMO
C4 photosynthesis is a biochemical pathway that operates across mesophyll and bundle sheath (BS) cells to increase CO2 concentration at the site of CO2 fixation. C4 plants benefit from high irradiance but their efficiency decreases under shade, causing a loss of productivity in crop canopies. We investigated shade acclimation responses of Setaria viridis, a model monocot of NADP-dependent malic enzyme subtype, focussing on cell-specific electron transport capacity. Plants grown under low light (LL) maintained CO2 assimilation rates similar to high light plants but had an increased chlorophyll and light-harvesting-protein content, predominantly in BS cells. Photosystem II (PSII) protein abundance, oxygen-evolving activity and the PSII/PSI ratio were enhanced in LL BS cells, indicating a higher capacity for linear electron flow. Abundances of PSI, ATP synthase, Cytochrome b6 f and the chloroplast NAD(P)H dehydrogenase complex, which constitute the BS cyclic electron flow machinery, were also increased in LL plants. A decline in PEP carboxylase activity in mesophyll cells and a consequent shortage of reducing power in BS chloroplasts were associated with a more oxidised plastoquinone pool in LL plants and the formation of PSII - light-harvesting complex II supercomplexes with an increased oxygen evolution rate. Our results suggest that the supramolecular composition of PSII in BS cells is adjusted according to the redox state of the plastoquinone pool. This discovery contributes to the understanding of the acclimation of PSII activity in C4 plants and will support the development of strategies for crop improvement, including the engineering of C4 photosynthesis into C3 plants.
Assuntos
Malato Desidrogenase (NADP+)/metabolismo , Fotossíntese/efeitos da radiação , Complexo de Proteína do Fotossistema II/metabolismo , Setaria (Planta)/fisiologia , Dióxido de Carbono/metabolismo , Clorofila/metabolismo , Proteínas de Cloroplastos/genética , Proteínas de Cloroplastos/metabolismo , Cloroplastos/enzimologia , Transporte de Elétrons , Luz , Malato Desidrogenase (NADP+)/genética , Células do Mesofilo/metabolismo , Complexo de Proteína do Fotossistema I/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Setaria (Planta)/genética , Setaria (Planta)/efeitos da radiação , Regulação para CimaRESUMO
In biological discovery and engineering research, there is a need to spatially and/or temporally regulate transgene expression. However, the limited availability of promoter sequences that are uniquely active in specific tissue-types and/or at specific times often precludes co-expression of multiple transgenes in precisely controlled developmental contexts. Here, we developed a system for use in rice that comprises synthetic designer transcription activator-like effectors (dTALEs) and cognate synthetic TALE-activated promoters (STAPs). The system allows multiple transgenes to be expressed from different STAPs, with the spatial and temporal context determined by a single promoter that drives expression of the dTALE. We show that two different systems-dTALE1-STAP1 and dTALE2-STAP2-can activate STAP-driven reporter gene expression in stable transgenic rice lines, with transgene transcript levels dependent on both dTALE and STAP sequence identities. The relative strength of individual STAP sequences is consistent between dTALE1 and dTALE2 systems but differs between cell-types, requiring empirical evaluation in each case. dTALE expression leads to off-target activation of endogenous genes but the number of genes affected is substantially less than the number impacted by the somaclonal variation that occurs during the regeneration of transformed plants. With the potential to design fully orthogonal dTALEs for any genome of interest, the dTALE-STAP system thus provides a powerful approach to fine-tune the expression of multiple transgenes, and to simultaneously introduce different synthetic circuits into distinct developmental contexts.
Assuntos
Oryza , Genes Reporter , Oryza/genética , Plantas/genética , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas/genética , Transgenes/genéticaRESUMO
ATP, produced by the light reactions of photosynthesis, acts as the universal cellular energy cofactor fuelling all life processes. Chloroplast ATP synthase produces ATP using the proton motive force created by solar energy-driven thylakoid electron transport reactions. Here we investigate how increasing abundance of ATP synthase affects leaf photosynthesis and growth of rice, Oryza sativa variety Kitaake. We show that overexpression of AtpD, the nuclear-encoded subunit of the chloroplast ATP synthase, stimulates both abundance of the complex, confirmed by immunodetection of thylakoid complexes separated by Blue Native-PAGE, and ATP synthase activity, detected as higher proton conductivity of the thylakoid membrane. Plants with increased AtpD content had higher CO2 assimilation rates when a stepwise increase in CO2 partial pressure was imposed on leaves at high irradiance. Fitting of the CO2 response curves of assimilation revealed that plants overexpressing AtpD had a higher electron transport rate (J) at high CO2, despite having wild-type-like abundance of the cytochrome b6f complex. A higher maximum carboxylation rate (Vcmax) and lower cyclic electron flow detected in transgenic plants both pointed to an increased ATP production compared with wild-type plants. Our results present evidence that the activity of ATP synthase modulates the rate of electron transport at high CO2 and high irradiance.
Assuntos
ATPases de Cloroplastos Translocadoras de Prótons , Oryza , ATPases de Cloroplastos Translocadoras de Prótons/genética , ATPases de Cloroplastos Translocadoras de Prótons/metabolismo , Oryza/genética , Oryza/metabolismo , Dióxido de Carbono/metabolismo , Fotossíntese/fisiologia , Transporte de Elétrons , Trifosfato de AdenosinaRESUMO
Xanthophylls are a class of carotenoids that are important micronutrients for humans. They are often found esterified with fatty acids in fruits, vegetables, and certain grains, including bread wheat (Triticum aestivum). Esterification promotes the sequestration and accumulation of carotenoids, thereby enhancing stability, particularly in tissues such as in harvested wheat grain. Here, we report on a plant xanthophyll acyltransferase (XAT) that is both necessary and sufficient for xanthophyll esterification in bread wheat grain. XAT contains a canonical Gly-Asp-Ser-Leu (GDSL) motif and is encoded by a member of the GDSL esterase/lipase gene family. Genetic evidence from allelic variants of wheat and transgenic rice (Oryza sativa) calli demonstrated that XAT catalyzes the formation of xanthophyll esters. XAT has broad substrate specificity and can esterify lutein, ß-cryptoxanthin, and zeaxanthin using multiple acyl donors, yet it has a preference for triacylglycerides, indicating that the enzyme acts via transesterification. A conserved amino acid, Ser-37, is required for activity. Despite xanthophylls being synthesized in plastids, XAT accumulated in the apoplast. Based on analysis of substrate preferences and xanthophyll ester formation in vitro and in vivo using xanthophyll-accumulating rice callus, we propose that disintegration of the cellular structure during wheat grain desiccation facilitates access to lutein-promoting transesterification.plantcell;31/12/3092/FX1F1fx1.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Luteína/metabolismo , Triticum/enzimologia , Xantofilas/metabolismo , Alelos , beta-Criptoxantina/metabolismo , Biocatálise , Hidrolases de Éster Carboxílico/genética , Carotenoides/metabolismo , Esterificação , Ésteres/metabolismo , Especificidade de Órgãos/genética , Oryza/metabolismo , Plantas Geneticamente Modificadas , Plastídeos/metabolismo , Triglicerídeos/metabolismo , Triticum/embriologia , Triticum/genética , Triticum/metabolismo , Zeaxantinas/metabolismoRESUMO
Photosynthesis is fundamental for plant growth and yield. The cytochrome b6 f complex catalyses a rate-limiting step in thylakoid electron transport and therefore represents an important point of regulation of photosynthesis. Here we show that overexpression of a single core subunit of cytochrome b6 f, the Rieske FeS protein, led to up to a 40% increase in the abundance of the complex in Nicotiana tabacum (tobacco) and was accompanied by an enhanced in vitro cytochrome f activity, indicating a full functionality of the complex. Analysis of transgenic plants overexpressing Rieske FeS by the light-induced fluorescence transients technique revealed a more oxidised primary quinone acceptor of photosystem II (QA ) and plastoquinone pool and faster electron transport from the plastoquinone pool to photosystem I upon changes in irradiance, compared to control plants. A faster establishment of qE , the energy-dependent component of nonphotochemical quenching, in transgenic plants suggests a more rapid buildup of the transmembrane proton gradient, also supporting the increased in vivo cytochrome b6 f activity. However, there was no consistent increase in steady-state rates of electron transport or CO2 assimilation in plants overexpressing Rieske FeS grown in either laboratory conditions or field trials, suggesting that the in vivo activity of the complex was only transiently increased upon changes in irradiance. Our results show that overexpression of Rieske FeS in tobacco enhances the abundance of functional cytochrome b6 f and may have the potential to increase plant productivity if combined with other traits.
Assuntos
Citocromos b , Nicotiana , Nicotiana/genética , Nicotiana/metabolismo , Citocromos b/metabolismo , Plastoquinona , Fotossíntese/fisiologia , Transporte de Elétrons/fisiologia , Complexo Citocromos b6f/genética , Complexo Citocromos b6f/metabolismo , Plantas Geneticamente Modificadas/metabolismoRESUMO
In oxygenic photosynthetic organisms, excluding angiosperms, flavodiiron proteins (FDPs) catalyze light-dependent reduction of O2 to H2 O. This alleviates electron pressure on the photosynthetic apparatus and protects it from photodamage. In Synechocystis sp. PCC 6803, four FDP isoforms function as hetero-oligomers of Flv1 and Flv3 and/or Flv2 and Flv4. An alternative electron transport pathway mediated by the NAD(P)H dehydrogenase-like complex (NDH-1) also contributes to redox hemostasis and the photoprotection of photosynthesis. Four NDH-1 types have been characterized in cyanobacteria: NDH-11 and NDH-12 , which function in respiration; and NDH-13 and NDH-14 , which function in CO2 uptake. All four types are involved in cyclic electron transport. Along with single FDP mutants (∆flv1 and Δflv3) and the double NDH-1 mutants (∆d1d2, which is deficient in NDH-11,2 and ∆d3d4, which is deficient in NDH-13,4 ), we studied triple mutants lacking one of Flv1 or Flv3, and NDH-11,2 or NDH-13,4 . We show that the presence of either Flv1/3 or NDH-11,2 , but not NDH-13,4 , is indispensable for survival during changes in growth conditions from high CO2 /moderate light to low CO2 /high light. Our results show functional redundancy between FDPs and NDH-11,2 under the studied conditions. We suggest that ferredoxin probably functions as a primary electron donor to both Flv1/3 and NDH-11,2 , allowing their functions to be dynamically coordinated for efficient oxidation of photosystem I and for photoprotection under variable CO2 and light availability.
Assuntos
Proteínas de Bactérias/metabolismo , Synechocystis/metabolismo , Proteínas de Bactérias/fisiologia , Luz , Oxirredução , Complexo de Proteína do Fotossistema I/metabolismo , Tilacoides/metabolismoRESUMO
The international C4 rice consortium aims to introduce into rice a high capacity photosynthetic mechanism, the C4 pathway, to increase yield. The C4 pathway is characterised by a complex combination of biochemical and anatomical specialisation that ensures high CO2 partial pressure at RuBisCO sites in bundle sheath (BS) cells. Here we report an update of the progress of the C4 rice project. Since its inception in 2008 there has been an exponential growth in synthetic biology and molecular tools. Golden Gate cloning and synthetic promoter systems have facilitated gene building block approaches allowing multiple enzymes and metabolite transporters to be assembled and expressed from single gene constructs. Photosynthetic functionalisation of the BS in rice remains an important step and there has been some success overexpressing transcription factors in the cytokinin signalling network which influence chloroplast volume. The C4 rice project has rejuvenated the research interest in C4 photosynthesis. Comparative anatomical studies now point to critical features essential for the design. So far little attention has been paid to the energetics. C4 photosynthesis has a greater ATP requirement, which is met by increased cyclic electron transport in BS cells. We hypothesise that changes in energy statues may drive this increased capacity for cyclic electron flow without the need for further modification. Although increasing vein density will ultimately be necessary for high efficiency C4 rice, our modelling shows that small amounts of C4 photosynthesis introduced around existing veins could already provide benefits of increased photosynthesis on the road to C4 rice.
Assuntos
Oryza/fisiologia , Fotossíntese , Melhoramento Vegetal/métodos , Cloroplastos/metabolismo , Transporte de Elétrons , Regulação da Expressão Gênica de Plantas , Folhas de Planta/anatomia & histologia , Folhas de Planta/fisiologia , Plantas Geneticamente Modificadas , Biologia Sintética/métodosRESUMO
Introduction of a C4 photosynthetic mechanism into C3 crops offers an opportunity to improve photosynthetic efficiency, biomass and yield in addition to potentially improving nitrogen and water use efficiency. To create a two-cell metabolic prototype for an NADP-malic enzyme type C4 rice, we transformed Oryza sativa spp. japonica cultivar Kitaake with a single construct containing the coding regions of carbonic anhydrase, phosphoenolpyruvate (PEP) carboxylase, NADP-malate dehydrogenase, pyruvate orthophosphate dikinase and NADP-malic enzyme from Zea mays, driven by cell-preferential promoters. Gene expression, protein accumulation and enzyme activity were confirmed for all five transgenes, and intercellular localization of proteins was analysed. 13 CO2 labelling demonstrated a 10-fold increase in flux though PEP carboxylase, exceeding the increase in measured in vitro enzyme activity, and estimated to be about 2% of the maize photosynthetic flux. Flux from malate via pyruvate to PEP remained low, commensurate with the low NADP-malic enzyme activity observed in the transgenic lines. Physiological perturbations were minor and RNA sequencing revealed no substantive effects of transgene expression on other endogenous rice transcripts associated with photosynthesis. These results provide promise that, with enhanced levels of the C4 proteins introduced thus far, a functional C4 pathway is achievable in rice.
Assuntos
Oryza , Malato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Oryza/genética , Oryza/metabolismo , Fosfoenolpiruvato Carboxilase/genética , Fosfoenolpiruvato Carboxilase/metabolismo , Fotossíntese , Piruvato Ortofosfato Diquinase/genética , Piruvato Ortofosfato Diquinase/metabolismo , Zea mays/metabolismoRESUMO
Various oxygen-utilizing electron sinks, including the soluble flavodiiron proteins (Flv1/3), and the membrane-localized respiratory terminal oxidases (RTOs), cytochrome c oxidase (Cox) and cytochrome bd quinol oxidase (Cyd), are present in the photosynthetic electron transfer chain of Synechocystis sp. PCC 6803. However, the role of individual RTOs and their relative importance compared with other electron sinks are poorly understood, particularly under light. Via membrane inlet mass spectrometry gas exchange, chlorophyll a fluorescence, P700 analysis, and inhibitor treatment of the wild type and various mutants deficient in RTOs, Flv1/3, and photosystem I, we investigated the contribution of these complexes to the alleviation of excess electrons in the photosynthetic chain. To our knowledge, for the first time, we demonstrated the activity of Cyd in oxygen uptake under light, although it was detected only upon inhibition of electron transfer at the cytochrome b6f site and in ∆flv1/3 under fluctuating light conditions, where linear electron transfer was drastically inhibited due to impaired photosystem I activity. Cox is mostly responsible for dark respiration and competes with P700 for electrons under high light. Only the ∆cox/cyd double mutant, but not single mutants, demonstrated a highly reduced plastoquinone pool in darkness and impaired gross oxygen evolution under light, indicating that thylakoid-based RTOs are able to compensate partially for each other. Thus, both electron sinks contribute to the alleviation of excess electrons under illumination: RTOs continue to function under light, operating on slower time ranges and on a limited scale, whereas Flv1/3 responds rapidly as a light-induced component and has greater capacity.
Assuntos
Oxirredutases/metabolismo , Synechocystis/enzimologia , Tilacoides/metabolismo , Transporte de Elétrons/efeitos da radiação , Fluorescência , Luz , Mutação/genética , Oxirredução/efeitos da radiação , Oxigênio/metabolismo , Fotossíntese/efeitos da radiação , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Plastoquinona/metabolismo , Synechocystis/crescimento & desenvolvimento , Synechocystis/metabolismo , Synechocystis/efeitos da radiação , Tilacoides/efeitos da radiaçãoRESUMO
Flavodiiron proteins are known to have crucial and specific roles in photoprotection of photosystems I and II in cyanobacteria. The filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 contains, besides the four flavodiiron proteins Flv1A, Flv2, Flv3A, and Flv4 present in vegetative cells, two heterocyst-specific flavodiiron proteins, Flv1B and Flv3B. Here, we demonstrate that Flv3B is responsible for light-induced O2 uptake in heterocysts, and that the absence of the Flv3B protein severely compromises the growth of filaments in oxic, but not in microoxic, conditions. It is further demonstrated that Flv3B-mediated photosynthetic O2 uptake has a distinct role in heterocysts which cannot be substituted by respiratory O2 uptake in the protection of nitrogenase from oxidative damage and, thus, in an efficient provision of nitrogen to filaments. In line with this conclusion, the Δflv3B strain has reduced amounts of nitrogenase NifHDK subunits and shows multiple symptoms of nitrogen deficiency in the filaments. The apparent imbalance of cytosolic redox state in Δflv3B heterocysts also has a pronounced influence on the amounts of different transcripts and proteins. Therefore, an O2-related mechanism for control of gene expression is suggested to take place in heterocysts.
Assuntos
Anabaena/metabolismo , Proteínas de Bactérias/metabolismo , Flavoproteínas/metabolismo , Metaloproteínas/metabolismo , Fotossíntese/fisiologia , Anabaena/genética , Proteínas de Bactérias/genética , Flavoproteínas/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Ferro/metabolismo , Metaloproteínas/genética , Nitrogenase/genética , Nitrogenase/metabolismo , OxirreduçãoRESUMO
Cyanobacterial flavodiiron proteins (FDPs; A-type flavoprotein, Flv) comprise, besides the ß-lactamase-like and flavodoxin domains typical for all FDPs, an extra NAD(P)H:flavin oxidoreductase module and thus differ from FDPs in other Bacteria and Archaea. Synechocystis sp. PCC 6803 has four genes encoding the FDPs. Flv1 and Flv3 function as an NAD(P)H:oxygen oxidoreductase, donating electrons directly to O2 without production of reactive oxygen species. Here we show that the Flv1 and Flv3 proteins are crucial for cyanobacteria under fluctuating light, a typical light condition in aquatic environments. Under constant-light conditions, regardless of light intensity, the Flv1 and Flv3 proteins are dispensable. In contrast, under fluctuating light conditions, the growth and photosynthesis of the Δflv1(A) and/or Δflv3(A) mutants of Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120 become arrested, resulting in cell death in the most severe cases. This reaction is mainly caused by malfunction of photosystem I and oxidative damage induced by reactive oxygen species generated during abrupt short-term increases in light intensity. Unlike higher plants that lack the FDPs and use the Proton Gradient Regulation 5 to safeguard photosystem I, the cyanobacterial homolog of Proton Gradient Regulation 5 is shown not to be crucial for growth under fluctuating light. Instead, the unique Flv1/Flv3 heterodimer maintains the redox balance of the electron transfer chain in cyanobacteria and provides protection for photosystem I under fluctuating growth light. Evolution of unique cyanobacterial FDPs is discussed as a prerequisite for the development of oxygenic photosynthesis.
Assuntos
Proteínas de Bactérias/metabolismo , Flavoproteínas/metabolismo , Synechocystis/crescimento & desenvolvimento , Synechocystis/metabolismo , Anabaena/genética , Anabaena/crescimento & desenvolvimento , Anabaena/metabolismo , Anabaena/efeitos da radiação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Dióxido de Carbono/metabolismo , Flavoproteínas/química , Flavoproteínas/genética , Genes Bacterianos , Luz , Mutação , Oxigênio/metabolismo , Fotossíntese , Multimerização Proteica , Synechocystis/genética , Synechocystis/efeitos da radiaçãoRESUMO
Flavodiiron (Flv) proteins are involved in detoxification of O(2) and NO in anaerobic bacteria and archaea. Cyanobacterial Flv proteins, on the contrary, function in oxygenic environment and possess an extra NAD(P)H:flavin oxidoreductase module. Synechocystis sp. PCC 6803 has four genes (sll1521, sll0219, sll0550, and sll0217) encoding Flv proteins (Flv1, Flv2, Flv3, and Flv4). Previous in vitro studies with recombinant Flv3 protein from Synechocystis provided evidence that it functions as a NAD(P)H:oxygen oxidoreductase, and subsequent in vivo studies with Synechocystis confirmed the role of Flv1 and Flv3 proteins in the Mehler reaction (photoreduction of O(2) to H(2)O). Interestingly, homologous proteins to Flv1 and Flv3 can be found also in green algae, mosses, and Selaginella. Here, we addressed the function of Flv1 and Flv3 in Synechocystis using the Δflv1, Δflv3, and Δflv1/Δflv3 mutants and applying inorganic carbon (C(i))-deprivation conditions. We propose that only the Flv1/Flv3 heterodimer form is functional in the Mehler reaction in vivo. (18)O(2) labeling was used to discriminate between O(2) evolution in photosynthetic water splitting and O(2) consumption. In wild type, â¼20% of electrons originated from water was targeted to O(2) under air level CO(2) conditions but increased up to 60% in severe limitation of C(i). Gas exchange experiments with Δflv1, Δflv3, and Δflv1/Δflv3 mutants demonstrated that a considerable amount of electrons in these mutants is directed to photorespiration under C(i) deprivation. This assumption is in line with increased transcript abundance of photorespiratory genes and accumulation of photorespiratory intermediates in the WT and to a higher extent in mutant cells under C(i) deprivation.