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Diatoms are central to the global carbon cycle. At the heart of diatom carbon fixation is an overlooked organelle called the pyrenoid, where concentrated CO 2 is delivered to densely packed Rubisco. Diatom pyrenoids fix approximately one-fifth of global CO 2 , but the protein composition of this organelle is largely unknown. Using fluorescence protein tagging and affinity purification-mass spectrometry, we generate a high-confidence spatially defined protein-protein interaction network for the diatom pyrenoid. Within our pyrenoid interaction network are 10 proteins with previously unknown functions. We show that six of these form a shell that encapsulates the Rubisco matrix and is critical for pyrenoid structural integrity, shape, and function. Although not conserved at a sequence or structural level, the diatom pyrenoid shares some architectural similarities to prokaryotic carboxysomes. Collectively, our results support the convergent evolution of pyrenoids across the two main plastid lineages and uncover a major structural and functional component of global CO 2 fixation. [Display omitted] • A spatial interaction network reveals 10 previously unknown diatom pyrenoid proteins • Six Shell proteins encapsulate the pyrenoids of diatoms • Shell1/2 and Shell4 are essential for efficient CO 2 fixation and pyrenoid architecture • Shell proteins are widely conserved in major phytoplankton primary producers A conserved family in the protein interaction network of the CO 2 -fixing pyrenoid of diatoms forms a pyrenoid-encapsulating layer that is essential for organelle architecture and efficient CO 2 fixation. [ABSTRACT FROM AUTHOR]

The bread wheat cultivar (Triticum aestivum L. cv. Sagittario) as a parental line and its mutant, drought-tolerant lines (Mutant lines 4 and 5) were subjected to polyethylene glycol (PEG)-induced drought. Drought stress resulted in decreased chlorophyll levels and the accumulation of proline and TBARS, despite increases in activities of catalase, peroxidase, and superoxide dismutase enzymes. Transcription of the genes encoding these enzymes and delta-1-pyrroline 5-carboxylase synthetase was induced by drought. 2-DE gel electrophoresis analysis identified differentially expressed proteins (DEPs) in the mutant lines, which are distinguished by "chloroplast", "mitochondrion", "pyruvate dehydrogenase complex", and "homeostatic process" terms. The drought tolerance of the mutant lines might be attributed to improved photosynthesis, efficient ATP synthesis, and modified antioxidant capacity. In addition to proteomics data, the drought tolerance of wheat genotypes might also be assessed by chlorophyll content and TaPOX gene expression. To our knowledge, this is the first proteomic analysis of gamma-induced mutants of bread wheat. These findings are expected to be utilized in plant breeding studies. [ABSTRACT FROM AUTHOR]

With the rising demand for sustainable renewable resources, microorganisms capable of producing bioproducts such as bioplastics are attractive. While many bioproduction systems are well-studied in model organisms, investigating non-model organisms is essential to expand the field and utilize metabolically versatile strains. This investigation centers on Rhodopseudomonas palustris TIE-1, a purple non-sulfur bacterium capable of producing bioplastics. To increase bioplastic production, genes encoding the putative regulatory protein PhaR and the depolymerase PhaZ of the polyhydroxyalkanoate (PHA) biosynthesis pathway were deleted. Genes associated with pathways that might compete with PHA production, specifically those linked to glycogen production and nitrogen fixation, were deleted. Additionally, RuBisCO form I and II genes were integrated into TIE-1's genome by a phage integration system, developed in this study. Our results show that deletion of phaR increases PHA production when TIE-1 is grown photoheterotrophically with butyrate and ammonium chloride (NH4Cl). Mutants unable to produce glycogen or fix nitrogen show increased PHA production under photoautotrophic growth with hydrogen and NH4Cl. The most significant increase in PHA production was observed when RuBisCO form I and form I & II genes were overexpressed, five times under photoheterotrophy with butyrate, two times with hydrogen and NH4Cl, and two times under photoelectrotrophic growth with N2. In summary, inserting copies of RuBisCO genes into the TIE-1 genome is a more effective strategy than deleting competing pathways to increase PHA production in TIE-1. The successful use of the phage integration system opens numerous opportunities for synthetic biology in TIE-1. [ABSTRACT FROM AUTHOR]

The Hyphomicrobiales (Rhizobiales) order contains soil bacteria with an irregular distribution of the Calvin–Benson–Bassham cycle (CBB). Key enzymes in the CBB cycle are ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), whose large and small subunits are encoded in cbbL and cbbS, and phosphoribulokinase (PRK), encoded by cbbP. These genes are often found in cbb operons, regulated by the LysR-type regulator CbbR. In Bradyrhizobium, pertaining to this order and bearing photosynthetic and non-photosynthetic species, the number of cbbL and cbbS copies varies, for example: zero in B. manausense, one in B. diazoefficiens, two in B. japonicum, and three in Bradyrhizobium sp. BTAi. Few studies addressed the role of CBB in Bradyrhizobium spp. symbiosis with leguminous plants. To investigate the horizontal transfer of the cbb operon among Hyphomicrobiales, we compared phylogenetic trees for concatenated cbbL-cbbP-cbbR and housekeeping genes (atpD-gyrB-recA-rpoB-rpoD). The distribution was consistent, indicating no horizontal transfer of the cbb operon in Hyphomicrobiales. We constructed a ΔcbbLS mutant in B. diazoefficiens, which lost most of the coding sequence of cbbL and has a frameshift creating a stop codon at the N-terminus of cbbS. This mutant nodulated normally but had reduced competitiveness for nodulation and long-term adhesion to soybean (Glycine max (L.) Merr.) roots, indicating a CBB requirement for colonizing soybean rhizosphere. [ABSTRACT FROM AUTHOR]

Balancing the ATP: NADPH demand from plant metabolism with supply from photosynthesis is essential for preventing photodamage and operating efficiently, so understanding its drivers is important for integrating metabolism with the light reactions of photosynthesis and for bioengineering efforts that may radically change this demand. It is often assumed that the C3 cycle and photorespiration consume the largest amount of ATP and reductant in illuminated leaves and as a result mostly determine the ATP: NADPH demand. However, the quantitative extent to which other energy consuming metabolic processes contribute in large ways to overall ATP: NADPH demand remains unknown. Here, we used the metabolic flux networks of numerous recently published isotopically non-stationary metabolic flux analyses (INST-MFA) to evaluate flux through the C3 cycle, photorespiration, the oxidative pentose phosphate pathway, the tricarboxylic acid cycle, and starch/sucrose synthesis and characterize broad trends in the demand of energy across different pathways and compartments as well as in the overall ATP:NADPH demand. These data sets include a variety of species including Arabidopsis thaliana, Nicotiana tabacum, and Camelina sativa as well as varying environmental factors including high/low light, day length, and photorespiratory levels. Examining these datasets in aggregate reveals that ultimately the bulk of the energy flux occurred in the C3 cycle and photorespiration, however, the energy demand from these pathways did not determine the ATP: NADPH demand alone. Instead, a notable contribution was revealed from starch and sucrose synthesis which might counterbalance photorespiratory demand and result in fewer adjustments in mechanisms which balance the ATP deficit. [ABSTRACT FROM AUTHOR]

Summary: The coordinated interspecific variation in leaf traits and leaf lifespan is known as the leaf economic spectrum (LES). The limitation of CO2 diffusion to chloroplasts within the lamina is significant in C3 photosynthesis, resulting in a shortage of CO2 for Rubisco. Although Rubisco CO2/O2 specificity (SC/O) should be adaptively adjusted in response to the interspecific variation in CO2 concentrations [CO2] associated with Rubisco, SC/O variations across species along the LES remain unknown.We investigated the coordination among leaf traits, including SC/O, CO2 conductance, leaf protein content, and leaf mass area, across 23 woody C3 species coexisting on an oceanic island through phylogenetic correlation analyses.A high SC/O indicates a high CO2 specificity of Rubisco. SC/O was negatively correlated with [CO2] at Rubisco and total CO2 conductance within lamina, while it was positively correlated with leaf protein across species, regardless of phylogenetic constraint. A simulation analysis shows that the optimal SC/O for maximizing photosynthesis depends on both [CO2] at Rubisco sites and leaf protein per unit leaf area.SC/O is a key parameter along the LES axis and is crucial for maximizing photosynthesis across species and the adaptation of woody plants. [ABSTRACT FROM AUTHOR]

Plant physiologists set about comprehending the genesis of the C4 photosynthetic pathway after its discovery by Hatch and Slack. They discovered that a sophisticated combination of morphological and biochemical adaptations allowed the plant to concentrate CO2 around RuBisCO to achieve maximum efficiency. We categorize the evolutionary events leading to C4 photosynthesis, beginning with anoxygenic photosynthesis and the evolution of RuBisCO to the cooling of Earth by the Great Oxygenation Event that led to the oxygenic photosynthesis. The evolutionary descent of the C4 plants is a phenomenon that occurred around 30 million years ago. Due to industrialization and population growth, improved photosynthetic efficiency and carbon fixation of C4 plants could contest the current global scenario of rising CO2 concentration. C3 crops engineered with C4 traits, implemented on a large scale, could impact the climate globally. Here we discuss the various strategies used to introduce C4 traits in the C3 plants and the potential techniques to be considered for successful hybridization. [ABSTRACT FROM AUTHOR]

The defensive role performed by exogenously supplied ascorbic acid in the cyanobacterium Nostoc muscorum Meg1 against damages produced by UV-C radiation exposure was assessed in this study. Exposure to UV-C (24 mJ/cm2) significantly enhanced reactive oxygen species (ROS) (50%) along with peroxidation of lipids (21%) and protein oxidation (22%) in the organism. But, addition of 0.5 mM ascorbic acid prior to UV-C exposure showed reduction in ROS production (1.7%) and damages to lipids and proteins (1.5 and 2%, respectively). Light and transmission electron microscopic studies revealed that ascorbic acid not only protected filament breakage but also restricted severe ultrastructural changes and cellular damages in the organism. Although the growth of the organism was repressed up to 9% under UV-C treatment within 15 days, a pre-treatment with ascorbic acid led to growth enhancement by 42% in the same period. Various growth parameters such as photo-absorbing pigments (phycoerythrin, phycocyanin, allophycocyanin, chlorophyll a, and carotenoids), water splitting complex (WSC), D1 protein, RuBisCO, glutamine synthetase and nitrogenase activities in the UV-C treated organism were seen to be relatively intact in the presence of ascorbic acid. Thus, a detailed analysis undertaken in the present study was able to demonstrate that ascorbic acid not only act as first responder against harmful UV-C radiation by down-regulating ROS production, it also accelerated the growth performance in the organism in the post UV-C incubation period as an immediate response to an adverse experience presented in the form of UV-C radiation exposure. [ABSTRACT FROM AUTHOR]

The enzyme rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyzes the majority of biological carbon fixation on Earth. Although the vast majority of rubiscos across the tree of life assemble as homo-oligomers, the globally predominant form I enzyme-found in plants, algae, and cyanobacteria-forms a unique hetero-oligomeric complex. The recent discovery of a homo-oligomeric sister group to form I rubisco (named form I') has filled a key gap in our understanding of the enigmatic origins of the form I clade. However, to elucidate the series of molecular events leading to the evolution of form I rubisco, we must examine more distantly related sibling clades to contextualize the molecular features distinguishing form I and form I' rubiscos. Here, we present a comparative structural study retracing the evolutionary history of rubisco that reveals a complex structural trajectory leading to the ultimate hetero-oligomerization of the form I clade. We structurally characterize the oligomeric states of deep-branching form Iα and I'' rubiscos recently discovered from metagenomes, which represent key evolutionary intermediates preceding the form I clade. We further solve the structure of form I'' rubisco, revealing the molecular determinants that likely primed the enzyme core for the transition from a homo-oligomer to a hetero-oligomer. Our findings yield new insight into the evolutionary trajectory underpinning the adoption and entrenchment of the prevalent assembly of form I rubisco, providing additional context when viewing the enzyme family through the broader lens of protein evolution.

Abstract Background Salinity is one major abiotic stress affecting photosynthesis, plant growth, and development, resulting in low-input crops. Although photosynthesis underlies the substantial productivity and biomass storage of crop yield, the response of the sunflower photosynthetic machinery to salinity imposition and how H2S mitigates the salinity-induced photosynthetic injury remains largely unclear. Seed priming with 0.5 mM NaHS, as a donor of H2S, was adopted to analyze this issue under NaCl stress. Primed and nonprime seeds were established in nonsaline soil irrigated with tape water for 14 d, and then the seedlings were exposed to 150 mM NaCl for 7 d under controlled growth conditions. Results Salinity stress significantly harmed plant growth, photosynthetic parameters, the structural integrity of chloroplasts, and mesophyll cells. H2S priming improved the growth parameters, relative water content, stomatal density and aperture, photosynthetic pigments, photochemical efficiency of PSII, photosynthetic performance, soluble sugar as well as soluble protein contents while reducing proline and ABA under salinity. H2S also boosted the transcriptional level of ribulose 1,5-bisphosphate carboxylase small subunit gene (HaRBCS). Further, the transmission electron microscope showed that under H2S priming and salinity stress, mesophyll cells maintained their cell membrane integrity and integrated chloroplasts with well-developed thylakoid membranes. Conclusion The results underscore the importance of H2S priming in maintaining photochemical efficiency, Rubisco activity, and preserving the chloroplast structure which participates in salinity stress adaptation, and possibly sunflower productivity under salinity imposition. This underpins retaining and minimizing the injury to the photosynthetic machinery to be a crucial trait in response of sunflower to salinity stress.

SUMMARY Photosynthetic and chemosynthetic extremophiles have evolved adaptations to thrive in challenging environments by finely adjusting their metabolic pathways through evolutionary processes. A prime adaptation target to allow autotrophy in extreme conditions is the enzyme Rubisco, which plays a central role in the conversion of inorganic to organic carbon. Here, we present an extensive compilation of Rubisco kinetic traits from a wide range of species of bacteria, archaea, algae, and plants, sorted by phylogenetic group, Rubisco type, and extremophile type. Our results show that Rubisco kinetics for the few extremophile organisms reported up to date are placed at the margins of the enzyme's natural variability. Form ID Rubisco from thermoacidophile rhodophytes and form IB Rubisco from halophile terrestrial plants exhibit higher specificity and affinity for CO2 than their non‐extremophilic counterparts, as well as higher carboxylation efficiency, whereas form ID Rubisco from psychrophile organisms possess lower affinity for O2. Additionally, form IB Rubisco from thermophile cyanobacteria shows enhanced CO2 specificity when compared to form IB non‐extremophilic cyanobacteria. Overall, these findings highlight the unique characteristics of extremophile Rubisco enzymes and provide useful clues to guide next explorations aimed at finding more efficient Rubiscos. [ABSTRACT FROM AUTHOR]

Throughout the tree of life, cells and organisms enter states of dormancy or hibernation as a key feature of their biology: from a bacterium arresting its growth in response to starvation, to a plant seed anticipating placement in fertile ground, to a human oocyte poised for fertilization to create a new life. Recent research shows that when cells hibernate, many of their essential enzymes hibernate too: they disengage from their substrates and associate with a specialized group of proteins known as hibernation factors. Here, we summarize how hibernation factors protect essential cellular enzymes from undesired activity or irreparable damage in hibernating cells. We show how molecular hibernation, once viewed as rare and exclusive to certain molecules like ribosomes, is in fact a widespread property of biological molecules that is required for the sustained persistence of life on Earth. [ABSTRACT FROM AUTHOR]

Autotrophy is the basis for complex life on Earth. Central to this process is rubisco—the enzyme that catalyzes almost all carbon fixation on the planet. Yet, with only a small fraction of rubisco diversity kinetically characterized so far, the underlying biological factors driving the evolution of fast rubiscos in nature remain unclear. We conducted a high-throughput kinetic characterization of over 100 bacterial form I rubiscos, the most ubiquitous group of rubisco sequences in nature, to uncover the determinants of rubisco's carboxylation velocity. We show that the presence of a carboxysome CO2 concentrating mechanism correlates with faster rubiscos with a median fivefold higher rate. In contrast to prior studies, we find that rubiscos originating from α-cyanobacteria exhibit the highest carboxylation rates among form I enzymes (≈10 s−1 median versus <7 s−1 in other groups). Our study systematically reveals biological and environmental properties associated with kinetic variation across rubiscos from nature. Synopsis: Form I rubisco, known for its slow kinetics in plants and algae, exhibits a great unexplored diversity in autotrophic bacteria. This article represents the first large-scale survey of bacterial form I rubisco kinetics and reveals unifying features of fast carboxylating rubiscos. Over 100 homologs were systematically screened, spanning the wide genetic diversity of form I rubisco enzymes across a variety of ecological niches and metabolic profiles. Phototrophy and carboxysome association are correlated with fast-carboxylating rubiscos. α-cyanobacteria emerges as the bacterial clade expressing the fastest form I rubiscos on Earth. Bacterial rubisco enzymes associated with carboxysomes have the fastest CO2-fixing rates. [ABSTRACT FROM AUTHOR]

Carbon assimilation by Rubisco is often a limitation to photosynthesis and therefore plant productivity. We have previously shown that transgenic co-expression of the Rubisco large (LS) and small (SS) subunits along with an essential Rubisco accumulation factor, Raf1, leads to faster growth, increased photosynthesis, and enhanced chilling tolerance in maize (Zea mays). Maize also requires Rubisco accumulation factor2 (Raf2) for full accumulation of Rubisco. Here we have analyzed transgenic maize lines with increased expression of Raf2 or Raf2 plus LS and SS. We show that increasing Raf2 expression alone had minor effects on photosynthesis, whereas expressing Raf2 with Rubisco subunits led to increased Rubisco content, more rapid carbon assimilation, and greater plant height, most notably in plants at least 6 weeks of age. The magnitude of the effects was similar to what was observed previously for expression of Raf1 together with Rubisco subunits. Taken together, this suggests that increasing the amount of either assembly factor with Rubisco subunits can independently enhance Rubisco abundance and some aspects of plant performance. These results could also imply either synergy or a degree of functional redundancy for Raf1 and Raf2, the latter of whose precise role in Rubisco assembly is currently unknown. [ABSTRACT FROM AUTHOR]

Wheat, a primary staple food in numerous countries, is extensively cultivated worldwide due to its adaptability. It constitutes a significant portion of the global calorie intake, accounting for approximately 20% of the total. Wheat cultivation spans a vast area of 215 million hectares, resulting in a worldwide production of 772 million metric tons. One of the major challenges in wheat breeding is the lengthy process of selecting homozygous genotypes. However, a valuable technique called speed breeding has been developed to address this bottleneck. Speed breeding significantly reduces the time required for variety development and selection, allowing the production of 5 to 6 wheat generations per year. Despite the accelerated growth achieved through speed breeding, the size of wheat plants remains shorter compared to traditional methods, although biomass has a positive correlation with plant yield. Biomass accumulation is influenced by the activity of the vital enzyme RuBisCO. An experiment was conducted on five wheat genotypes under normal sunlight and controlled conditions using light-emitting diodes (LEDs) to evaluate RuBisCO activity. The experiment followed a factorial, completely randomized design with three replications. Various traits, including plant height, flag leaf area, spike length, stomata size, chlorophyll content, biomass, and 1000-grain weight, were measured and showed significant differences among the genotypes and treatments. Proteomics analysis using SDS-PAGE revealed variability in RuBisCO protein expression between normal and controlled plants. Light intensity was also measured for sunlight and the speed of breeding chamber lights. The findings highlight the critical role of light intensity, which remains an indispensable factor in studies about biomass-related research. [ABSTRACT FROM AUTHOR]

Heliotropium thermophilum (Boraginaceae) plants have strong antioxidant properties. This study investigated the effectiveness of the antioxidant system in protecting the photosynthetic machinery of H. thermophilum. Plants were obtained from Kızıldere geothermal area in Buharkent district, Aydın, Turkey. Plants in the geothermal area that grew at 25–35°C were regarded as the low temperature group, while those that grew at 55–65°C were regarded as the high temperature group. We analysed the physiological changes of these plants at the two temperature conditions at stage pre-flowering and flowering. We meaured the effect of high soil temperature on water potential, malondialdehyde, cell membrane stability, and hydrogen peroxide analysis to determine stress levels on leaves and roots. Changes in antioxidant enzyme activities, ascorbate and chlorophyll content, chlorophyll fluorescence, photosynthetic gas exchange parameters, and photosynthetic enzymes (Rubisco and invertase) activities were also determined. Our results showed minimal changes to stress levels, indicating that plants were tolerant to high soil temperatures. In general, an increase in antioxidant enzyme activities, ascorbat levels, and all chlorophyll fluorescence parameters except for non-photochemical quenching (NPQ) and F v/ F m were observed. The pre-flowering and flowering stages were both characterised by decreased NPQ, despite F v/ F m not changing. Additionally, there was a rise in the levels of photosynthetic gas exchange parameters, Rubisco, and invertase activities. High temperature did not affect photosynthetic yield because H. thermophilum was found to stimulate antioxidant capacity, which reduces oxidative damage and maintains its photosynthetic machinery in high temperature conditions and therefore, it is tolerant to high soil temperature. Heliotropium thermophilum did not exhibit significant stress parameters under high soil temperature conditions, and it was a highly tolerant plant to high temperature. In the measurements taken at pre-flowering and flowering, plants in the pre-flowering stage were slightly more tolerant to high temperatures. The protection of PSII function in plants at higher temperatures could be linked to an increase in antioxidant activities. [ABSTRACT FROM AUTHOR]

The effect of elevated CO2 (eCO2) on changes in the morphophysiological, biochemical, and molecular genetic parameters of the C4-NADP halophyte Kochia prostrata (L.) Schrad. under short-term water deficit (WD) and/or elevated temperatures (eT) was studied. The changes in the parameters of growth, water–salt balance, CO2/H2O gas exchange, PSII efficiency, PSI cyclic electron transport (CET) activity, photosynthetic gene expression, key carboxylation enzyme content, and antioxidative system activity were studied. Plants reacted more negatively to water deficit than to other individual factors (eCO2 or eT). The expression of genes encoding components of PSI (psaA, psaB), ribulose-1,5-bisphosphate carboxylase/oxygenase Rubisco (rbcL), and pyruvate phosphate dikinase (PPDK) was downregulated, and a decrease was observed in the content of the photosynthetic enzymes Rubisco and phosphoenolpyruvate carboxylase (PEPС) and fresh biomass, PSII efficiency, apparent photosynthesis, and transpiration rates, indicating a stomatal and metabolic limitation of photosynthesis associated with insufficient functioning of the C4 carbon-concentrating mechanism. eT did not significantly affect the growth parameters and water–salt balance of K. prostrata. Decreased apparent photosynthesis intensity at eT was mainly associated with stomatal limitation (decreased transpiration). The most severe stress was caused by eT + WD. A decrease in psaA, psaB, rbcL, and PPDK expression, PSII efficiency and PSI CET activity, photosynthesis and transpiration intensity, fresh biomass and water content and an increase in proline accumulation and oxidative stress indices were observed. The effect of eCO2 mitigated the negative effect of individual and combined water deficit and temperature stress on CO2/H2O gas exchanges (apparent photosynthesis, transpiration) but enhanced their negative effect on PSII functioning. Additionally, under eT + WD, eCO2 contributed to a significant increase in proline content and the activation of antioxidant defense, with the participation of catalase, phenolic compounds, and PSI CET. Overall, eCO2 affected the mechanisms of homeostatic water balance and antioxidant defense and the ratio of light and dark reactions of photosynthesis during the adaptation of K. prostrata to drought and/or eT compared with ambient CO2 conditions. [ABSTRACT FROM AUTHOR]

Ribulose‐1,5‐bisphosphate carboxylase/oxygenase (RuBisCO) is the enzyme responsible for the first step of carbon dioxide (CO2) fixation in plants, which proceeds via the carboxylation of ribulose 1,5‐biphosphate. Because of the enormous importance of this reaction in agriculture and the environment, there is considerable interest in the mechanism of fixation of CO2 by RuBisCO. Here, a serial synchrotron crystallography structure of spinach RuBisCO is reported at 2.3 Å resolution. This structure is consistent with earlier single‐crystal X‐ray structures of this enzyme and the results are a good starting point for a further push towards time‐resolved serial synchrotron crystallography in order to better understand the mechanism of the reaction. [ABSTRACT FROM AUTHOR]

Ribulose bisphosphate carboxylase/oxygenase (RuBisCO), is a widely available plant protein receiving great interest because of its nutritional and functional properties. It can be a valuable source of protein for vegetarians. However, it has not received commercial significance due to the lack of a streamlined extraction process at the industrial scale, including its potential health benefits. In this review, we have summarized the literature on the biochemical characteristics of RuBisCO and compared its nutritional value with other plant proteins, as well as highlighted its digestibility, allergic traits, and potential health benefits. Moreover, the existing literature on the extraction of RuBisCO, associated challenges in industrial-scale RuBisCO purification, and recent innovations that occurred in this context are compiled. We believe this review will provide insights into RuBisCO's nutritional value and techno-functionality. Altogether, RuBisCO can be a sustainable source of protein in the future, especially for vegetarians. [ABSTRACT FROM AUTHOR]

SUMMARY: The introduction of the carboxysome‐based CO2 concentrating mechanism (CCM) into crop plants has been modelled to significantly increase crop yields. This projection serves as motivation for pursuing this strategy to contribute to global food security. The successful implementation of this engineering challenge is reliant upon the transfer of a microcompartment that encapsulates cyanobacterial Rubisco, known as the carboxysome, alongside active bicarbonate transporters. To date, significant progress has been achieved with respect to understanding various aspects of the cyanobacterial CCM, and more recently, different components of the carboxysome have been successfully introduced into plant chloroplasts. In this Perspective piece, we summarise recent findings and offer new research avenues that will accelerate research in this field to ultimately and successfully introduce the carboxysome into crop plants for increased crop yields. Significance Statement: The efficacy of the cyanobacterial CO2‐concentrating mechanism (CCM) relies on its ability to concentrate CO2 around the Rubisco enzyme, which is natively encapsulated within a carboxysome. As our understanding of carboxysome biogenesis and functionality grows, there is optimism that this microcompartment and the cyanobacterial CCM as a whole, can contribute to addressing global food security. [ABSTRACT FROM AUTHOR]

Photosynthesis is a process where solar energy is utilized to convert atmospheric CO2 into carbohydrates, which forms the basis for plant productivity. The increasing demand for food has created a global urge to enhance yield. Earlier, the plant breeding program was targeting the yield and yield-associated traits to enhance the crop yield. However, the yield cannot be further improved without improving the leaf photosynthetic rate. Hence, in this review, various strategies to enhance leaf photosynthesis were presented. The most promising strategies were the optimization of Rubisco carboxylation efficiency, the introduction of a CO2 concentrating mechanism in C3 plants, and the manipulation of photorespiratory bypasses in C3 plants, which are discussed in detail. Improving Rubisco's carboxylation efficiency is possible by engineering targets such as Rubisco subunits, chaperones, and Rubisco activase enzyme activity. Carbon-concentrating mechanisms can be introduced in C3 plants by the adoption of pyrenoid and carboxysomes, which can increase the CO2 concentration around the Rubisco enzyme. Photorespiration is the process by which the fixed carbon is lost through an oxidative process. Different approaches to reduce carbon and nitrogen loss were discussed. Overall, the potential approaches to improve the photosynthetic process and the way forward were discussed in detail. [ABSTRACT FROM AUTHOR]

Rice blast, caused by the fungal pathogen Magnaporthe oryzae (M. oryzae), is a highly destructive disease that significantly impacts rice yield and quality. During the infection, M. oryzae secretes effector proteins to subvert the host immune response. However, the interaction between the effector protein AvrPik-D and its target proteins in rice, and the mechanism by which AvrPik-D exacerbates disease severity to facilitate infection, remains poorly understood. In this study, we found that the M. oryzae effector AvrPik-D interacts with the Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) small subunit OsRBCS4. The overexpression of the OsRBCS4 gene in transgenic rice not only enhances resistance to M. oryzae but also induces more reactive oxygen species following chitin treatment. OsRBCS4 localizes to chloroplasts and co-localizes with AvrPik-D within these organelles. AvrPik-D suppresses the transcriptional expression of OsRBCS4 and inhibits Rubisco activity in rice. In conclusion, our results demonstrate that the M. oryzae effector AvrPik-D targets the Rubisco small subunit OsRBCS4 and inhibits its carboxylase and oxygenase activity, thereby suppressing rice innate immunity to facilitate infection. This provides a novel mechanism for the M. oryzae effector to subvert the host immunity to promote infection. [ABSTRACT FROM AUTHOR]

Carbon fixation is the process by which CO2 is converted from a gas into biomass. The Calvin-Benson-Bassham cycle (CBB) is the dominant carbon-consuming pathway on Earth, driving >99.5% of the ∼120 billion tons of carbon that are converted to sugar by plants, algae, and cyanobacteria. The carboxylase enzyme in the CBB, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco), fixes one CO2 molecule per turn of the cycle into bioavailable sugars. Despite being critical to the assimilation of carbon, rubisco's kinetic rate is not very fast, limiting flux through the pathway. This bottleneck presents a paradox: Why has rubisco not evolved to be a better catalyst? Many hypothesize that the catalytic mechanism of rubisco is subject to one or more trade-offs and that rubisco variants have been optimized for their native physiological environment. Here, we review the evolution and biochemistry of rubisco through the lens of structure and mechanism in order to understand what trade-offs limit its improvement. We also review the many attempts to improve rubisco itself and thereby promote plant growth.

The history of Earth's carbon cycle reflects trends in atmospheric composition convolved with the evolution of photosynthesis. Fortunately, key parts of the carbon cycle have been recorded in the carbon isotope ratios of sedimentary rocks. The dominant model used to interpret this record as a proxy for ancient atmospheric CO2 is based on carbon isotope fractionations of modern photoautotrophs, and longstanding questions remain about how their evolution might have impacted the record. Therefore, we measured both biomass (εp) and enzymatic (εRubisco) carbon isotope fractionations of a cyanobacterial strain (Synechococcus elongatus PCC 7942) solely expressing a putative ancestral Form 1B rubisco dating to ≫1 Ga. This strain, nicknamed ANC, grows in ambient pCO2 and displays larger εp values than WT, despite having a much smaller εRubisco (17.23 ± 0.61‰ vs. 25.18 ± 0.31‰, respectively). Surprisingly, ANC εp exceeded ANC εRubisco in all conditions tested, contradicting prevailing models of cyanobacterial carbon isotope fractionation. Such models can be rectified by introducing additional isotopic fractionation associated with powered inorganic carbon uptake mechanisms present in Cyanobacteria, but this amendment hinders the ability to accurately estimate historical pCO2 from geological data. Understanding the evolution of rubisco and the CO2 concentrating mechanism is therefore critical for interpreting the carbon isotope record, and fluctuations in the record may reflect the evolving efficiency of carbon fixing metabolisms in addition to changes in atmospheric CO2.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a pivotal enzyme that mediates the fixation of CO2. As the most abundant protein on earth, Rubisco has a significant impact on global carbon, water, and nitrogen cycles. However, the significantly low carboxylation activity and competing oxygenase activity of Rubisco greatly impede high carbon fixation efficiency. This review first summarizes the current efforts in directly or indirectly modifying plant Rubisco, which has been challenging due to its high conservation and limitations in chloroplast transformation techniques. However, recent advancements in understanding Rubisco biogenesis with the assistance of chaperones have enabled successful heterologous expression of all Rubisco forms, including plant Rubisco, in microorganisms. This breakthrough facilitates the acquisition and evaluation of modified proteins, streamlining the measurement of their activity. Moreover, the establishment of a screening system in E. coli opens up possibilities for obtaining high-performance mutant enzymes through directed evolution. Finally, this review emphasizes the utilization of Rubisco in microorganisms, not only expanding their carbon-fixing capabilities but also holding significant potential for enhancing biotransformation processes.

Form I rubiscos evolved in Cyanobacteria ≥ 2.5 billion years ago and are enzymatically unique due to the presence of small subunits (RbcS) capping both ends of an octameric large subunit (RbcL) rubisco assembly to form a hexadecameric (L8S8) holoenzyme. Although RbcS was previously thought to be integral to Form I rubisco stability, the recent discovery of a closely related sister clade of octameric rubiscos (Form I'; L8) demonstrates that the L8 complex can assemble without small subunits (Banda et al. 2020). Rubisco also displays a kinetic isotope effect (KIE) where the 3PG product is depleted in 13C relative to 12C. In Cyanobacteria, only two Form I KIE measurements exist, making interpretation of bacterial carbon isotope data difficult. To aid comparison, we measured in vitro the KIEs of Form I' (Candidatus Promineofilum breve) and Form I (Synechococcus elongatus PCC 6301) rubiscos and found the KIE to be smaller in the L8 rubisco (16.25 ± 1.36‱ vs. 22.42 ± 2.37‱, respectively). Therefore, while small subunits may not be necessary for protein stability, they may affect the KIE. Our findings may provide insight into the function of RbcS and allow more refined interpretation of environmental carbon isotope data.

BackgroundPermanently stratified lakes contain diverse microbial communities that vary with depth and so serve as useful models for studying the relationships between microbial community structure and geochemistry. Recent work has shown that these lakes can also harbor numerous bacteria and archaea from novel lineages, including those from the Candidate Phyla Radiation (CPR). However, the extent to which geochemical stratification differentially impacts carbon metabolism and overall genetic potential in CPR bacteria compared to other organisms is not well defined.ResultsHere, we determine the distribution of microbial lineages along an oxygen gradient in Lac Pavin, a deep, stratified lake in central France, and examine the influence of this gradient on their metabolism. Genome-based analyses revealed an enrichment of distinct C1 and CO2 fixation pathways in the oxic lake interface and anoxic zone/sediments, suggesting that oxygen likely plays a role in structuring metabolic strategies in non-CPR bacteria and archaea. Notably, we find that the oxidation of methane and its byproducts is largely spatially separated from methane production, which is mediated by diverse communities of sediment methanogens that vary on the centimeter scale. In contrast, we detected evidence for RuBisCO throughout the water column and sediments, including form II/III and form III-related enzymes encoded by CPR bacteria in the water column and DPANN archaea in the sediments. On the whole, though, CPR bacteria and phages did not show strong signals of gene content differentiation by depth, despite the fact that distinct species groups populate different lake and sediment compartments.ConclusionsOverall, our analyses suggest that environmental gradients in Lac Pavin select for capacities of CPR bacteria and phages to a lesser extent than for other bacteria and archaea. This may be due to the fact that selection in the former groups is indirect and depends primarily on host characteristics. Video Abstract.

Engineering bioenergy crops to accumulate coproducts in planta can increase the value of lignocellulosic biomass and enable a sustainable bioeconomy. In this study, we engineered sorghum with a bacterial gene encoding a chorismate pyruvate-lyase (ubiC) to reroute the plastidial pool of chorismate from the shikimate pathway into the valuable compound 4-hydroxybenzoic acid (4-HBA). A gene encoding a feedback-resistant version of 3-deoxy-d-arabino-heptulonate-7-phosphate synthase (aroG) was also introduced in an attempt to increase the carbon flux through the shikimate pathway. At the full maturity and senesced stage, two independent lines that co-express ubiC and aroG produced 1.5 and 1.7 dw% of 4-HBA in biomass, which represents 36- and 40-fold increases compared to the titer measured in wildtype. The two transgenic lines showed no obvious phenotypes, growth defects, nor alteration of cell wall polysaccharide content when cultivated under controlled conditions. In the field, when harvested before grain maturity, transgenic lines contained 0.8 and 1.2 dw% of 4-HBA, which represent economically relevant titers based on recent technoeconomic analysis. Only a slight reduction (11-15%) in biomass yield was observed in transgenics grown under natural environment. This work provides the first metabolic engineering steps toward 4-HBA overproduction in the bioenergy crop sorghum to improve the economics of biorefineries by accumulating a value-added coproduct that can be recovered from biomass and provide an additional revenue stream.

Many organisms that utilize the Calvin-Benson-Bassham (CBB) cycle for autotrophic growth harbor metabolic pathways to remove and/or salvage 2-phosphoglycolate, the product of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been presumed that the occurrence of 2-phosphoglycolate salvage is linked to the CBB cycle, and in particular, the C2 pathway to the CBB cycle and oxygenic photosynthesis. Here, we examined 2-phosphoglycolate salvage in the hyperthermophilic archaeon Thermococcus kodakarensis, an obligate anaerobe that harbors a Rubisco that functions in the pentose bisphosphate pathway. T. kodakarensis harbors enzymes that have the potential to convert 2-phosphoglycolate to glycine and serine, and their genes were identified by biochemical and/or genetic analyses. 2-phosphoglycolate phosphatase activity increased 1.6-fold when cells were grown under microaerobic conditions compared to anaerobic conditions. Among two candidates, TK1734 encoded a phosphatase specific for 2-phosphoglycolate, and the enzyme was responsible for 80% of the 2-phosphoglycolate phosphatase activity in T. kodakarensis cells. The TK1734 disruption strain displayed growth impairment under microaerobic conditions, which was relieved upon addition of sodium sulfide. In addition, glycolate was detected in the medium when T. kodakarensis was grown under microaerobic conditions. The results suggest that T. kodakarensis removes 2-phosphoglycolate via a phosphatase reaction followed by secretion of glycolate to the medium. As the Rubisco in T. kodakarensis functions in the pentose bisphosphate pathway and not in the CBB cycle, mechanisms to remove 2-phosphoglycolate in this archaeon emerged independent of the CBB cycle. [ABSTRACT FROM AUTHOR]

Terpenes are high-value chemicals which can be produced by engineered cyanobacteria from sustainable resources, solar energy, water and CO2. We previously reported that the euryhaline unicellular cyanobacteria Synechocystis sp. PCC 6803 (S.6803) and Synechococcus sp. PCC 7002 (S.7002) produce farnesene and limonene, respectively, more efficiently than other terpenes. In the present study, we attempted to enhance farnesene production in S.6803 and limonene production in S.7002. Practically, we tested the influence of key cyanobacterial enzymes acting in carbon fixation (RubisCO, PRK, CcmK3 and CcmK4), utilization (CrtE, CrtR and CruF) and storage (PhaA and PhaB) on terpene production in S.6803, and we compared some of the findings with the data obtained in S.7002. We report that the overproduction of RubisCO from S.7002 and PRK from Cyanothece sp. PCC 7425 increased farnesene production in S.6803, but not limonene production in S.7002. The overexpression of the crtE genes (synthesis of terpene precursors) from S.6803 or S.7002 did not increase farnesene production in S.6803. In contrast, the overexpression of the crtE gene from S.6803, but not S.7002, increased farnesene production in S.7002, emphasizing the physiological difference between these two model cyanobacteria. Furthermore, the deletion of the crtR and cruF genes (carotenoid synthesis) and phaAB genes (carbon storage) did not increase the production of farnesene in S.6803. Finally, as a containment strategy of genetically modified strains of S.6803, we report that the deletion of the ccmK3K4 genes (carboxysome for CO2 fixation) did not affect the production of limonene, but decreased the production of farnesene in S.6803. [ABSTRACT FROM AUTHOR]

Summary: The kinetic properties of Rubisco, the most important carbon‐fixing enzyme, have been assessed in a small fraction of the estimated existing biodiversity of photosynthetic organisms. Until recently, one of the most significant gaps of knowledge in Rubisco kinetics was marine macrophytes, an ecologically relevant group including brown (Ochrophyta), red (Rhodophyta) and green (Chlorophyta) macroalgae and seagrasses (Streptophyta). These organisms express various Rubisco types and predominantly possess CO2‐concentrating mechanisms (CCMs), which facilitate the use of bicarbonate for photosynthesis. Since bicarbonate is the most abundant form of dissolved inorganic carbon in seawater, CCMs allow marine macrophytes to overcome the slow gas diffusion and low CO2 availability in this environment. The present review aims to compile and integrate recent findings on the biochemical diversity of Rubisco and CCMs in the main groups of marine macrophytes. The Rubisco kinetic data provided demonstrate a more relaxed relationship among catalytic parameters than previously reported, uncovering a variability in Rubisco catalysis that has been hidden by a bias in the literature towards terrestrial vascular plants. The compiled data indicate the existence of convergent evolution between Rubisco and biophysical CCMs across the polyphyletic groups of marine macrophytes and suggest a potential role for oxygen in shaping such relationship. [ABSTRACT FROM AUTHOR]

Physaria fendleri is a member of the Brassicaceae that produces in its embryos hydroxy fatty acids, constituents of oils that are very valuable and widely used by industry for cosmetics, lubricants, biofuels, etc. Free of toxins and rich in hydroxy fatty acids, Physaria provides a promising alternative to imported castor oil and is on the verge of being commercialized. This study aims to identify important biochemical step(s) for oil synthesis in Physaria, which may serve as target(s) for future crop improvement. To advance towards this goal, the endosperm composition was analysed by LC-MS/MS to develop and validate culture conditions that mimic the development of the embryos in planta. Using developing Physaria embryos in culture and 13C-labeling, our studies revealed that: (i) Physaria embryos metabolize carbon into biomass with an efficiency significantly lower than other photosynthetic embryos; (ii) the plastidic malic enzyme provides 42% of the pyruvate used for de novo fatty acid synthesis, which is the highest measured so far in developing ‘green’ oilseed embryos; and (iii) Physaria uses non-conventional pathways to channel carbon into oil, namely the Rubisco shunt, which fixes CO2 released in the plastid, and the reversibility of isocitrate dehydrogenase, which provides additional carbon for fatty acid elongation. [ABSTRACT FROM AUTHOR]

Rubisco is the primary entry point for carbon into the biosphere. However, rubisco is widely regarded as inefficient leading many to question whether the enzyme can adapt to become a better catalyst. Through a phylogenetic investigation of the molecular and kinetic evolution of Form I rubisco we uncover the evolutionary trajectory of rubisco kinetic evolution in angiosperms. We show that rbcL is among the 1% of slowest-evolving genes and enzymes on Earth, accumulating one nucleotide substitution every 0.9 My and one amino acid mutation every 7.2 My. Despite this, rubisco catalysis has been continually evolving toward improved CO2/O2 specificity, carboxylase turnover, and carboxylation efficiency. Consistent with this kinetic adaptation, increased rubisco evolution has led to a concomitant improvement in leaf-level CO2 assimilation. Thus, rubisco has been slowly but continually evolving toward improved catalytic efficiency and CO2 assimilation in plants. [ABSTRACT FROM AUTHOR]

Pyrenoids are microcompartments that are universally found in the photosynthetic plastids of various eukaryotic algae. They contain ribulose-1,5-bisphosphate carbox-ylase/oxygenase (Rubisco) and play a pivotal role in facilitating CO2 assimilation via CO2-concentrating mechanisms (CCMs). Recent investigations involving model algae have revealed that pyrenoid-associated proteins participate in pyrenoid biogenesis and CCMs. However, these organisms represent only a small part of algal lineages, which limits our comprehensive understanding of the diversity and evolution of pyrenoid-based CCMs. Here we report a pyrenoid proteome of the chlorarachniophyte alga Amorphochlora amoebiformis, which possesses complex plastids acquired through secondary endosymbiosis with green algae. Proteomic analysis using mass spectrom-etry resulted in the identification of 154 potential pyrenoid components. Subsequent localization experiments demonstrated the specific targeting of eight proteins to pyrenoids. These included a putative Rubisco-binding linker, carbonic anhydrase, membrane transporter, and uncharacterized GTPase proteins. Notably, most of these proteins were unique to this algal lineage. We suggest a plausible scenario in which pyrenoids in chlorarachniophytes have evolved independently, as their components are not inherited from green algal pyrenoids. [ABSTRACT FROM AUTHOR]

The kinetic properties of Rubisco, a key enzyme for photosynthesis, have been examined in numerous plant species. However, this information on some plant groups, such as ferns, is scarce. This study examined Rubisco carboxylase activity and leaf Rubisco levels in seven ferns, including four Equisetum plants (E. arvense, E. hyemale, E. praealtum, and E. variegatum), considered living fossils. The turnover rates of Rubisco carboxylation (kcatc) in E. praealtum and E. hyemale were comparable to those in the C4 plants maize (Zea mays) and sorghum (Sorghum bicolor), whose kcatc values are high. Rubisco CO2 affinity, estimated from the percentage of Rubisco carboxylase activity under CO2 unsaturated conditions in kcatc in these Equisetum plants, was low and also comparable to that in maize and sorghum. In contrast, kcatc and CO2 affinities of Rubisco in other ferns, including E. arvense and E. variegatum were comparable with those in C3 plants. The N allocation to Rubisco in the ferns examined was comparable to that in the C3 plants. These results indicate that E. praealtum and E. hyemale have abundant Rubisco with high kcatc and low CO2 affinity, whereas the carboxylase activity and abundance of Rubisco in other ferns were similar to those in C3 plants. Herein, the Rubisco properties of E. praealtum and E. hyemale were discussed regarding their evolution and physiological implications. [ABSTRACT FROM AUTHOR]

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a pivotal enzyme that mediates the fixation of CO2. As the most abundant protein on earth, Rubisco has a significant impact on global carbon, water, and nitrogen cycles. However, the significantly low carboxylation activity and competing oxygenase activity of Rubisco greatly impede high carbon fixation efficiency. This review first summarizes the current efforts in directly or indirectly modifying plant Rubisco, which has been challenging due to its high conservation and limitations in chloroplast transformation techniques. However, recent advancements in understanding Rubisco biogenesis with the assistance of chaperones have enabled successful heterologous expression of all Rubisco forms, including plant Rubisco, in microorganisms. This breakthrough facilitates the acquisition and evaluation of modified proteins, streamlining the measurement of their activity. Moreover, the establishment of a screening system in E. coli opens up possibilities for obtaining high-performance mutant enzymes through directed evolution. Finally, this review emphasizes the utilization of Rubisco in microorganisms, not only expanding their carbon-fixing capabilities but also holding significant potential for enhancing biotransformation processes. [ABSTRACT FROM AUTHOR]

This study aimed to determine the photomixotrophic and physiological responses using different temporary immersion systems (TIS) during in vitro multiplication of agave Tobalá. The culture systems were SETIS™ bioreactor, Temporary Immersion Bioreactor (TIB), Monobloc Advance Temporary Immersion System, and semisolid culture medium as the control. At six weeks of culture, different physiological variables were evaluated: chlorophyll content, stomatal index, percentage of closed stomata, Phosphoenolpyruvate (PEP) and Rubisco during the multiplication stage, and survival percentage in the acclimatization stage. TIS increased multiplication rate (41%), stomatal index (44%), percentage of closed stomata (11%) and chlorophyll content (45%) with respect to the semisolid culture medium system. The highest PEP content (> 35%) was observed in temporary immersion (TI), whereas, Rubisco content, showed no differences among the culture systems evaluated. Regarding survival percentage during acclimatization, the highest shoot survival was obtained in TI, and all regenerate shoots were rooted ex vitro. This study shows that in vitro photomixotrophism was induced in TIS during the multiplication stage. In conclusion, SETIS™ bioreactor and TIB systems are an alternative for mass multiplication of A. potatorum; however, all TIS evaluated guarantee a high survival rate during acclimatization. [ABSTRACT FROM AUTHOR]

Photosynthesis is responsible for sustained plant productivity and ensures food supply. The change in global climatic patterns affects photosynthesis that subsequently reduces plant yield and poses threat to food security. Photosynthesis relies on a dual nature enzyme ribulose 1, 5 bisphosphate carboxylase oxygenase (Rubisco), which can fix CO2 as well as O2. The fixation rate of CO2 to O2 depends upon the relative concentration of CO2 inside chloroplast. Higher level of CO2 results in improved photosynthesis, however, its concentration depends upon environmental conditions. Under adverse climate conditions, the CO2 level drops down that leads to increased oxygenation which impedes the photosynthesis and reduces plant productivity. The impact is more significant and apparent specifically in C3 plants. Attempts have been made to address the loss in photosynthesis and multiple strategies have been adapted to date that focus on improvement of photosynthesis in C3 plants. In this review, we have discussed the multiple strategies being employed by different researchers to date for improvement of photosynthesis. The strategies discussed in this review include: improving the performance of Rubisco, engineering CO2-concentrating mechanism of C4 photosynthesis into C3 species, transformation of bicarbonate transporters from cyanobacteria into chloroplasts of C3 plants, and establishment of photorespiratory bypasses to catabolise toxic glycolate in shortest possible pathway. [ABSTRACT FROM AUTHOR]

Tropospheric ozone (O3) is a significant abiotic stressor whose rising concentration negatively influences plant growth. Studies related to the differential response of Abelmoschus cytotypes to elevatedO3 treatment are scarce and need further exploration to recognise the role of polyploidisation in stress tolerance. In this study, we analysed the changes in growth pattern, ultrastructure, physiology and foliar protein profile occurring under O3 stress in Abelmoschus moschatus (monoploid), Abelmoschus esculentus (diploid) and Abelmoschus caillei (triploid). Our findings showed that higher stomatal conductance in A. moschatus triggered higher O3 intake, causing damage to stomatal cells and photosynthetic pigments. Additionally, it caused a reduction in photosynthetic rates, leading to reduced plant growth, total biomass and economic yield. ThisO3-induced toxicity was less in diploid and triploid cytotypes of Abelmoschus. Protein profiling by sodium dodecyl sulpate-polyacrylamide gel electrophoresis showed a significant decrease in the commonly found RuBisCO larger and smaller subunits. The decrease was more prominent in monoploid compared to diploid and triploid. This study provides crucial data for research that aim to enhance plant ability to withstand O3 induced oxidative stress. Our findings may help in developing a tolerant variety through plant breeding techniques, which will be economically more advantageous in reaching the objective of sustainable production at the high O3 levels projected under a climate change scenario. [ABSTRACT FROM AUTHOR]

Ribulose bisphosphate carboxylase/oxygenase (RuBisCO), is a widely available plant protein receiving great interest because of its nutritional and functional properties. It can be a valuable source of protein for vegetarians. However, it has not received commercial significance due to the lack of a streamlined extraction process at the industrial scale, including its potential health benefits. In this review, we have summarized the literature on the biochemical characteristics of RuBisCO and compared its nutritional value with other plant proteins, as well as highlighted its digestibility, allergic traits, and potential health benefits. Moreover, the existing literature on the extraction of RuBisCO, associated challenges in industrial-scale RuBisCO purification, and recent innovations that occurred in this context are compiled. We believe this review will provide insights into RuBisCO’s nutritional value and techno-functionality. Altogether, RuBisCO can be a sustainable source of protein in the future, especially for vegetarians.

Throughout the tree of life, cells and organisms enter states of dormancy or hibernation as a key feature of their biology: from a bacterium arresting its growth in response to starvation, to a plant seed anticipating placement in fertile ground, to a human oocyte poised for fertilization to create a new life. Recent research shows that when cells hibernate, many of their essential enzymes hibernate too: they disengage from their substrates and associate with a specialized group of proteins known as hibernation factors. Here, we summarize how hibernation factors protect essential cellular enzymes from undesired activity or irreparable damage in hibernating cells. We show how molecular hibernation, once viewed as rare and exclusive to certain molecules like ribosomes, is in fact a widespread property of biological molecules that is required for the sustained persistence of life on Earth.

The Hyphomicrobiales (Rhizobiales) order contains soil bacteria with an irregular distribution of the Calvin–Benson–Bassham cycle (CBB). Key enzymes in the CBB cycle are ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), whose large and small subunits are encoded in cbbL and cbbS, and phosphoribulokinase (PRK), encoded by cbbP. These genes are often found in cbb operons, regulated by the LysR-type regulator CbbR. In Bradyrhizobium, pertaining to this order and bearing photosynthetic and non-photosynthetic species, the number of cbbL and cbbS copies varies, for example: zero in B. manausense, one in B. diazoefficiens, two in B. japonicum, and three in Bradyrhizobium sp. BTAi. Few studies addressed the role of CBB in Bradyrhizobium spp. symbiosis with leguminous plants. To investigate the horizontal transfer of the cbb operon among Hyphomicrobiales, we compared phylogenetic trees for concatenated cbbL-cbbP-cbbR and housekeeping genes (atpD-gyrB-recA-rpoB-rpoD). The distribution was consistent, indicating no horizontal transfer of the cbb operon in Hyphomicrobiales. We constructed a ΔcbbLS mutant in B. diazoefficiens, which lost most of the coding sequence of cbbL and has a frameshift creating a stop codon at the N-terminus of cbbS. This mutant nodulated normally but had reduced competitiveness for nodulation and long-term adhesion to soybean (Glycine max (L.) Merr.) roots, indicating a CBB requirement for colonizing soybean rhizosphere.

The pyrenoid is a chloroplastic microcompartment in which most algae and some terrestrial plants condense the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) as part of a CO2-concentrating mechanism that improves the efficiency of CO2 capture. Engineering a pyrenoid-based CO2-concentrating mechanism (pCCM) into C3 crop plants is a promising strategy to enhance yield capacities and resilience to the changing climate. Many pyrenoids are characterized by a sheath of starch plates that is proposed to act as a barrier to limit CO2 diffusion. Recently, we have reconstituted a phase-separated "proto-pyrenoid" Rubisco matrix in the model C3 plant Arabidopsis thaliana using proteins from the alga with the most well-studied pyrenoid, Chlamydomonas reinhardtii [N. Atkinson, Y. Mao, K. X. Chan, A. J. McCormick, Nat. Commun. 11, 6303 (2020)]. Here, we describe the impact of introducing the Chlamydomonas proteins StArch Granules Abnormal 1 (SAGA1) and SAGA2, which are associated with the regulation of pyrenoid starch biogenesis and morphology. We show that SAGA1 localizes to the proto-pyrenoid in engineered Arabidopsis plants, which results in the formation of atypical spherical starch granules enclosed within the proto-pyrenoid condensate and adjacent plate-like granules that partially cover the condensate, but without modifying the total amount of chloroplastic starch accrued. Additional expression of SAGA2 further increases the proportion of starch synthesized as adjacent plate-like granules that fully encircle the proto-pyrenoid. Our findings pave the way to assembling a diffusion barrier as part of a functional pCCM in vascular plants, while also advancing our understanding of the roles of SAGA1 and SAGA2 in starch sheath formation and broadening the avenues for engineering starch morphology. [ABSTRACT FROM AUTHOR]

SUMMARY: Plants grown under low magnesium (Mg) soils are highly susceptible to encountering light intensities that exceed the capacity of photosynthesis (A), leading to a depression of photosynthetic efficiency and eventually to photooxidation (i.e., leaf chlorosis). Yet, it remains unclear which processes play a key role in limiting the photosynthetic energy utilization of Mg‐deficient leaves, and whether the plasticity of A in acclimation to irradiance could have cross‐talk with Mg, hence accelerating or mitigating the photodamage. We investigated the light acclimation responses of rapeseed (Brassica napus) grown under low‐ and adequate‐Mg conditions. Magnesium deficiency considerably decreased rapeseed growth and leaf A, to a greater extent under high than under low light, which is associated with higher level of superoxide anion radical and more severe leaf chlorosis. This difference was mainly attributable to a greater depression in dark reaction under high light, with a higher Rubisco fallover and a more limited mesophyll conductance to CO2 (gm). Plants grown under high irradiance enhanced the content and activity of Rubisco and gm to optimally utilize more light energy absorbed. However, Mg deficiency could not fulfill the need to activate the higher level of Rubisco and Rubisco activase in leaves of high‐light‐grown plants, leading to lower Rubisco activation and carboxylation rate. Additionally, Mg‐deficient leaves under high light invested more carbon per leaf area to construct a compact leaf structure with smaller intercellular airspaces, lower surface area of chloroplast exposed to intercellular airspaces, and CO2 diffusion conductance through cytosol. These caused a more severe decrease in within‐leaf CO2 diffusion rate and substrate availability. Taken together, plant plasticity helps to improve photosynthetic energy utilization under high light but aggravates the photooxidative damage once the Mg nutrition becomes insufficient. Significance Statement: Plants grown under low Mg soils are highly susceptible to excessive light intensity, resulting in photooxidation and subsequent leaf chlorosis. The results indicated that plant plasticity, such as increasing Rubisco content and gm, helps to improve photosynthetic energy utilization under high light but aggravates the photooxidative damage once the Mg nutrition becomes insufficient. [ABSTRACT FROM AUTHOR]

Summary: Efficient plant acclimation to changing environmental conditions relies on fast adjustments of the transcriptome, proteome, and metabolome. Regulation of enzyme activity depends on the activity of specific chaperones, chemical post‐translational modifications (PTMs) of amino acid residues, and changes in the cellular and organellar microenvironment. Central to carbon assimilation, and thus plant growth and yield, Rubisco activity is regulated by its chaperone Rubisco activase (Rca) and by adjustments in the chloroplast stroma environment. Focused on crops, this review highlights the main PTMs and stromal ions and metabolites affecting Rubisco and Rca in response to environmental stimuli. Rca isoforms differ in regulatory properties and heat sensitivity, with expression changing according to the surrounding environment. Much of the physiological relevance of Rubisco and Rca PTMs is still poorly understood, though some PTMs have been associated with Rubisco regulation in response to stress. Ion and metabolite concentrations in the chloroplast change in response to variations in light and temperature. Some of these changes promote Rubisco activation while others inhibit activation, deactivate the enzyme, or change the rates of catalysis. Understanding these regulatory mechanisms will aid the development of strategies to improve carbon fixation by Rubisco under rapidly changing environments as experienced by crop plants. [ABSTRACT FROM AUTHOR]

Species distributed across a wide elevation range have broad environmental tolerance and adopt specific adaptation strategies to cope with varying climatic conditions. The aim of this study is to understand the patterns of variation in leaf eco-physiological traits that are related to the adaptation of species with a wide distribution in different climatic conditions. We studied the variability in eco-physiological traits of two co-occurring species of Western Himalaya (Rumex nepalensis and Taraxacum officinale), along elevational gradients. We conducted our study in elevations ranging from 1000 to 4000 m a.s.l. in three transects separated in an eco-region spanning 2.5° latitudes and 2.3° longitudes in the Western Himalaya. We hypothesized substantial variation in eco-physiological traits, especially increased net rate of photosynthesis (PN), Rubisco specific activity (RSA), and biochemicals at higher elevations, enabling species to adapt to varying environmental conditions. Therefore, the photosynthetic measurements along with leaf sampling were carried out during the months of June–August and the variations in photosynthetic performance and other leaf traits were assessed. Data was analyzed using a linear mixed effect model with 'species,' 'elevation' as fixed and 'transect' as random factor. Elevation had a significant effect on majority of traits. It was found that PN and maximum carboxylation rate of Rubisco (Vcmax) have unimodal or declining trend along increasing elevations. High RSA was observed at higher elevations in all the three transects. Trends for biochemical traits such as total soluble sugars, total soluble proteins, proline, and total phenolics content suggested an increase in these traits for the survival of plants in harsh environments of higher elevations. Our study reveals that although there is considerable variation in the eco-physiological traits of the two species across elevational gradients of different transects, there are certain similarities in the patterns that depict their high adaptive potential in varying climatic conditions. [ABSTRACT FROM AUTHOR]

The bacterial chaperonin GroEL-GroES promotes protein folding through ATP-regulated cycles of substrate protein binding, encapsulation, and release. Here, we have used cryoEM to determine structures of GroEL, GroEL-ADP ·BeF3, and GroEL-ADP ·AlF3-GroES all complexed with the model substrate Rubisco. Our structures provide a series of snapshots that show how the conformation and interactions of non-native Rubisco change as it proceeds through the GroEL-GroES reaction cycle. We observe specific charged and hydrophobic GroEL residues forming strong initial contacts with non-native Rubisco. Binding of ATP or ADP·BeF3 to GroEL-Rubisco results in the formation of an intermediate GroEL complex displaying striking asymmetry in the ATP/ADP·BeF3-bound ring. In this ring, four GroEL subunits bind Rubisco and the other three are in the GroES-accepting conformation, suggesting how GroEL can recruit GroES without releasing bound substrate. Our cryoEM structures of stalled GroEL-ADP ·AlF3-Rubisco-GroES complexes show Rubisco folding intermediates interacting with GroEL-GroES via different sets of residues. [ABSTRACT FROM AUTHOR]