Carbon isotope discrimination as a diagnostic tool for C₄ photosynthesis in C₃-C₄ intermediate species

The presence and activity of the C₄ cycle in C₃-C₄ intermediate species have proven difficult to analyze, especially when such activity is low. This study proposes a strategy to detect C₄ activity and estimate its contribution to overall photosynthesis in intermediate plants, by using tunable diode laser absorption spectroscopy (TDLAS) coupled to gas exchange systems to simultaneously measure the CO₂ responses of CO₂ assimilation (A) and carbon isotope discrimination (Δ) under low O₂ partial pressure. Mathematical models of C₃-C₄ photosynthesis and Δ are then fitted concurrently to both responses using the same set of constants. This strategy was applied to the intermediate species Flaveria floridana and F. brownii, and to F. pringlei and F. bidentis as C₃ and C₄ controls, respectively. Our results support the presence of a functional C4 cycle in F. floridana, that can fix 12-21% of carbon. In F. brownii, 75-100% of carbon is fixed via the C₄ cycle, and the contribution of mesophyll Rubisco to overall carbon assimilation increases with CO₂ partial pressure in both intermediate plants. Combined gas exchange and Δ measurement and modeling is a powerful diagnostic tool for C₄ photosynthesis.This research was supported by the Bill and Melinda Gates Foundation’s funding for the C₄ Rice consortium and by the Australian Research Council Centre of Excellence for Translational Photosynthesis (CE140100015)

Determining RuBisCO activation kinetics and other rate and equilibrium constants by simultaneous multiple non-linear regression of a kinetic model

The forward and reverse rate constants involved in carbamylation, activation, carboxylation, and inhibition of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) have been estimated by a new technique of simultaneous non-linear regression of a differential equation kinetic model to multiple experimental data. Parameters predicted by the model fitted to data from purified spinach enzyme in vitro included binding affinity constants for non-substrate CO2 and Mg2+ of 200±80 μM and 700±200 μM, respectively, as well as a turnover number (kcat) of 3.3±0.5 s-1, a Michaelis half-saturation constant for carboxylation (KM,C) of 10±4 μM and a Michaelis constant for RuBP binding (KM,RuBP) of 1.5±0.5 μM. These and other constants agree well with previously measured values where they exist. The model is then used to show that slow inactivation of RuBisCO (fallover) in oxygen-free conditions at low concentrations of CO2 and Mg2+ is due to decarbamylation and binding of RuBP to uncarbamylated enzyme. In spite of RuBP binding more tightly to uncarbamylated enzyme than to the activated form, RuBisCO is activated at high concentrations of CO2 and Mg2+. This apparent paradox is resolved by considering activation kinetics and the fact that while RuBP binds tightly but slowly to uncarbamylated enzyme, it binds fast and loosely to activated enzyme. This modelling technique is presented as a new method for determining multiple kinetic data simultaneously from a limited experimental data set. The method can be used to compare the properties of RuBisCO from different species quickly and easily

Photosynthesis and food security: the evolving story of C4 rice

Traditional “Green Revolution” cereal breeding strategies to improve yield are now reaching a plateau in our principal global food crop rice. Photosynthesis has now become a major target of international consortia to increase yield potential. Synthetic biology is being used across multiple large projects to improve photosynthetic efficiency. This review follows the genesis and progress of one of the first of these consortia projects, now in its 13th year; the Bill and Melinda Gates funded C4 Rice Project. This project seeks to install the biochemical and anatomical attributes necessary to support C4 photosynthesis in the C3 crop rice. Here we address the advances made thus far in installing the biochemical pathway and some of the key targets yet to be reached

Rubisco carboxylase/oxygenase: From the enzyme to the globe: A gas exchange perspective

Rubisco is the primary carboxylase of the photosynthetic process, the most abundant enzyme in the biosphere, and also one of the best-characterized enzymes. Rubisco also functions as an oxygenase, a discovery made 50 years ago by Bill Ogren. Carboxylation of ribulose bisphosphate (RuBP) is the first step of the photosynthetic carbon reduction cycle and leads to the assimilation of CO2, whereas the oxygenase activity necessitates the recycling of phosphoglycolate through the photorespiratory carbon oxidation cycle with concomitant loss of CO2. Since the discovery of Rubisco's dual function, the biochemical properties of Rubisco have underpinned the mechanistic mathematical models of photosynthetic CO2 fixation which link Rubisco kinetic properties to gas exchange of leaves. This has allowed assessments of global CO2 exchange and predictions of how Rubisco has and will shape the environmental responses of crop and global photosynthesis in future climates. Rubisco's biochemical properties, including its slow catalytic turnover and poor affinity for CO2, constrain crop growth and therefore improving its activity and regulation and minimising photorespiration are key targets for crop improvement.The Research was funded by the Australian Research Council Centre of Excellence for Translational Photosynthesis (CE140100015

Growth of the C4 dicot Flaveria bidentis: photosynthetic acclimation to low light through shifts in leaf anatomy and biochemistry

In C4 plants, acclimation to growth at low irradiance by means of anatomical and biochemical changes to leaf tissue is considered to be limited by the need for a close interaction and coordination between bundle sheath and mesophyll cells. Here differences in relative growth rate (RGR), gas exchange, carbon isotope discrimination, photosynthetic enzyme activity, and leaf anatomy in the C4 dicot Flaveria bidentis grown at a low (LI; 150 μmol quanta m2 s−1) and medium (MI; 500 μmol quanta m2 s−1) irradiance and with a 12 h photoperiod over 36 d were examined. RGRs measured using a 3D non-destructive imaging technique were consistently higher in MI plants. Rates of CO2 assimilation per leaf area measured at 1500 μmmol quanta m2 s−1 were higher for MI than LI plants but did not differ on a mass basis. LI plants had lower Rubisco and phosphoenolpyruvate carboxylase activities and chlorophyll content on a leaf area basis. Bundle sheath leakiness of CO2 (ϕ) calculated from real-time carbon isotope discrimination was similar for MI and LI plants at high irradiance. ϕ increased at lower irradiances, but more so in MI plants, reflecting acclimation to low growth irradiance. Leaf thickness and vein density were greater in MI plants, and mesophyll surface area exposed to intercellular airspace (Sm) and bundle sheath surface area per unit leaf area (Sb) measured from leaf cross-sections were also both significantly greater in MI compared with LI leaves. Both mesophyll and bundle sheath conductance to CO2 diffusion were greater in MI compared with LI plants. Despite being a C4 species, F. bidentis is very plastic with respect to growth irradiance

Correlation between carbon isotope discrimination and transpiration efficiency in lines of the C4 species Sorghum bicolor in the glasshouse and the field

Transpiration efficiency, W, the ratio of plant carbon produced to water transpired and carbon isotope discrimination of leaf dry matter, Δd, were measured together on 30 lines of the C4 species, Sorghum bicolor, in the glasshouse and on eight lines grown in the field. In the glasshouse, the mean W observed was 4.9 mmol C mol-1 H2O and the range was 0.8 mmol C mol -1 H2O. The mean Δd was 3.0 and the observed range was 0.4‰. In the field, the mean W was lower at 2.8 mmol C mol-1 H2O and the mean Δd was 4.6‰. Significant positive correlations between W and Δd were observed for plants grown in the glasshouse and in the field. The observed correlations were consistent with theory, opposite to those for C3 species, and showed that variation in Δd was an integrated measure of long-term variation in the ratio of intercellular to ambient CO2 partial pressure, pi/pa. Detailed gas exchange measurements of carbon isotope discrimination during CO2 uptake, ΔA, and pi/pa were made on leaves of eight S. bicolorlines. The observed relationship between ΔA and pi/pa was linear with a negative slope of 3.7‰ in ΔA for a unit change in pi/pa. The slope of this linear relationship between ΔA and pi/pa in C4 species is dependent on the leakiness of the CO2 concentrating mechanism of the C4 pathway. We estimated the leakiness (defined as the fraction of CO2 released in the bundle sheath by C4 acid decarboxylations, which is lost by leakage) to be 0.2. We conclude that, although variation in Δd observed in the 30 lines of S. bicolor is smaller than that commonly observed in C3 species, it also reflects variation in transpiration efficiency, W. Among the eight lines examined in detail and in the environments used, there was considerable genotype × environment interaction

C4 photosynthesis: 50 years of discovery and innovation

It is now over half a century since the biochemical characterization of the C4 photosynthetic pathway, and this special issue highlights the sheer breadth of current knowledge. New genomic and transcriptomic information shows that multi-level regulation of gene expression is required for the pathway to function, yet we know it to be one of the most dynamic examples of convergent evolution. Now, a focus on the molecular transition from C3– C4 intermediates, together with improved mathematical models, experimental tools and transformation systems, holds great promise for improving C4 photosynthesis in crops