Expression of a CO2-permeable aquaporin enhances mesophyll conductance in the C4 species Setaria viridis.

A fundamental limitation of photosynthetic carbon fixation is the availability of CO2. In C4 plants, primary carboxylation occurs in mesophyll cytosol, and little is known about the role of CO2 diffusion in facilitating C4 photosynthesis. We have examined the expression, localization, and functional role of selected plasma membrane intrinsic aquaporins (PIPs) from Setaria italica (foxtail millet) and discovered that SiPIP2;7 is CO2-permeable. When ectopically expressed in mesophyll cells of Setaria viridis (green foxtail), SiPIP2;7 was localized to the plasma membrane and caused no marked changes in leaf biochemistry. Gas exchange and C18O16O discrimination measurements revealed that targeted expression of SiPIP2;7 enhanced the conductance to CO2 diffusion from the intercellular airspace to the mesophyll cytosol. Our results demonstrate that mesophyll conductance limits C4 photosynthesis at low pCO2 and that SiPIP2;7 is a functional CO2 permeable aquaporin that can improve CO2 diffusion at the airspace/mesophyll interface and enhance C4 photosynthesis.

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.

Diffusion of CO2 across the Mesophyll-Bundle Sheath Cell Interface in a C4 Plant with Genetically Reduced PEP Carboxylase Activity.

Phosphoenolpyruvate carboxylase (PEPC), localized to the cytosol of the mesophyll cell, catalyzes the first carboxylation step of the C4 photosynthetic pathway. Here, we used RNA interference to target the cytosolic photosynthetic PEPC isoform in Setaria viridis and isolated independent transformants with very low PEPC activities. These plants required high ambient CO2 concentrations for growth, consistent with the essential role of PEPC in C4 photosynthesis. The combination of estimating direct CO2 fixation by the bundle sheath using gas-exchange measurements and modeling C4 photosynthesis with low PEPC activity allowed the calculation of bundle sheath conductance to CO2 diffusion (gbs ) in the progeny of these plants. Measurements made at a range of temperatures suggested no or negligible effect of temperature on gbs depending on the technique used to calculate gbs Anatomical measurements revealed that plants with reduced PEPC activity had reduced cell wall thickness and increased plasmodesmata (PD) density at the mesophyll-bundle sheath (M-BS) cell interface, whereas we observed little difference in these parameters at the mesophyll-mesophyll cell interface. The increased PD density at the M-BS interface was largely driven by an increase in the number of PD pit fields (cluster of PDs) rather than an increase in PD per pit field or the size of pit fields. The correlation of gbs with bundle sheath surface area per leaf area and PD area per M-BS area showed that these parameters and cell wall thickness are important determinants of gbs It is intriguing to speculate that PD development is responsive to changes in C4 photosynthetic flux.

A sorghum (Sorghum bicolor) mutant with altered carbon isotope ratio.

Recent efforts to engineer C4 photosynthetic traits into C3 plants such as rice demand an understanding of the genetic elements that enable C4 plants to outperform C3 plants. As a part of the C4 Rice Consortium's efforts to identify genes needed to support C4 photosynthesis, EMS mutagenized sorghum populations were generated and screened to identify genes that cause a loss of C4 function. Stable carbon isotope ratio (δ13C) of leaf dry matter has been used to distinguishspecies with C3 and C4 photosynthetic pathways. Here, we report the identification of a sorghum (Sorghum bicolor) mutant with a low δ13C characteristic. A mutant (named Mut33) with a pale phenotype and stunted growth was identified from an EMS treated sorghum M2 population. The stable carbon isotope analysis of the mutants showed a decrease of 13C uptake capacity. The noise of random mutation was reduced by crossing the mutant and its wildtype (WT). The back-cross (BC1F1) progenies were like the WT parent in terms of 13C values and plant phenotypes. All the BC1F2 plants with low δ13C died before they produced their 6th leaf. Gas exchange measurements of the low δ13C sorghum mutants showed a higher CO2 compensation point (25.24 µmol CO2.mol-1air) and the maximum rate of photosynthesis was less than 5µmol.m-2.s-1. To identify the genetic determinant of this trait, four DNA pools were isolated; two each from normal and low δ13C BC1F2 mutant plants. These were sequenced using an Illumina platform. Comparison of allele frequency of the single nucleotide polymorphisms (SNPs) between the pools with contrasting phenotype showed that a locus in Chromosome 10 between 57,941,104 and 59,985,708 bps had an allele frequency of 1. There were 211 mutations and 37 genes in the locus, out of which mutations in 9 genes showed non-synonymous changes. This finding is expected to contribute to future research on the identification of the causal factor differentiating C4 from C3 species that can be used in the transformation of C3 to C4 plants.

Effects of reduced carbonic anhydrase activity on CO2 assimilation rates in Setaria viridis: a transgenic analysis.

In C4 species, the major ß-carbonic anhydrase (ß-CA) localized in the mesophyll cytosol catalyses the hydration of CO2 to HCO3-, which phosphoenolpyruvate carboxylase uses in the first step of C4 photosynthesis. To address the role of CA in C4 photosynthesis, we generated transgenic Setaria viridis depleted in ß-CA. Independent lines were identified with as little as 13% of wild-type CA. No photosynthetic defect was observed in the transformed lines at ambient CO2 partial pressure (pCO2). At low pCO2, a strong correlation between CO2 assimilation rates and CA hydration rates was observed. C18O16O isotope discrimination was used to estimate the mesophyll conductance to CO2 diffusion from the intercellular air space to the mesophyll cytosol (gm) in control plants, which allowed us to calculate CA activities in the mesophyll cytosol (Cm). This revealed a strong relationship between the initial slope of the response of the CO2 assimilation rate to cytosolic pCO2 (ACm) and cytosolic CA activity. However, the relationship between the initial slope of the response of CO2 assimilation to intercellular pCO2 (ACi) and cytosolic CA activity was curvilinear. This indicated that in S. viridis, mesophyll conductance may be a contributing limiting factor alongside CA activity to CO2 assimilation rates at low pCO2.

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.

A new set of ESTs and cDNA clones from full-length and normalized libraries for gene discovery and functional characterization in citrus.

BACKGROUND: Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. Indeed, nearly 40% of genes in plants encode proteins of unknown function. Functional characterization of these genes is one of the main challenges in modern biology. In this regard, the availability of full-length cDNA clones may fill in the gap created between sequence information and biological knowledge. Full-length cDNA clones facilitate functional analysis of the corresponding genes enabling manipulation of their expression in heterologous systems and the generation of a variety of tagged versions of the native protein. In addition, the development of full-length cDNA sequences has the power to improve the quality of genome annotation. RESULTS: We developed an integrated method to generate a new normalized EST collection enriched in full-length and rare transcripts of different citrus species from multiple tissues and developmental stages. We constructed a total of 15 cDNA libraries, from which we isolated 10,898 high-quality ESTs representing 6142 different genes. Percentages of redundancy and proportion of full-length clones range from 8 to 33, and 67 to 85, respectively, indicating good efficiency of the approach employed. The new EST collection adds 2113 new citrus ESTs, representing 1831 unigenes, to the collection of citrus genes available in the public databases. To facilitate functional analysis, cDNAs were introduced in a Gateway-based cloning vector for high-throughput functional analysis of genes in planta. Herein, we describe the technical methods used in the library construction, sequence analysis of clones and the overexpression of CitrSEP, a citrus homolog to the Arabidopsis SEP3 gene, in Arabidopsis as an example of a practical application of the engineered Gateway vector for functional analysis. CONCLUSION: The new EST collection denotes an important step towards the identification of all genes in the citrus genome. Furthermore, public availability of the cDNA clones generated in this study, and not only their sequence, enables testing of the biological function of the genes represented in the collection. Expression of the citrus SEP3 homologue, CitrSEP, in Arabidopsis results in early flowering, along with other phenotypes resembling the over-expression of the Arabidopsis SEPALLATA genes. Our findings suggest that the members of the SEP gene family play similar roles in these quite distant plant species.

Antagonistic interactions between Arabidopsis K-homology domain genes uncover PEPPER as a positive regulator of the central floral repressor FLOWERING LOCUS C.

Plant floral transition is a major developmental switch regulated by an integrated network of pathways. Arabidopsis FLOWERING LOCUS K (FLK), a protein with three KH RNA-binding domains, operates in the autonomous flowering-promotive pathway by decreasing the transcript levels of the key flowering repressor FLOWERING LOCUS C (FLC). Here we report that PEPPER (PEP), an FLK paralog previously shown to affect vegetative and pistil development, antagonizes FLK by positively regulating FLC. Lack of PEP function rescues the flk late-flowering phenotype with a concomitant decrease in FLC RNA levels. Loss of HUA2, another FLC activator encoding an RNA-binding protein, further rescues flk, being flk hua2 pep triple mutants virtually wild-type regarding flowering time. Consistently, PEP overexpression determines high levels of FLC transcripts and flowering delay. Genetic and molecular analyses indicate that FLK and PEP act independently of FCA, another important FLC repressor in the autonomous pathway. In addition, we present data suggesting that PEP may affect FLC expression at both transcriptional and post-transcriptional levels. Overall, our results uncover PEP as a new factor for FLC upregulation, underscoring the importance of RNA-binding activities during developmental timing of flowering.

Common regulatory networks in leaf and fruit patterning revealed by mutations in the Arabidopsis ASYMMETRIC LEAVES1 gene.

Carpels and leaves are evolutionarily related organs, as the former are thought to be modified leaves. Therefore, developmental pathways that play crucial roles in patterning both organs are presumably conserved. In leaf primordia of Arabidopsis thaliana, the ASYMMETRIC LEAVES1 (AS1) gene interacts with AS2 to repress the class I KNOTTED1-like homeobox (KNOX) genes BREVIPEDICELLUS (BP), KNAT2 and KNAT6, restricting the expression of these genes to the meristem. In this report, we describe how AS1, presumably in collaboration with AS2, patterns the Arabidopsis gynoecium by repressing BP, which is expressed in the replum and valve margin, interacts in the replum with REPLUMLESS (RPL), an essential gene for replum development, and positively regulates the expression of this gene. Misexpression of BP in the gynoecium causes an increase in replum size, while the valve width is slightly reduced, and enhances the effect of mutations in FRUITFULL (FUL), a gene with an important function in valve development. Altogether, these findings strongly suggest that BP plays a crucial role in replum development. We propose a model for pattern formation along the mediolateral axis of the ovary, whereby three domains (replum, valve margin and valve) are specified by the opposing gradients of two antagonistic factors, valve factors and replum factors, the class I KNOX genes working as the latter.