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

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

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

Multiple mechanisms for enhanced plasmodesmata density in disparate subtypes of C4 grasses

Proliferation of plasmodesmata (PD) connections between bundle sheath (BS) and mesophyll (M) cells has been proposed as a key step in the evolution of two-cell C4 photosynthesis; However, a lack of quantitative data has hampered further exploration and validation of this hypothesis. In this study, we quantified leaf anatomical traits associated with metabolite transport in 18 species of BEP and PACMAD grasses encompassing four origins of C4 photosynthesis and all three C4 subtypes (NADP-ME, NAD-ME, and PCK). We demonstrate that C4 leaves have greater PD density between M and BS cells than C3 leaves. We show that this greater PD density is achieved by increasing either the pit field (cluster of PD) area or the number of PD per pit field area. NAD-ME species had greater pit field area per M–BS interface than NADP-ME or PCK species. In contrast, NADP-ME and PCK species had lower pit field area with increased number of PD per pit field area than NAD-ME species. Overall, PD density per M–BS cell interface was greatest in NAD-ME species while PD density in PCK species exhibited the largest variability. Finally, the only other anatomical characteristic that clearly distinguished C4 from C3 species was their greater Sb value, the BS surface area to subtending leaf area ratio. In contrast, BS cell volume was comparable between the C3 and C4 grass species examined.FRD is supported by scholarship awards from the Lee Foundation (IRRI) and the Australian Research Council Centre of Excellence for Translational Photosynthesis (CE140100015). SK is a Royal Society University Research Fellow. Work in SK’s lab is supported by the European Union’s Horizon 2020 research and innovation programme under grant agreement number 637765