C4 Grasses Employ Distinct Strategies to Acclimate Rubisco Activase to Heat Stress.

Rising temperatures due to the current climate crisis will soon have devastating impacts on crop performance and resilience. In particular, CO2 assimilation is dramatically limited at high temperatures. CO2 assimilation is accomplished by rubisco, which is inhibited by the binding of inhibitory sugar phosphates to its active site. Plants therefore utilize the essential chaperone rubisco activase (RCA) to remove these inhibitors and enable continued CO2 fixation. However, RCA does not function at moderately high temperatures (42oC), resulting in impaired rubisco activity and reduced CO2 assimilation. We set out to understand temperature-dependent RCA regulation in four different C4 plants, with a focus on the crop plants maize (two cultivars) and sorghum, as well as the model grass Setaria viridis (setaria) using gas exchange measurements, which confirm that CO2 assimilation is limited by carboxylation in these organisms at high temperatures (42oC). All three species express distinct complements of RCA isoforms and each species alters the isoform and proteoform abundances in response to heat; however, the changes are species-specific. We also examine whether the heat-mediated inactivation of RCA is due to biochemical regulation rather than simple thermal denaturation. We reveal that biochemical regulation affects RCA function differently in different C4 species, and differences are apparent even between different cultivars of the same species. Our results suggest that each grass evolved different strategies to maintain RCA function during stress and we conclude that a successful engineering approach aimed at improving carbon capture in C4 grasses will need to accommodate these individual regulatory mechanisms.

A thaumatin-like effector protein suppresses the rust resistance of wheat and promotes the pathogenicity of Puccinia triticina by targeting TaRCA.

Thaumatin-like proteins (TLPs) in plants play a crucial role in combating stress, and they have been proven to possess antifungal properties. However, the role of TLPs in pathogens has not been reported. We identified a effector protein, Pt9029, which contained a Thaumatin domain in Puccinia triticina (Pt), possessing a chloroplast transit peptide and localized in the chloroplasts. Silencing Pt9029 in the Pt physiological race THTT resulted in a notable reduction in virulence and stunted growth and development of Pt hypha in near-isogenic wheat line TcLr2b. Overexpression of Pt9029 in wheat exerted a suppressive effect on H2O2 production, consequently impeding the wheat's disease resistance mechanisms. The TLP domain of Pt9029 targets the Rubisco activase (TaRCA) in chloroplasts. This interaction effectively inhibited the function of TaRCA, subsequently leading to a decrease in Rubisco enzyme activity. Therefore, this indicates that TLPs in Pt can inhibit host defense mechanisms during the pathogenic process of Pt. Moreover, TaRCA silencing resulted in reduced resistance of TcLr2b against Pt race THTT. This clearly demonstrated that TaRCA positively regulates wheat resistance to leaf rust. These findings reveal a novel strategy exploited by Pt to manipulate wheat rust resistance and promote pathogenicity.

Antibody Design for the Quantification of Photosynthetic Proteins and Their Isoforms.

Antibodies are a valuable research tool, with uses including detection and quantification of specific proteins. By using peptide fragments to raise antibodies, they can be designed to differentiate between structurally similar proteins, or to bind conserved motifs in divergent proteins. Peptide sequence selection and antibody validation are crucial to ensure reliable results from antibody-based experiments. This chapter describes the steps for the identification of peptide sequences to produce protein- or isoform-specific antibodies using recombinant technologies as well as the subsequent validation of such antibodies. The photosynthetic protein Rubisco activase is used as a case study to explain the various steps involved and key aspects to take into consideration.

Extraction of Soluble Proteins from Leaves.

Protein biochemistry can provide valuable answers to better understand plant performance and responses to the surrounding environment. In this chapter, we describe the process of extracting proteins from plant leaf samples. We highlight the key aspects to take into consideration to preserve protein integrity, from sample collection to extraction and preparation or storage for subsequent analysis of protein abundance and/or enzymatic activities.

Down-regulation of wheat Rubisco activase isoforms expression by virus-induced gene silencing.

Rubisco activase (Rca) is an essential photosynthetic enzyme that removes inhibitors from the catalytic sites of the carboxylating enzyme Rubisco. In wheat, Rca is composed of one longer 46 kDa α-isoform and two shorter 42 kDa ß-isoforms encoded by the genes TaRca1 and TaRca2. TaRca1 produces a single transcript from which a short 1ß-isoform is expressed, whereas two alternative transcripts are generated from TaRca2 directing expression of either a long 2α-isoform or a short 2ß-isoform. The 2ß isoform is similar but not identical to 1ß. Here, virus-induced gene silencing (VIGS) was used to silence the different TaRca transcripts. Abundance of the transcripts and the respective protein isoforms was then evaluated in the VIGS-treated and control plants. Remarkably, treatment with the construct specifically targeting TaRca1 efficiently decreased expression not only of TaRca1 but also of the two alternative TaRca2 transcripts. Similarly, specific targeting of the TaRca2 transcript encoding a long isoform TaRca2α resulted in silencing of both TaRca2 alternative transcripts. The corresponding protein isoforms decreased in abundance. These findings indicate concomitant down-regulation of TaRca1 and TaRca2 at both transcript and protein levels and may impact the feasibility of altering the relative abundance of Rca isoforms in wheat.

Jasmonic acid improves barley photosynthetic efficiency through a possible regulatory module, MYC2-RcaA, under combined drought and salinity stress.

The combined stress of drought and salinity is prevalent in various regions of the world, affects several physiological and biochemical processes in crops, and causes their yield to decrease. Photosynthesis is one of the main processes that are disturbed by combined stress. Therefore, improving the photosynthetic efficiency of crops is one of the most promising strategies to overcome environmental stresses, making studying the molecular basis of regulation of photosynthesis a necessity. In this study, we sought a potential mechanism that regulated a major component of the combined stress response in the important crop barley (Hordeum vulgare L.), namely the Rubisco activase A (RcaA) gene. Promoter analysis of the RcaA gene led to identifying Jasmonic acid (JA)-responsive elements with a high occurrence. Specifically, a Myelocytomatosis oncogenes 2 (MYC2) transcription factor binding site was highlighted as a plausible functional promoter motif. We conducted a controlled greenhouse experiment with an abiotic stress-susceptible barley genotype and evaluated expression profiling of the RcaA and MYC2 genes, photosynthetic parameters, plant water status, and cell membrane damages under JA, combined drought and salinity stress (CS) and JA + CS treatments. Our results showed that applying JA enhances barley's photosynthetic efficiency and water relations and considerably compensates for the adverse effects of combined stress. Significant association was observed among gene expression profiles and evaluated physiochemical characteristics. The results showed a plausible regulatory route through the JA-dependent MYC2-RcaA module involved in photosynthesis regulation and combined stress tolerance. These findings provide valuable knowledge for further functional studies of the regulation of photosynthesis under abiotic stresses toward the development of multiple-stress-tolerant crops.

Regulation of Rubisco activity in crops.

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.

The challenge of engineering Rubisco for improving photosynthesis.

Photosynthesis uses the energy of sunlight to convert water and atmospheric CO2 into sugars, providing food and oxygen for life. The fixation of atmospheric CO2 in this crucial biological process is mediated by the enzyme Rubisco. The inefficiencies of Rubisco have inspired researchers for decades to explore ways to improve its function with the goal of increasing crop yields [1-4], and more recently to combat global warming [5]. In this graphical review we highlight the challenges involved in engineering plant Rubisco, with a focus on the extensive chaperone requirement for its biogenesis. We discuss strategies for engineering the catalytic properties of Rubisco and for sequestering the enzyme in membraneless compartments to increase CO2 fixation.

Effects of OsRCA Overexpression on Rubisco Activation State and Photosynthesis in Maize.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the rate-limiting enzyme for photosynthesis. Rubisco activase (RCA) can regulate the Rubisco activation state, influencing Rubisco activity and photosynthetic rate. We obtained transgenic maize plants that overproduced rice RCA (OsRCAOE) and evaluated photosynthesis in these plants by measuring gas exchange, energy conversion efficiencies in photosystem (PS) I and PSII, and Rubisco activity and activation state. The OsRCAOE lines showed significantly higher initial Rubisco activity and activation state, net photosynthetic rate, and PSII photochemical quantum yield than wild-type plants. These results suggest that OsRCA overexpression can promote maize photosynthesis by increasing the Rubisco activation state.

Recent developments in the engineering of Rubisco activase for enhanced crop yield.

Rubisco activase (RCA) catalyzes the release of inhibitory sugar phosphates from ribulose-1,6-biphosphate carboxylase/oxygenase (Rubisco) and can play an important role in biochemical limitations of photosynthesis under dynamic light and elevated temperatures. There is interest in increasing RCA activity to improve crop productivity, but a lack of understanding about the regulation of photosynthesis complicates engineering strategies. In this review, we discuss work relevant to improving RCA with a focus on advances in understanding the structural cause of RCA instability under heat stress and the regulatory interactions between RCA and components of photosynthesis. This reveals substantial variation in RCA thermostability that can be influenced by single amino acid substitutions, and that engineered variants can perform better in vitro and in vivo under heat stress. In addition, there are indications RCA activity is controlled by transcriptional, post-transcriptional, post-translational, and spatial regulation, which may be important for balancing between carbon fixation and light capture. Finally, we provide an overview of findings from recent field experiments and consider the requirements for commercial validation as part of efforts to increase crop yields in the face of global climate change.

Molecular mechanism of Rubisco activase: Dynamic assembly and Rubisco remodeling.

Ribulose-1,5-bisphosphate (RuBP) carboxylase-oxygenase (Rubisco) enzyme is the limiting step of photosynthetic carbon fixation, and its activation is regulated by its co-evolved chaperone, Rubisco activase (Rca). Rca removes the intrinsic sugar phosphate inhibitors occupying the Rubisco active site, allowing RuBP to split into two 3-phosphoglycerate (3PGA) molecules. This review summarizes the evolution, structure, and function of Rca and describes the recent findings regarding the mechanistic model of Rubisco activation by Rca. New knowledge in these areas can significantly enhance crop engineering techniques used to improve crop productivity.

Dynamics of Rubisco regulation by sugar phosphate derivatives and their phosphatases.

Regulating the central CO2-fixing enzyme Rubisco is as complex as its ancient reaction mechanism and involves interaction with a series of cofactors and auxiliary proteins that activate catalytic sites and maintain activity. A key component among the regulatory mechanisms is the binding of sugar phosphate derivatives that inhibit activity. Removal of inhibitors via the action of Rubisco activase is required to restore catalytic competency. In addition, specific phosphatases dephosphorylate newly released inhibitors, rendering them incapable of binding to Rubisco catalytic sites. The best studied inhibitor is 2-carboxy-d-arabinitol 1-phosphate (CA1P), a naturally occurring nocturnal inhibitor that accumulates in most species during darkness and low light, progressively binding to Rubisco. As light increases, Rubisco activase removes CA1P from Rubisco, and the specific phosphatase CA1Pase dephosphorylates CA1P to CA, which cannot bind Rubisco. Misfire products of Rubisco's complex reaction chemistry can also act as inhibitors. One example is xylulose-1,5-bisphosphate (XuBP), which is dephosphorylated by XuBPase. Here we revisit key findings related to sugar phosphate derivatives and their specific phosphatases, highlighting outstanding questions and how further consideration of these inhibitors and their role is important for better understanding the regulation of carbon assimilation.

Phase Separation of Rubisco by the Folded SSUL Domains of CcmM in Beta-Carboxysome Biogenesis.

Carboxysomes are large, cytosolic bodies present in all cyanobacteria and many proteobacteria that function as the sites of photosynthetic CO2 fixation by the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The carboxysome lumen is enriched with Rubisco and carbonic anhydrase (CA). The polyhedral proteinaceous shell allows the passage of HCO3- ions into the carboxysome, where they are converted to CO2 by CA. Thus, the carboxysome functions as a CO2-concentrating mechanism (CCM), enhancing the efficiency of Rubisco in CO2 fixation. In ß-cyanobacteria, carboxysome biogenesis first involves the aggregation of Rubisco by CcmM, a scaffolding protein that exists in two isoforms. Both isoforms contain a minimum of three Rubisco small subunit-like (SSUL) domains, connected by flexible linkers. Multivalent interaction between these linked SSUL domains with Rubisco results in phase separation and condensate formation. Here, we use Rubisco and the short isoform of CcmM (M35) of the ß-cyanobacterium Synechococcus elongatus PCC7942 to describe the methods used for in vitro analysis of the mechanism of condensate formation driven by the SSUL domains. The methods include turbidity assays, bright-field and fluorescence microscopy, as well as transmission electron microscopy (TEM) in both negative staining and cryo-conditions.

Improving plant heat tolerance through modification of Rubisco activase in C3 plants to secure crop yield and food security in a future warming world.

The world's population may reach 10 billion by 2050, but 10% still suffer from food shortages. At the same time, global warming threatens food security by decreasing crop yields, so it is necessary to develop crops with enhanced resistance to high temperatures in order to secure the food supply. In this review, the role of Rubisco activase as an important factor in plant heat tolerance is summarized, based on the conclusions of recent findings. Rubisco activase is a molecular chaperone determining the activation of Rubisco, whose heat sensitivity causes reductions of photosynthesis at high temperatures. Thus, the thermostability of Rubisco activase is considered to be critical for improving plant heat tolerance. It has been shown that the introduction of thermostable Rubisco activase through gene editing into Arabidopsis thaliana and from heat-adapted wild Oryza species or C4Zea mays into Oryza sativa improves Rubisco activation, photosynthesis, and plant growth at high temperatures. We propose that developing a universal thermostable Rubisco activase could be a promising direction for further studies.

Regulation of Calvin-Benson cycle enzymes under high temperature stress.

The Calvin-Benson cycle (CBC) consists of three critical processes, including fixation of CO2 by Rubisco, reduction of 3-phosphoglycerate (3PGA) to triose phosphate (triose-P) with NADPH and ATP generated by the light reactions, and regeneration of ribulose 1,5-bisphosphate (RuBP) from triose-P. The activities of photosynthesis-related proteins, mainly from the CBC, were found more significantly affected and regulated in plants challenged with high temperature stress, including Rubisco, Rubisco activase (RCA) and the enzymes involved in RuBP regeneration, such as sedoheptulose-1,7-bisphosphatase (SBPase). Over the past years, the regulatory mechanism of CBC, especially for redox-regulation, has attracted major interest, because balancing flux at the various enzymatic reactions and maintaining metabolite levels in a range are of critical importance for the optimal operation of CBC under high temperature stress, providing insights into the genetic manipulation of photosynthesis. Here, we summarize recent progress regarding the identification of various layers of regulation point to the key enzymes of CBC for acclimation to environmental temperature changes along with open questions are also discussed.

Removal of redox-sensitive Rubisco Activase does not alter Rubisco regulation in soybean.

Rubisco activase (Rca) facilitates the catalytic repair of Rubisco, the CO2-fixing enzyme of photosynthesis, following periods of darkness, low to high light transitions or stress. Removal of the redox-regulated isoform of Rubisco activase, Rca-α, enhances photosynthetic induction in Arabidopsis and has been suggested as a strategy for the improvement of crops, which may experience frequent light transitions in the field; however, this has never been tested in a crop species. Therefore, we used RNAi to reduce the Rca-α content of soybean (Glycine max cv. Williams 82) below detectable levels and then characterized the growth, photosynthesis, and Rubisco activity of the resulting transgenics, in both growth chamber and field conditions. Under a 16 h sine wave photoperiod, the reduction of Rca-α contents had no impact on morphological characteristics, leaf expansion rate, or total biomass. Photosynthetic induction rates were unaltered in both chamber-grown and field-grown plants. Plants with reduced Rca-α content maintained the ability to regulate Rubisco activity in low light just as in control plants. This result suggests that in soybean, Rca-α is not as centrally involved in the regulation of Rca oligomer activity as it is in Arabidopsis. The isoform stoichiometry supports this conclusion, as Rca-α comprises only ~ 10% of the Rubisco activase content of soybean, compared to ~ 50% in Arabidopsis. This is likely to hold true in other species that contain a low ratio of Rca-α to Rca-ß isoforms.

Downregulation of a gibberellin 3ß-hydroxylase enhances photosynthesis and increases seed yield in soybean.

Seed yield, determined mainly by seed numbers and seed weight, is the primary target of soybean breeding. Identifying the genes underlying yield-related traits is of great significance. Through joint linkage mapping and a genome-wide association study for 100-seed weight, we cloned GmGA3ox1, a gene encoding gibberellin 3ß-hydroxylase, which is the key enzyme in the gibberellin synthesis pathway. Genome resequencing identified a beneficial GmGA3ox1 haplotype contributing to high seed weight, which was further confirmed by soybean transformants. CRISPR/Cas9-generated gmga3ox1 mutants showed lower seed weight, but promoted seed yield by increasing seed numbers. The gmga3ox1 mutants reduced gibberellin biosynthesis while enhancing photosynthesis. Knockout of GmGA3ox1 resulted in the upregulation of numerous photosynthesis-related genes, particularly the GmRCA family encoding ribulose-1,5-bispho-sphate carboxylase-oxygenase (Rubisco) activases. The basic leucine zipper transcription factors GmbZIP97 and GmbZIP159, which were both upregulated in the gmga3ox1 mutants and induced by the gibberellin synthesis inhibitor uniconazole, could bind to the promoter of GmRCAß and activate its expression. Analysis of genomic sequences with over 2700 soybean accessions suggested that GmGA3ox1 is being gradually utilized in modern breeding. Our results elucidated the important role of GmGA3ox1 in soybean yield. These findings reveal important clues for future high-yield breeding in soybean and other crops.

The reliance of glycerol utilization by Cupriavidus necator on CO2 fixation and improved glycerol catabolism.

While crude glycerol is a cheap carbon source for industrial-scale cultivation of microorganisms, its application relies on fast growth and conversion. The biopolymer producing Cupriavidus necator H16 (synonym: Ralstonia eutropha H16) grows poorly on glycerol. The heterologous expression of glycerol facilitator glpF, glycerol kinase glpK, and glycerol dehydrogenase glpD from E. coli accelerated the growth considerably. The naturally occurring glycerol utilization is inhibited by low glycerol kinase activity. A limited heterotrophic growth promotes the dependency on autotrophic growth by carbon dioxide (CO2) fixation and refixation. As mixotrophic growth occurs in the wildtype due to low consumption rates of glycerol, CO2 fixation by the Calvin-Benson-Bassham (CBB) cycle is essential. The deletion of both cbbX copies encoding putative RuBisCO-activases (AAA + ATPase) resulted in a sharp slowdown of growth and glycerol consumption. Activase activity is necessary for functioning carboxylation by RuBisCO. Each of the two copies compensates for the loss of the other, as suggested by observed expression levels. The strong tendency towards autotrophy supports previous investigations of glycerol growth and emphasizes the versatility of the metabolism of C. necator H16. Mixotrophy with glycerol-utilization and CO2 fixation with a high dependence on the CBB is automatically occurring unless transportation and degradation of glycerol are optimized. Parallel engineering of CO2 fixation and glycerol degradation is suggested towards application for value-added production from crude glycerol. KEY POINTS: • Growth on glycerol is highly dependent on efficient carbon fixation via CBB cycle. • CbbX is essential for the efficiency of RuBisCO in C. necator H16. • Expression of glycerol degradation pathway enzymes accelerates glycerol utilization.

Expression of flavodiiron protein rescues defects in electron transport around PSI resulting from overproduction of Rubisco activase in rice.

Fragility of photosystem I has been observed in transgenic rice plants that overproduce Rubisco activase (RCA). In this study, we examined the effects of RCA overproduction on the sensitivity of PSI to photoinhibition in three lines of plants overexpressing RCA (RCA-ox). In all the RCA-ox plants the quantum yield of PSI [Y(I)] decreased whilst in contrast the quantum yield of acceptor-side limitation of PSI [Y(NA)] increased, especially under low light conditions. In the transgenic line with the highest RCA content (RCA-ox 1), the quantum yield of PSII [Y(II)] and CO2 assimilation also decreased under low light. When leaves were exposed to high light (2000 µmol photon m-2 s-1) for 60 min, the maximal activity of PSI (Pm) drastically decreased in RCA-ox 1. These results suggested that overproduction of RCA disturbs PSI electron transport control, thus increasing the susceptibility of PSI to photoinhibition. When flavodiiron protein (FLV), which functions as a large electron sink downstream of PSI, was expressed in the RCA-ox 1 background (RCA-FLV), PSI and PSII parameters, and CO2 assimilation were recovered to wild-type levels. Thus, expression of FLV restored the robustness of PSI in RCA-ox plants.