Differences in stomatal sensitivity to CO2 and light influence variation in water use efficiency and leaf carbon isotope composition in two genotypes of the C4 plant Zea mays.

The ratio of net CO2 uptake (Anet) and stomatal conductance (gs) is an intrinsic measurement of leaf water use efficiency (WUEi) however its measurement can be challenging for large phenotypic screens. Measurements of leaf carbon isotope composition (δ13Cleaf) may be a scalable tool to approximate WUEi for screening because it in part reflects the competing influences of Anet and gs on the CO2 partial pressure (pCO2) inside the leaf over time. However, in C4 photosynthesis the CO2 concentrating mechanism complicates the relationship between δ13Cleaf and WUEi. Despite this complicated relationship, several studies have shown genetic variation in δ13Cleaf across C4 plants. Yet there has not been a clear demonstration of whether Anet or gs are the causal mechanisms controlling WUEi and δ13Cleaf. Our approach was to characterize leaf photosynthetic traits of two Zea mays recombinant inbred lines (Z007E0067 and Z007E0150) which consistently differ for δ13Cleaf even though they have minimal confounding genetic differences. We demonstrate that these two genotypes contrasted in WUEi driven by differences in the speed of stomatal responses to changes in pCO2 and light that lead to unproductive leaf water loss. These findings provide support that differences in δ13Cleaf in closely related genotypes do reflect greater WUEi and further suggests that differences in stomatal kinetic response to changing environmental conditions is a key target to improve WUEi.

Probing the in situ volumes of Arabidopsis leaf plastids using three-dimensional confocal and scanning electron microscopy.

Leaf plastids harbor a plethora of biochemical reactions including photosynthesis, one of the most important metabolic pathways on Earth. Scientists are eager to unveil the physiological processes within the organelle but also their interconnection with the rest of the plant cell. An increasingly important feature of this venture is to use experimental data in the design of metabolic models. A remaining obstacle has been the limited in situ volume information of plastids and other cell organelles. To fill this gap for chloroplasts, we established three microscopy protocols delivering in situ volumes based on: (i) chlorophyll fluorescence emerging from the thylakoid membrane, (ii) a CFP marker embedded in the envelope, and (iii) calculations from serial block-face scanning electron microscopy (SBFSEM). The obtained data were corroborated by comparing wild-type data with two mutant lines affected in the plastid division machinery known to produce small and large mesophyll chloroplasts, respectively. Furthermore, we also determined the volume of the much smaller guard cell plastids. Interestingly, their volume is not governed by the same components of the division machinery which defines mesophyll plastid size. Based on our three approaches, the average volume of a mature Col-0 wild-type mesophyll chloroplasts is 93 µm3 . Wild-type guard cell plastids are approximately 18 µm3 . Lastly, our comparative analysis shows that the chlorophyll fluorescence analysis can accurately determine chloroplast volumes, providing an important tool to research groups without access to transgenic marker lines expressing genetically encoded fluorescence proteins or costly SBFSEM equipment.

Multiple highly expressed phosphoenolpyruvate carboxylase genes have divergent enzyme kinetic properties in two C4 grasses.

BACKGROUND AND AIMS: Phosphoenolpyruvate (PEP) carboxylase (PEPC) catalyses the irreversible carboxylation of PEP with bicarbonate to produce oxaloacetate. This reaction powers the carbon-concentrating mechanism (CCM) in plants that perform C4 photosynthesis. This CCM is generally driven by a single PEPC gene product that is highly expressed in the cytosol of mesophyll cells. We found two C4 grasses, Panicum miliaceum and Echinochloa colona, that each have two highly expressed PEPC genes. We characterized the kinetic properties of the two most abundant PEPCs in E. colona and P. miliaceum to better understand how the enzyme's amino acid structure influences its function. METHODS: Coding sequences of the two most abundant PEPC proteins in E. colona and P. miliaceum were synthesized by GenScript and were inserted into bacteria expression plasmids. Point mutations resulting in substitutions at conserved amino acid residues (e.g. N-terminal serine and residue 890) were created via site-directed PCR mutagenesis. The kinetic properties of semi-purified plant PEPCs from Escherichia coli were analysed using membrane-inlet mass spectrometry and a spectrophotometric enzyme-coupled reaction. KEY RESULTS: The two most abundant P. miliaceum PEPCs (PmPPC1 and PmPPC2) have similar sequence identities (>95 %), and as a result had similar kinetic properties. The two most abundant E. colona PEPCs (EcPPC1 and EcPPC2) had identities of ~78 % and had significantly different kinetic properties. The PmPPCs and EcPPCs had different responses to allosteric inhibitors and activators, and substitutions at the conserved N-terminal serine and residue 890 resulted in significantly altered responses to allosteric regulators. CONCLUSIONS: The two, significantly expressed C4Ppc genes in P. miliaceum were probably the result of genomes combining from two closely related C4Panicum species. We found natural variation in PEPC's sensitivity to allosteric inhibition that seems to bypass the conserved 890 residue, suggesting alternative evolutionary pathways for increased malate tolerance and other kinetic properties.

Altered cell wall hydroxycinnamate composition impacts leaf- and canopy-level CO2 uptake and water use in rice.

Cell wall properties play a major role in determining photosynthetic carbon uptake and water use through their impact on mesophyll conductance (CO2 diffusion from substomatal cavities into photosynthetic mesophyll cells) and leaf hydraulic conductance (water movement from xylem, through leaf tissue, to stomata). Consequently, modification of cell wall (CW) properties might help improve photosynthesis and crop water use efficiency (WUE). We tested this using 2 independent transgenic rice (Oryza sativa) lines overexpressing the rice OsAT10 gene (encoding a "BAHD" CoA acyltransferase), which alters CW hydroxycinnamic acid content (more para-coumaric acid and less ferulic acid). Plants were grown under high and low water levels, and traits related to leaf anatomy, CW composition, gas exchange, hydraulics, plant biomass, and canopy-level water use were measured. Alteration of hydroxycinnamic acid content led to statistically significant decreases in mesophyll CW thickness (-14%) and increased mesophyll conductance (+120%) and photosynthesis (+22%). However, concomitant increases in stomatal conductance negated the increased photosynthesis, resulting in no change in intrinsic WUE (ratio of photosynthesis to stomatal conductance). Leaf hydraulic conductance was also unchanged; however, transgenic plants showed small but statistically significant increases in aboveground biomass (AGB) (+12.5%) and canopy-level WUE (+8.8%; ratio of AGB to water used) and performed better under low water levels than wild-type plants. Our results demonstrate that changes in CW composition, specifically hydroxycinnamic acid content, can increase mesophyll conductance and photosynthesis in C3 cereal crops such as rice. However, attempts to improve photosynthetic WUE will need to enhance mesophyll conductance and photosynthesis while maintaining or decreasing stomatal conductance.

Leaf cell wall properties and stomatal density influence oxygen isotope enrichment of leaf water.

Measurements of oxygen isotope enrichment of leaf water above source water (Δ18 OLW ) can improve our understanding of the interaction between leaf anatomy and physiology on leaf water transport. Models have been developed to predict Δ18 OLW such as the string-of-lakes model, which describes the mixing of leaf water pools, and the Péclet effect model, which incorporates transpiration rate and the mixing length between unenriched xylem and enriched mesophyll water in the mesophyll (Lm ) or veins (Lv ). Here we compare measurements and models of Δ18 OLW on two cell wall composition mutants grown under two light intensities and relative humidities to evaluate cell wall properties on leaf water transport. In maize (Zea mays), the compromised ultrastructure of the suberin lamellae in the bundle sheath of the ALIPHATIC SUBERIN FERULOYL TRANSFERASE mutant (Zmasft) reduced barriers to apoplastic water movement, resulting in higher E and, potentially, Lv and, consequently, lower Δ18 OLW . The difference in Δ18 OLW in cellulose synthase-like F6 (CslF6) mutants and wild-type of rice (Oryza sativa) grown under two light intensities co-varied with stomatal density. These results show that cell wall composition and stomatal density influence Δ18 OLW and that stable isotopes can facilitate the development of a physiologically and anatomically explicit water transport model.

Mesophyll conductance response to short-term changes in pCO2 is related to leaf anatomy and biochemistry in diverse C4 grasses.

Mesophyll CO2 conductance (gm ) in C3 species responds to short-term (minutes) changes in environment potentially due to changes in leaf anatomical and biochemical properties and measurement artefacts. Compared with C3 species, there is less information on gm responses to short-term changes in environmental conditions such as partial pressure of CO2 (pCO2 ) across diverse C4 species and the potential determinants of these responses. Using 16 C4 grasses we investigated the response of gm to short-term changes in pCO2 and its relationship with leaf anatomy and biochemistry. In general, gm increased as pCO2 decreased (statistically significant increase in 12 species), with percentage increases in gm ranging from +13% to +250%. Greater increase in gm at low pCO2 was observed in species exhibiting relatively thinner mesophyll cell walls along with greater mesophyll surface area exposed to intercellular air spaces, leaf N, photosynthetic capacity and activities of phosphoenolpyruvate carboxylase and Rubisco. Species with greater CO2 responses of gm were also able to maintain their leaf water-use efficiencies (TEi ) under low CO2 . Our study advances understanding of CO2 response of gm in diverse C4 species, identifies the key leaf traits related to this response and has implications for improving C4 photosynthetic models and TEi through modification of gm .

A Rapid Method for Detecting Normal or Modified Plant and Algal Carbonic Anhydrase Activity Using Saccharomyces cerevisiae.

In recent years, researchers have attempted to improve photosynthesis by introducing components from cyanobacterial and algal CO2-concentrating mechanisms (CCMs) into terrestrial C3 plants. For these attempts to succeed, we need to understand the CCM components in more detail, especially carbonic anhydrase (CA) and bicarbonate (HCO3−) transporters. Heterologous complementation systems capable of detecting carbonic anhydrase activity (i.e., catalysis of the pH-dependent interconversion between CO2 and HCO3−) or active HCO3− transport can be of great value in the process of introducing CCM components into terrestrial C3 plants. In this study, we generated a Saccharomyces cerevisiae CA knock-out (ΔNCE103 or ΔCA) that has a high-CO2-dependent phenotype (5% (v/v) CO2 in air). CAs produce HCO3− for anaplerotic pathways in S. cerevisiae; therefore, the unavailability of HCO3− for neutral lipid biosynthesis is a limitation for the growth of ΔCA in ambient levels of CO2 (0.04% (v/v) CO2 in air). ΔCA can be complemented for growth at ambient levels of CO2 by expressing a CA from human red blood cells. ΔCA was also successfully complemented for growth at ambient levels of CO2 through the expression of CAs from Chlamydomonas reinhardtii and Arabidopsis thaliana. The ΔCA strain is also useful for investigating the activity of modified CAs, allowing for quick screening of modified CAs before putting them into the plants. CA activity in the complemented ΔCA strains can be probed using the Wilbur−Anderson assay and by isotope exchange membrane-inlet mass spectrometry (MIMS). Other potential uses for this new ΔCA-based screening system are also discussed.

Pyruvate, phosphate dikinase regulatory protein impacts light response of C4 photosynthesis in Setaria viridis.

In C4 plants, the pyruvate (Pyr), phosphate dikinase regulatory protein (PDRP) regulates the activity of the C4 pathway enzyme Pyr, phosphate dikinase (PPDK) in a light-/dark-dependent manner. The importance of this regulatory action to C4 pathway function and overall C4 photosynthesis is unknown. To resolve this question, we assessed in vivo PPDK phospho-regulation and whole leaf photophysiology in a CRISPR-Cas9 PDRP knockout (KO) mutant of the NADP-ME C4 grass green millet (Setaria viridis). PDRP enzyme activity was undetectable in leaf extracts from PDRP KO lines. Likewise, PPDK phosphorylated at the PDRP-regulatory Thr residue was immunologically undetectable in leaf extracts. PPDK enzyme activity in rapid leaf extracts was constitutively high in the PDRP KO lines, irrespective of light or dark pretreatment of leaves. Gas exchange analysis of net CO2 assimilation revealed PDRP KO leaves had markedly slower light induction kinetics when leaves transition from dark to high-light or low-light to high-light. In the initial 30 min of the light induction phase, KO leaves had an ∼15% lower net CO2 assimilation rate versus the wild-type (WT). Despite the impaired slower induction kinetics, we found growth and vigor of the KO lines to be visibly indistinguishable from the WT when grown in normal air and under standard growth chamber conditions. However, the PDRP KO plants grown under a fluctuating light regime exhibited a gradual multi-day decline in Fv/Fm, indicative of progressive photosystem II damage due to the absence of PDRP. Collectively, our results demonstrate that one of PDRP's functions in C4 photosynthesis is to ensure optimal photosynthetic light induction kinetics during dynamic changes in incident light.

Increased signal-to-noise ratios within experimental field trials by regressing spatially distributed soil properties as principal components.

Environmental variability poses a major challenge to any field study. Researchers attempt to mitigate this challenge through replication. Thus, the ability to detect experimental signals is determined by the degree of replication and the amount of environmental variation, noise, within the experimental system. A major source of noise in field studies comes from the natural heterogeneity of soil properties which create microtreatments throughout the field. In addition, the variation within different soil properties is often nonrandomly distributed across a field. We explore this challenge through a sorghum field trial dataset with accompanying plant, microbiome, and soil property data. Diverse sorghum genotypes and two watering regimes were applied in a split-plot design. We describe a process of identifying, estimating, and controlling for the effects of spatially distributed soil properties on plant traits and microbial communities using minimal degrees of freedom. Importantly, this process provides a method with which sources of environmental variation in field data can be identified and adjusted, improving our ability to resolve effects of interest and to quantify subtle phenotypes.

Drought Tolerance Strategies and Autophagy in Resilient Wheat Genotypes.

Drought resiliency strategies combine developmental, physiological, cellular, and molecular mechanisms. Here, we compare drought responses in two resilient spring wheat (Triticum aestivum) genotypes: a well-studied drought-resilient Drysdale and a resilient genotype from the US Pacific North-West Hollis. While both genotypes utilize higher water use efficiency through the reduction of stomatal conductance, other mechanisms differ. First, Hollis deploys the drought escape mechanism to a greater extent than Drysdale by accelerating the flowering time and reducing root growth. Second, Drysdale uses physiological mechanisms such as non-photochemical quenching (NPQ) to dissipate the excess of harvested light energy and sustain higher Fv/Fm and ϕPSII, whereas Hollis maintains constant NPQ but lower Fv/Fm and ϕPSII values. Furthermore, more electron donors of the electron transport chain are in the oxidized state in Hollis than in Drysdale. Third, many ROS homeostasis parameters, including peroxisome abundance, transcription of peroxisome biogenesis genes PEX11 and CAT, catalase protein level, and enzymatic activity, are higher in Hollis than in Drysdale. Fourth, transcription of autophagy flux marker ATG8.4 is upregulated to a greater degree in Hollis than in Drysdale under drought, whereas relative ATG8 protein abundance under drought stress is lower in Hollis than in Drysdale. These data demonstrate the activation of autophagy in both genotypes and a greater autophagic flux in Hollis. In conclusion, wheat varieties utilize different drought tolerance mechanisms. Combining these mechanisms within one genotype offers a promising strategy to advance crop resiliency.

Lack of leaf carbonic anhydrase activity eliminates the C4 carbon-concentrating mechanism requiring direct diffusion of CO2 into bundle sheath cells.

Carbonic anhydrase (CA) performs the first enzymatic step of C4 photosynthesis by catalysing the reversible hydration of dissolved CO2 that diffuses into mesophyll cells from intercellular airspaces. This CA-catalysed reaction provides the bicarbonate used by phosphoenolpyruvate carboxylase to generate products that flow into the C4 carbon-concentrating mechanism (CCM). It was previously demonstrated that the Zea mays ca1ca2 double mutant lost 97% of leaf CA activity, but there was little difference in the growth phenotype under ambient CO2 partial pressures (pCO2 ). We hypothesise that since CAs are among the fastest enzymes, minimal activity from a third CA, CA8, can provide the inorganic carbon needed to drive C4 photosynthesis. We observed that removing CA8 from the maize ca1ca2 background resulted in plants that had 0.2% of wild-type leaf CA activity. These ca1ca2ca8 plants had reduced photosynthetic parameters and could only survive at elevated pCO2 . Photosynthetic and carbon isotope analysis combined with modelling of photosynthesis and carbon isotope discrimination was used to determine if ca1ca2ca8 plants had a functional C4 cycle or were relying on direct CO2 diffusion to ribulose 1,5-bisphosphate carboxylase/oxygenase within bundle sheath cells. The results suggest that leaf CA activity in ca1ca2ca8 plants was not sufficient to sustain the C4 CCM.

Limitation of C4 photosynthesis by low carbonic anhydrase activity increases with temperature but does not influence mesophyll CO2 conductance.

The CO2-concentrating mechanism (CCM) in C4 plants is initiated by the uptake of bicarbonate (HCO3-) via phosphoenolpyruvate carboxylase (PEPC). Generation of HCO3- for PEPC is determined by the interaction between mesophyll CO2 conductance and the hydration of CO2 to HCO3- by carbonic anhydrase (CA). Genetic reduction of CA was previously shown not to limit C4 photosynthesis under ambient atmospheric partial pressures of CO2 (pCO2). However, CA activity varies widely across C4 species and it is unknown if there are specific environmental conditions (e.g. high temperature) where CA may limit HCO3- production for C4 photosynthesis. Additionally, CA activity has been suggested to influence mesophyll conductance, but this has not been experimentally tested. We hypothesize that CA activity can limit PEPC at high temperatures, particularly at low pCO2, but does not directly influence gm. Here we tested the influence of genetically reduced CA activity on photosynthesis and gm in the C4 plant Zea mays under a range of pCO2 and temperatures. Reduced CA activity limited HCO3- production for C4 photosynthesis at low pCO2 as temperatures increased, but did not influence mesophyll conductance. Therefore, high leaf CA activity may enhance C4 photosynthesis under high temperature when stomatal conductance restricts the availability of atmospheric CO2.

The genetic architecture of leaf stable carbon isotope composition in Zea mays and the effect of transpiration efficiency on leaf elemental accumulation.

With increased demand on freshwater resources for agriculture, it is imperative that more water-use efficient crops are developed. Leaf stable carbon isotope composition, δ13C, is a proxy for transpiration efficiency and a possible tool for breeders, but the underlying mechanisms effecting δ13C in C4 plants are not known. It has been suggested that differences in specific leaf area (SLA), which potentially reflects variation in internal CO2 diffusion, can impact leaf δ13C. Furthermore, although it is known that water movement is important for elemental uptake, it is not clear how manipulation of transpiration for increased water-use efficiency may impact nutrient accumulation. Here, we characterize the genetic architecture of leaf δ13C and test its relationship to SLA and the ionome in five populations of maize. Five significant QTL for leaf δ13C were identified, including novel QTL as well as some that were identified previously in maize kernels. One of the QTL regions contains an Erecta-like gene, the ortholog of which has been shown to regulate transpiration efficiency and leaf δ13C in Arabidopsis. QTL for δ13C were located in the same general chromosome region, but slightly shifted, when comparing data from two different years. Our data does not support a relationship between δ13C and SLA, and of the 19 elements analyzed, only a weak correlation between molybdenum and δ13C was detected. Together these data add to the genetic understanding of leaf δ13C in maize and suggest that improvements to plant water use may be possible without significantly influencing elemental homeostasis.

Engineering chloroplast development in rice through cell-specific control of endogenous genetic circuits.

The engineering of C4 photosynthetic activity into the C3 plant rice has the potential to nearly double rice yields. To engineer a two-cell photosynthetic system in rice, the rice bundle sheath (BS) must be rewired to enhance photosynthetic capacity. Here, we show that BS chloroplast biogenesis is enhanced when the transcriptional activator, Oryza sativa Cytokinin GATA transcription factor 1 (OsCGA1), is driven by a vascular specific promoter. Ectopic expression of OsCGA1 resulted in increased BS chloroplast planar area and increased expression of photosynthesis-associated nuclear genes (PhANG), required for the biogenesis of photosynthetically active chloroplasts in BS cells of rice. A further refinement using a DNAse dead Cas9 (dCas9) activation module driven by the same cell-type specific promoter, directed enhanced chloroplast development of the BS cells when gRNA sequences were delivered by the dCas9 module to the promoter of the endogenous OsCGA1 gene. Single gRNA expression was sufficient to mediate the transactivation of both the endogenous gene and a transgenic GUS reporter fused with OsCGA1 promoter. Our results illustrate the potential for tissue-specific dCas9-activation and the co-regulation of genes needed for multistep engineering of C4 rice.

Predicting photosynthetic capacity in tobacco using shortwave infrared spectral reflectance.

Plateauing yield and stressful environmental conditions necessitate selecting crops for superior physiological traits with untapped potential to enhance crop performance. Plant productivity is often limited by carbon fixation rates that could be improved by increasing maximum photosynthetic carboxylation capacity (Vcmax). However, Vcmax measurements using gas exchange and biochemical assays are slow and laborious, prohibiting selection in breeding programs. Rapid hyperspectral reflectance measurements show potential for predicting Vcmax using regression models. While several hyperspectral models have been developed, contributions from different spectral regions to predictions of Vcmax have not been clearly identified or linked to biochemical variation contributing to Vcmax. In this study, hyperspectral reflectance data from 350-2500 nm were used to build partial least squares regression models predicting in vivo and in vitro Vcmax. Wild-type and transgenic tobacco plants with antisense reductions in Rubisco content were used to alter Vcmax independent from chlorophyll, carbon, and nitrogen content. Different spectral regions were used to independently build partial least squares regression models and identify key regions linked to Vcmax and other leaf traits. The greatest Vcmax prediction accuracy used a portion of the shortwave infrared region from 2070 nm to 2470 nm, where the inclusion of fewer spectral regions resulted in more accurate models.

Differences in leaf anatomy determines temperature response of leaf hydraulic and mesophyll CO2 conductance in phylogenetically related C4 and C3 grass species.

Leaf hydraulic and mesophyll CO2 conductance are both influenced by leaf anatomical traits, however it is poorly understood how the temperature response of these conductances differs between C4 and C3 species with distinct leaf anatomy. This study investigated the temperature response of leaf hydraulic conductance (Kleaf ), stomatal (gs ) and mesophyll (gm ) conductance to CO2 , and leaf anatomical traits in phylogenetically related Panicum antidotale (C4 ) and P. bisulcatum (C3 ) grasses. The C4 species had lower hydraulic conductance outside xylem (Kox ) and Kleaf compared with the C3 species. However, the C4 species had higher gm compared with the C3 species. Traits associated with leaf water movement, Kleaf and Kox , increased with temperature more in the C3 than in the C4 species, whereas traits related to carbon uptake, Anet and gm , increased more with temperature in the C4 than the C3 species. Our findings demonstrate that, in addition to a CO2 concentrating mechanism, outside-xylem leaf anatomy in the C4 species P. antidotale favours lower water movement through the leaf and stomata that provides an additional advantage for greater leaf carbon uptake relative to water loss with increasing leaf temperature than in the C3 species P. bisulcatum.

Leaf temperature impacts canopy water use efficiency independent of changes in leaf level water use efficiency.

Canopy water use efficiency (above-ground biomass over lifetime water loss, WUEcanopy) can influence yield in wheat and other crops. Breeding for WUEcanopy is difficult because it is influenced by many component traits. For example, intrinsic water use efficiency (WUEi), the ratio of net carbon assimilation (Anet) over stomatal conductance, contributes to WUEcanopy and can be estimated from carbon isotope discrimination (Δ). However, Δ is not sensitive to differences in the water vapor pressure deficit between the air and leaf (VPDleaf). Alternatively, measurements of instantaneous leaf water use efficiency (WUEleaf) are defined as Anet over transpiration and can be determined with gas exchange, but the dynamic nature of field conditions are not represented. Specifically, fluctuations in canopy temperature lead to changes in VPDleaf that impact transpiration but not Anet. This alters WUEleaf and in turn affects WUEcanopy. To test this relationship, WUEcanopy was measured in conjunction with WUEi, WUEcanopy, and canopy temperature under well-watered and water-limited conditions in two drought-tolerant wheat cultivars that differ in canopy architecture. In this experiment, boundary layer conductance was low and significant changes in leaf temperature occurred between cultivars and treatments that correlated with WUEcanopy likely because of the effect of canopy temperature on VPDleaf driving T. However, deviations between WUEi, WUEleaf, and WUEcanopy were present because measurements made at the leaf level do not account for variations in leaf temperature. This uncoupled the relationship of measured WUEleaf and WUEi from WUEcanopy and emphasizes the importance of canopy temperature on carbon uptake and transpired water loss.

Kinetic variation in grass phosphoenolpyruvate carboxylases provides opportunity to enhance C4 photosynthetic efficiency.

The high rates of photosynthesis and the carbon-concentrating mechanism (CCM) in C4 plants are initiated by the enzyme phosphoenolpyruvate (PEP) carboxylase (PEPC). The flow of inorganic carbon into the CCM of C4 plants is driven by PEPC's affinity for bicarbonate (KHCO3 ), which can be rate limiting when atmospheric CO2 availability is restricted due to low stomatal conductance. We hypothesize that natural variation in KHCO3 across C4 plants is driven by specific amino acid substitutions to impact rates of C4 photosynthesis under environments such as drought that restrict stomatal conductance. To test this hypothesis, we measured KHCO3 from 20 C4 grasses to compare kinetic properties with specific amino acid substitutions. There was nearly a twofold range in KHCO3 across these C4 grasses (24.3 ± 1.5 to 46.3 ± 2.4 µm), which significantly impacts modeled rates of C4 photosynthesis. Additionally, molecular engineering of a low-HCO3- affinity PEPC identified key domains that confer variation in KHCO3 . This study advances our understanding of PEPC kinetics and builds the foundation for engineering increased-HCO3- affinity and C4 photosynthetic efficiency in important C4 crops.

The Rice Plastidial Phosphorylase Participates Directly in Both Sink and Source Processes.

The plastidial starch phosphorylase (Pho1) functions in starch metabolism. A distinctive structural feature of the higher Pho1 is a 50-82-amino-acid long peptide (L50-L82), which is absent in phosphorylases from non-plant organisms. To study the function of the rice Pho1 L80 peptide, we complemented a pho1- rice mutant (BMF136) with the wild-type Pho1 gene or with a Pho1 gene lacking the L80 region (Pho1ΔL80). While expression of Pho1 in BMF136 restored normal wild-type phenotype, the introduction of Pho1ΔL80 enhanced the growth rate and plant productivity above wild-type levels. Mass spectrometry analysis of proteins captured by anti-Pho1 showed the surprising presence of PsaC, the terminal electron acceptor/donor subunit of photosystem I (PSI). This unexpected interaction was substantiated by reciprocal immobilized protein pull-down assays of seedling extracts and supported by the presence of Pho1 on isolated PSI complexes resolved by blue-native gels. Spectrophotometric studies showed that Pho1ΔL80 plants exhibited modified PSI and enhanced CO2 assimilation properties. Collectively, these findings indicate that the higher plant Pho1 has dual roles as a potential modulator of source and sink processes.

Installation of C4 photosynthetic pathway enzymes in rice using a single construct.

Introduction of a C4 photosynthetic mechanism into C3 crops offers an opportunity to improve photosynthetic efficiency, biomass and yield in addition to potentially improving nitrogen and water use efficiency. To create a two-cell metabolic prototype for an NADP-malic enzyme type C4 rice, we transformed Oryza sativa spp. japonica cultivar Kitaake with a single construct containing the coding regions of carbonic anhydrase, phosphoenolpyruvate (PEP) carboxylase, NADP-malate dehydrogenase, pyruvate orthophosphate dikinase and NADP-malic enzyme from Zea mays, driven by cell-preferential promoters. Gene expression, protein accumulation and enzyme activity were confirmed for all five transgenes, and intercellular localization of proteins was analysed. 13 CO2 labelling demonstrated a 10-fold increase in flux though PEP carboxylase, exceeding the increase in measured in vitro enzyme activity, and estimated to be about 2% of the maize photosynthetic flux. Flux from malate via pyruvate to PEP remained low, commensurate with the low NADP-malic enzyme activity observed in the transgenic lines. Physiological perturbations were minor and RNA sequencing revealed no substantive effects of transgene expression on other endogenous rice transcripts associated with photosynthesis. These results provide promise that, with enhanced levels of the C4 proteins introduced thus far, a functional C4 pathway is achievable in rice.