The importance of species-specific and temperature-sensitive parameterisation of A/C models: A case study using cotton (Gossypium hirsutum L.) and the automated 'OptiFitACi' R-package.

Leaf gas exchange measurements are an important tool for inferring a plant's photosynthetic biochemistry. In most cases, the responses of photosynthetic CO assimilation to variable intercellular CO concentrations (A/C response curves) are used to model the maximum (potential) rate of carboxylation by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, V) and the rate of photosynthetic electron transport at a given incident photosynthetically active radiation flux density (PAR; J). The standard Farquhar-von Caemmerer-Berry model is often used with default parameters of Rubisco kinetic values and mesophyll conductance to CO (g) derived from tobacco that may be inapplicable across species.

Grafting Rhodobacter sphaeroides with red algae Rubisco to accelerate catalysis and plant growth.

Improving the carboxylation properties of Rubisco has primarily arisen from unforeseen amino acid substitutions remote from the catalytic site. The unpredictability has frustrated rational design efforts to enhance plant Rubisco towards the prized growth-enhancing carboxylation properties of red algae Griffithsia monilis GmRubisco. To address this, we determined the crystal structure of GmRubisco to 1.

Perspectives on improving crop Rubisco by directed evolution.

Rubisco catalyses the entry of almost all CO into the biosphere and is often the rate-limiting step in plant photosynthesis and growth. Its notoriety as the most abundant protein on Earth stems from the slow and error-prone catalytic properties that require plants, cyanobacteria, algae and photosynthetic bacteria to produce it in high amounts. Efforts to improve the CO-fixing properties of plant Rubisco has been spurred on by the discovery of more effective isoforms in some algae with the potential to significantly improve crop productivity.

Potential abiotic stress targets for modern genetic manipulation.

Research into crop yield and resilience has underpinned global food security, evident in yields tripling in the past 5 decades. The challenges that global agriculture now faces are not just to feed 10+ billion people within a generation, but to do so under a harsher, more variable, and less predictable climate, and in many cases with less water, more expensive inputs, and declining soil quality. The challenges of climate change are not simply to breed for a "hotter drier climate," but to enable resilience to floods and droughts and frosts and heat waves, possibly even within a single growing season.

Escherichia coli expressing chloroplast chaperones as a proxy to test heterologous Rubisco production in leaves.

Rubisco is a fundamental enzyme in photosynthesis and therefore for life. Efforts to improve plant Rubisco performance have been hindered by the enzymes' complex chloroplast biogenesis requirements. New Synbio approaches, however, now allow the production of some plant Rubisco isoforms in Escherichia coli.

A cross-scale analysis to understand and quantify the effects of photosynthetic enhancement on crop growth and yield across environments.

Photosynthetic manipulation provides new opportunities for enhancing crop yield. However, understanding and quantifying the importance of individual and multiple manipulations on the seasonal biomass growth and yield performance of target crops across variable production environments is limited. Using a state-of-the-art cross-scale model in the APSIM platform we predicted the impact of altering photosynthesis on the enzyme-limited (A ) and electron transport-limited (A ) rates, seasonal dynamics in canopy photosynthesis, biomass growth, and yield formation via large multiyear-by-location crop growth simulations.

Rubisco Engineering by Plastid Transformation and Protocols for Assessing Expression.

The assimilation of CO within chloroplasts is catalyzed by the bifunctional enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, Rubisco. Within higher plants the Rubisco large subunit gene, rbcL, is encoded in the plastid genome, while the Rubisco small subunit gene, RbcS is coded in the nucleus by a multigene family. Rubisco is considered a poor catalyst due to its slow turnover rate and its additional fixation of O that can result in wasteful loss of carbon through the energy requiring photorespiratory cycle.

Rubisco Adaptation Is More Limited by Phylogenetic Constraint Than by Catalytic Trade-off.

Rubisco assimilates CO2 to form the sugars that fuel life on earth. Correlations between rubisco kinetic traits across species have led to the proposition that rubisco adaptation is highly constrained by catalytic trade-offs. However, these analyses did not consider the phylogenetic context of the enzymes that were analyzed.

The dependency of red Rubisco on its cognate activase for enhancing plant photosynthesis and growth.

Plant photosynthesis and growth are often limited by the activity of the CO-fixing enzyme Rubisco. The broad kinetic diversity of Rubisco in nature is accompanied by differences in the composition and compatibility of the ancillary proteins needed for its folding, assembly, and metabolic regulation. Variations in the protein folding needs of catalytically efficient red algae Rubisco prevent their production in plants.

Modifying Plant Photosynthesis and Growth via Simultaneous Chloroplast Transformation of Rubisco Large and Small Subunits.

Engineering improved Rubisco for the enhancement of photosynthesis is challenged by the alternate locations of the chloroplast gene and nuclear genes. Here we develop an RNAi- tobacco () master-line, tobRrΔS, for producing homogenous plant Rubisco by L-S operon chloroplast transformation. Four genotypes encoding alternative genes and adjoining 5'-intergenic sequences revealed that Rubisco production was highest (50% of the wild type) in the lines incorporating a gene whose codon use and 5' untranslated-region matched Additional tobacco genotypes produced here incorporated differing potato () - operons that either encoded one of three mesophyll small subunits (pS1, pS2, and pS3) or the potato trichome pS-subunit.

BSD2 is a Rubisco-specific assembly chaperone, forms intermediary hetero-oligomeric complexes, and is nonlimiting to growth in tobacco.

The folding and assembly of Rubisco large and small subunits into L S holoenzyme in chloroplasts involves many auxiliary factors, including the chaperone BSD2. Here we identify apparent intermediary Rubisco-BSD2 assembly complexes in the model C plant tobacco. We show BSD2 and Rubisco content decrease in tandem with leaf age with approximately half of the BSD2 in young leaves (~70 nmol BSD2 protomer.

Directed -in vitro- evolution of Precambrian and extant Rubiscos.

Rubisco is an ancient, catalytically conserved yet slow enzyme, which plays a central role in the biosphere's carbon cycle. The design of Rubiscos to increase agricultural productivity has hitherto relied on the use of in vivo selection systems, precluding the exploration of biochemical traits that are not wired to cell survival. We present a directed -in vitro- evolution platform that extracts the enzyme from its biological context to provide a new avenue for Rubisco engineering.

Short-term thermal photosynthetic responses of C4 grasses are independent of the biochemical subtype.

C4 photosynthesis evolved independently many times, resulting in multiple biochemical pathways; however, little is known about how these different pathways respond to temperature. We investigated the photosynthetic responses of eight C4 grasses belonging to three biochemical subtypes (NADP-ME, PEP-CK, and NAD-ME) to four leaf temperatures (18, 25, 32, and 40 °C). We also explored whether the biochemical subtype influences the thermal responses of (i) in vitro PEPC (Vpmax) and Rubisco (Vcmax) maximal activities, (ii) initial slope (IS) and CO2-saturated rate (CSR) derived from the A-Ci curves, and (iii) CO2 leakage out of the bundle sheath estimated from carbon isotope discrimination.

An improved screen for Rubisco identifies a protein-protein interface that can enhance CO-fixation kinetics.

An overarching goal of photosynthesis research is to identify how components of the process can be improved to benefit crop productivity, global food security, and renewable energy storage. Improving carbon fixation has mostly focused on enhancing the CO fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). This grand challenge has mostly proved ineffective because of catalytic mechanism constraints and required chaperone complementarity that hinder Rubisco biogenesis in alternative hosts.

The role of Rubisco kinetics and pyrenoid morphology in shaping the CCM of haptophyte microalgae.

The haptophyte algae are a cosmopolitan group of primary producers that contribute significantly to the marine carbon cycle and play a major role in paleo-climate studies. Despite their global importance, little is known about carbon assimilation in haptophytes, in particular the kinetics of their Form 1D CO2-fixing enzyme, Rubisco. Here we examine Rubisco properties of three haptophytes with a range of pyrenoid morphologies (Pleurochrysis carterae, Tisochrysis lutea, and Pavlova lutheri) and the diatom Phaeodactylum tricornutum that exhibit contrasting sensitivities to the trade-offs between substrate affinity (Km) and turnover rate (kcat) for both CO2 and O2.

Rubisco Extraction and Purification from Diatoms.

This protocol describes a method to extract ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) from diatoms () to determine catalytic performance. This protocol has been adapted from use in cyanobacteria and higher plants (Andrews, 1988; Whitney and Sharwood, 2007). First part (steps A1-A3) of the extraction provides a crude extract of Rubisco that is sufficient for carboxylation assays to measure the Michaelis constant for CO (K) and the catalytic turnover rate ( ).

Biogenesis and Metabolic Maintenance of Rubisco.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) mediates the fixation of atmospheric CO in photosynthesis by catalyzing the carboxylation of the 5-carbon sugar ribulose-1,5-bisphosphate (RuBP). Rubisco is a remarkably inefficient enzyme, fixing only 2-10 CO molecules per second. Efforts to increase crop yields by bioengineering Rubisco remain unsuccessful, owing in part to the complex cellular machinery required for Rubisco biogenesis and metabolic maintenance.

Linking photosynthesis and leaf N allocation under future elevated CO2 and climate warming in Eucalyptus globulus.

Leaf-level photosynthetic processes and their environmental dependencies are critical for estimating CO2 uptake from the atmosphere. These estimates use biochemical-based models of photosynthesis that require accurate Rubisco kinetics. We investigated the effects of canopy position, elevated atmospheric CO2 [eC; ambient CO2 (aC)+240 ppm] and elevated air temperature (eT; ambient temperature (aT)+3 °C) on Rubisco content and activity together with the relationship between leaf N and Vcmax (maximal Rubisco carboxylation rate) of 7 m tall, soil-grown Eucalyptus globulus trees.

Temperature responses of Rubisco from Paniceae grasses provide opportunities for improving C photosynthesis.

Enhancing the catalytic properties of the CO-fixing enzyme Rubisco is a target for improving agricultural crop productivity. Here, we reveal extensive diversity in the kinetic response between 10 and 37 °C by Rubisco from C and C species within the grass tribe Paniceae. The CO fixation rate (kcatc) for Rubisco from the C grasses with nicotinamide adenine dinucleotide (NAD) phosphate malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PCK) photosynthetic pathways was twofold greater than the kcatc of Rubisco from NAD-ME species across all temperatures.

Prospects for improving CO2 fixation in C3-crops through understanding C4-Rubisco biogenesis and catalytic diversity.

By operating a CO2 concentrating mechanism, C4-photosynthesis offers highly successful solutions to remedy the inefficiency of the CO2-fixing enzyme Rubisco. C4-plant Rubisco has characteristically evolved faster carboxylation rates with low CO2 affinity. Owing to high CO2 concentrations in bundle sheath chloroplasts, faster Rubisco enhances resource use efficiency in C4 plants by reducing the energy and carbon costs associated with photorespiration and lowering the nitrogen investment in Rubisco.

Large variation in the Rubisco kinetics of diatoms reveals diversity among their carbon-concentrating mechanisms.

While marine phytoplankton rival plants in their contribution to global primary productivity, our understanding of their photosynthesis remains rudimentary. In particular, the kinetic diversity of the CO2-fixing enzyme, Rubisco, in phytoplankton remains unknown. Here we quantify the maximum rates of carboxylation (k cat (c)), oxygenation (k cat (o)), Michaelis constants (K m) for CO2 (K C) and O2 (K O), and specificity for CO2 over O2 (SC/O) for Form I Rubisco from 11 diatom species.

Improved analysis of C4 and C3 photosynthesis via refined in vitro assays of their carbon fixation biochemistry.

Plants operating C3 and C4 photosynthetic pathways exhibit differences in leaf anatomy and photosynthetic carbon fixation biochemistry. Fully understanding this underpinning biochemical variation is requisite to identifying solutions for improving photosynthetic efficiency and growth. Here we refine assay methods for accurately measuring the carboxylase and decarboxylase activities in C3 and C4 plant soluble protein.

Evolving Methanococcoides burtonii archaeal Rubisco for improved photosynthesis and plant growth.

In photosynthesis Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyses the often rate limiting CO2-fixation step in the Calvin cycle. This makes Rubisco both the gatekeeper for carbon entry into the biosphere and a target for functional improvement to enhance photosynthesis and plant growth. Encumbering the catalytic performance of Rubisco is its highly conserved, complex catalytic chemistry.

Improving recombinant Rubisco biogenesis, plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone.

Enabling improvements to crop yield and resource use by enhancing the catalysis of the photosynthetic CO2-fixing enzyme Rubisco has been a longstanding challenge. Efforts toward realization of this goal have been greatly assisted by advances in understanding the complexities of Rubisco's biogenesis in plastids and the development of tailored chloroplast transformation tools. Here we generate transplastomic tobacco genotypes expressing Arabidopsis Rubisco large subunits (AtL), both on their own (producing tob(AtL) plants) and with a cognate Rubisco accumulation factor 1 (AtRAF1) chaperone (producing tob(AtL-R1) plants) that has undergone parallel functional coevolution with AtL.