Rubisco activase (Rca) is an ATP-dependent chaperone that facilitates dissociation of inhibitory sugar phosphates from the catalytic sites of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) during photosynthesis. In Arabidopsis, Rca is negatively regulated by dark-dependent phosphorylation of Thr78. Here we identify chloroplast localized SHEWENELLA-LIKE PROTEIN PHOSPHATASE 1 (SLP1) as the major phosphatase responsible for light-dependent Rca dephosphorylation. The prevalence of Thr78 in Rca was investigated across sequences from 91 plant species, finding 29 (~32%) species shared a threonine in the same position. Analysis of seven C3 species with an antibody raised against a Thr78 phospho-peptide demonstrated that this position is phosphorylated in multiple genera. However, light-dependent dephosphorylation of Thr78 was observed only in Arabidopsis. Further, phosphorylation of Thr78 could not be detected in any of the four C4 grass species examined. The results suggest that despite conservation of Thr78 in Rca from a wide range of species, a regulatory role for phosphorylation at this site may be limited. This provides a case study for how variation in post-translational regulation can amplify functional divergence across the phylogeny of plants beyond what is explained by sequence variation in a metabolically important protein. ### Competing Interest Statement The authors have declared no competing interest.

As the demand for renewable energy sources with reduced carbon emissions to the atmosphere increases, bioenergy crops remain an attractive alternative to fossil fuels. Another appeal of bioenergy crops is the potential utilization of marginal lands not suitable for food crops. Prairie cordgrass is a perennial warm-season polyploid grass species that is considered a bioenergy crop that also exhibits a high tolerance for salinity and flooding. The ploidy level of prairie cordgrass has previously been suggested to correlate with agronomic traits. However, little is known about the agricultural performance of prairie cordgrass on marginal lands and if a correlation of ploidy level and stress tolerance exists. In this experiment, the productivity of tetra-, hexa-, and octoploids was investigated when grown on three different marginal sites, including waterlogged, saline, and low-nutrient gravelly soils. Here, we found that prairie cordgrass populations annually produced dry biomass on a wet marginal site comparable to normal cropland. However, the productivity was remarkedly decreased at both a salt-affected site and a limestone mine reclamation site. We were unable to conclude that different ploidy levels affected the biomass yield of prairie cordgrass grown under abiotic stress. However, there was a differential response of populations to various types of marginal sites. These results indicate potential genetic resources for developing breeding targets and identifying mechanisms of abiotic stress tolerance. Moreover, we highlight the need to generate diverse ploidy levels of prairie cordgrass in the same genetic backgrounds for future testing. Using marginal lands for bioenergy crop production can avoid competition with food crops.Prairie cordgrass was well established on a wet marginal site, producing an average of 18 Mg ha-1 of biomass.Prairie cordgrass productivity was decreased at both a saline site and a low-nutrient gravelly soil site.Populations of prairie cordgrass responded differently to various marginal sites.

Volume and surface area of chloroplasts and surface area of plasmodesmata pit fields are presented for two C-4 species, maize and sugarcane, with respect to cell surface area and cell volume.Serial block face scanning electron microscopy (SBF-SEM) and confocal laser scanning microscopy with the Airyscan system (LSM) were used. Chloroplast size estimates were much faster and easier using LSM than with SBF-SEM; however, the results were more variable than SBF-SEM.Mesophyll cells were lobed where chloroplasts were located, facilitating cell-to-cell connections while allowing for greater intercellular airspace exposure. Bundle sheath cells were cylindrical with chloroplasts arranged centrifugally. Chloroplasts occupied c. 30-50% of mesophyll cell volume, and 60-70% of bundle sheath cell volume. Roughly 2-3% of each cell surface area was covered by plasmodesmata pit fields for both bundle sheath and mesophyll cells.This work will aid future research to develop SBF-SEM methodologies with the aim to better understand the effect of cell structure on C-4 photosynthesis.

Chlorophyll is the major light-absorbing pigment for plant photosynthesis. While evolution has been selected for high chlorophyll content in leaves, previous work suggests that domesticated crops grown in modern high-density agricultural environments overinvest in chlorophyll production, thereby lowering light use and nitrogen use efficiency. To investigate the potential benefits of reducing chlorophyll levels, we created ethanol-inducible RNAi tobacco mutants that suppress Mg-chelatase subunit I (CHLI) with small RNA within 3 h of induction and reduce chlorophyll within 5 days in field conditions. We initiated chlorophyll reduction later in plant development to avoid the highly sensitive seedling stage and to allow young plants to have full green leaves to maximise light interception before canopy formation. This study demonstrated that leaf chlorophyll reduction >60% during seed-filling stages increased tobacco seed nitrogen concentration by as much as 17% while canopy photosynthesis, biomass and seed yields were maintained. These results indicate that time-specific reduction of chlorophyll could be a novel strategy that decouples the inverse relationship between yield and seed nitrogen by utilising saved nitrogen from the reduction of chlorophyll while maintaining full carbon assimilation capacity.

Demand for energy and the environmental consequences associated with fossil fuel emissions has created a need for alternative energy sources. To avoid conflict over the conversion of existing agricultural land from food production to fuel production, bioenergy crops could instead be cultivated on marginal land. This study compared the performances, biomass yield, tiller density, and lignocellulosic bioenergy feedstock composition of four prairie cordgrass populations with two-row spacings cultivated on a waterlog-prone marginal land, as well as three other bioenergy candidate crops used as controls: big bluestem, M. × giganteus , and switchgrass. Across all populations and spacings, annual biomass yield of prairie cordgrass was 13 Mg ha −1 . Row spacing had significant impacts on prairie cordgrass productivity, with higher biomass yields observed in the 45 cm spacing than in the 90 cm spacing. Feedstock composition (cellulose, hemicellulose, ADL, and ash concentration) was not influenced by row spacing and did not deviate from expected values for growth on agricultural lands. However, biomass yields of the control species M . × giganteus and switchgrass planted in 45 cm spacing were higher than the prairie cordgrass. Our results provide evidence that prairie cordgrass could be a good energy crop with comparable biomass yield production to the energy crops M . × giganteus and switchgrass on waterlogged marginal land.

Bioenergy grass species are a renewable energy source, but their productivity has not been fully realized. Improving photosynthetic efficiency has been proposed as a mechanism to increase the productivity of bioenergy grass species. Fluctuating light, experienced by all field grown crops, is known to reduce photosynthetic efficiency. This experiment aimed to evaluate the photosynthetic performance of both C3 and C4 bioenergy grass species under steady state and fluctuating light conditions by examining leaf gas exchange. The fluctuating light regime used here decreased carbon assimilation across all species when compared to expected steady state values. Overall, C4 species assimilated more carbon than C3 species during the fluctuating light regime, with both photosynthetic types assimilating about 16% less carbon than expected based on steady state measurements. Little diversity was observed in response to fluctuating light among C3 species, and photorespiration partially contributed to the rapid decreases in net photosynthetic rates during high to low light transitions. In C4 species, differences among the four NADP‐ME species were apparent. Diversity observed among C4 species in this experiment provides evidence that photosynthetic efficiency in response to fluctuating light may be targeted to increase C4 bioenergy grass productivity.

Summary Use of a complete dynamic model of NADP‐malic enzyme C 4 photosynthesis indicated that, during transitions from dark or shade to high light, induction of the C 4 pathway was more rapid than that of C 3 , resulting in a predicted transient increase in bundle‐sheath CO 2 leakiness ( ϕ ). Previously, ϕ has been measured at steady state; here we developed a new method, coupling a tunable diode laser absorption spectroscope with a gas‐exchange system to track ϕ in sorghum and maize through the nonsteady‐state condition of photosynthetic induction. In both species, ϕ showed a transient increase to > 0.35 before declining to a steady state of 0.2 by 1500 s after illumination. Average ϕ was 60% higher than at steady state over the first 600 s of induction and 30% higher over the first 1500 s. The transient increase in ϕ , which was consistent with model prediction, indicated that capacity to assimilate CO 2 into the C 3 cycle in the bundle sheath failed to keep pace with the rate of dicarboxylate delivery by the C 4 cycle. Because nonsteady‐state light conditions are the norm in field canopies, the results suggest that ϕ in these major crops in the field is significantly higher and energy conversion efficiency lower than previous measured values under steady‐state conditions.

Enhancement of Rubisco kinetics could improve photosynthetic efficiency, ultimately resulting in increased crop yield. However, imprecise knowledge of the reaction mechanism and the individual rate constants limits our ability to optimize the enzyme. Membrane inlet mass spectrometry (MIMS) may offer benefits over traditional methods for determining individual rate constants of the Rubisco reaction mechanism, as it can directly monitor concentration changes in CO2, O2, and their isotopologs during assays. However, a direct comparison of MIMS with the traditional radiolabel method of determining Rubisco kinetic parameters has not been made. Here, the temperature responses of Rubisco kinetic parameters from Arabidopsis thaliana were measured using radiolabel and MIMS methods. The two methods provided comparable parameters above 25 °C, but temperature responses deviated at low temperature as MIMS-derived catalytic rates of carboxylation, oxygenation, and CO2/O2 specificity showed thermal breakpoints. Here, we discuss the variability and uncertainty surrounding breakpoints in the Rubisco temperature response and the relevance of individual rate constants of the reaction mechanisms to potential breakpoints.

Mesophyll conductance (gm ) is an important factor limiting rates of C3 photosynthesis. However, its role in C4 photosynthesis is poorly understood because it has been historically difficult to estimate. We use two methods to derive the temperature responses of gm in C4 species. The first (Δ18 O) combines measurements of gas exchange with models and measurements of 18 O discrimination. The second method (in vitro Vpmax ) derives gm by retrofitting models of C4 photosynthesis and 13 C discrimination with gas exchange, kinetic constants and in vitro Vpmax measurements. The two methods produced similar gm for Setaria viridis and Zea mays. Additionally, we present the first temperature response (10-40°C) of C4 gm in S. viridis, Z. mays and Miscanthus × giganteus. Values for gm at 25°C ranged from 2.90 to 7.85 μmol m-2  s-1  Pa-1 . Our study demonstrated that: the two described methods are suitable to calculate gm in C4 species; gm values in C4 are similar to high-end values reported for C3 species; and gm increases with temperature analogous to reports for C3 species and the response is species specific. These results improve our mechanistic understanding of C4 photosynthesis.

The photosynthetic assimilation of CO2 in C-4 plants is potentially limited by the enzymatic rates of Rubisco, phosphoenolpyruvate carboxylase (PEPc), and carbonic anhydrase (CA). Therefore, the activity and kinetic properties of these enzymes are needed to accurately parameterize C-4 biochemical models of leaf CO2 exchange in response to changes in CO2 availability and temperature. There are currently no published temperature responses of both Rubisco carboxylation and oxygenation kinetics from a C-4 plant, nor are there known measurements of the temperature dependency of the PEPc Michaelis-Menten constant for its substrate HCO3-, and there is little information on the temperature response of plant CA activity. Here, we used membrane inlet mass spectrometry to measure the temperature responses of Rubisco carboxylation and oxygenation kinetics, PEPc carboxylation kinetics, and the activity and first-order rate constant for the CA hydration reaction from 10 degrees C to 40 degrees C using crude leaf extracts from the C-4 plant Setaria viridis. The temperature dependencies of Rubisco, PEPc, and CA kinetic parameters are provided. These findings describe a new method for the investigation of PEPc kinetics, suggest an HCO3- limitation imposed by CA, and show similarities between the Rubisco temperature responses of previously measured C-3 species and the C-4 plant S. viridis.