Mining for allelic gold: finding genetic variation in photosynthetic traits in crops and wild relatives.

Improvement of photosynthetic traits in crops to increase yield potential and crop resilience has recently become a major breeding target. Synthetic biology and genetic technologies offer unparalleled opportunities to create new genetics for photosynthetic traits driven by existing fundamental knowledge. However, large 'gene bank' collections of germplasm comprising historical collections of crop species and their relatives offer a wealth of opportunities to find novel allelic variation in the key steps of photosynthesis, to identify new mechanisms and to accelerate genetic progress in crop breeding programmes. Here we explore the available genetic resources in food and fibre crops, strategies to selectively target allelic variation in genes underpinning key photosynthetic processes, and deployment of this variation via gene editing in modern elite material.

Lateral transfers of large DNA fragments spread functional genes among grasses.

A fundamental tenet of multicellular eukaryotic evolution is that vertical inheritance is paramount, with natural selection acting on genetic variants transferred from parents to offspring. This lineal process means that an organism's adaptive potential can be restricted by its evolutionary history, the amount of standing genetic variation, and its mutation rate. Lateral gene transfer (LGT) theoretically provides a mechanism to bypass many of these limitations, but the evolutionary importance and frequency of this process in multicellular eukaryotes, such as plants, remains debated. We address this issue by assembling a chromosome-level genome for the grass Alloteropsis semialata, a species surmised to exhibit two LGTs, and screen it for other grass-to-grass LGTs using genomic data from 146 other grass species. Through stringent phylogenomic analyses, we discovered 57 additional LGTs in the A. semialata nuclear genome, involving at least nine different donor species. The LGTs are clustered in 23 laterally acquired genomic fragments that are up to 170 kb long and have accumulated during the diversification of Alloteropsis. The majority of the 59 LGTs in A. semialata are expressed, and we show that they have added functions to the recipient genome. Functional LGTs were further detected in the genomes of five other grass species, demonstrating that this process is likely widespread in this globally important group of plants. LGT therefore appears to represent a potent evolutionary force capable of spreading functional genes among distantly related grass species.

Variation between rice accessions in photosynthetic induction in flag leaves and underlying mechanisms.

Several breeding initiatives have sought to improve flag leaf performance as its health and physiology are closely correlated to rice yield. Previous studies have described natural variation of photosynthesis for flag leaves; however, none has examined their performance under the non-steady-state conditions that prevail in crop fields. Photosynthetic induction is the transient response of photosynthesis to a change from low to high light. Rice flag leaf photosynthesis was measured in both steady- and non-steady-state conditions to characterize natural variation. Between the lowest and highest performing accession, there was a 152% difference for average CO2 assimilation during induction (A300), a 77% difference for average intrinsic water use efficiency during induction (iWUEavg), and a 185% difference for the speed of induction (IT50), indicating plentiful variation. No significant correlation was found between steady- and non-steady-state photosynthetic traits. Additionally, measures of neither steady-state nor non-steady-state photosynthesis of flag leaves correlated with the same measures of leaves in the vegetative growth stage, with the exception of iWUEavg. Photosynthetic induction was measured at six [CO2], to determine biochemical and diffusive limitations to photosynthesis in vivo. Photosynthetic induction in rice flag leaves was limited primarily by biochemistry.

Overexpression of the chloroplastic 2-oxoglutarate/malate transporter disturbs carbon and nitrogen homeostasis in rice.

The chloroplastic 2-oxaloacetate (OAA)/malate transporter (OMT1 or DiT1) takes part in the malate valve that protects chloroplasts from excessive redox poise through export of malate and import of OAA. Together with the glutamate/malate transporter (DCT1 or DiT2), it connects carbon with nitrogen assimilation, by providing 2-oxoglutarate for the GS/GOGAT (glutamine synthetase/glutamate synthase) reaction and exporting glutamate to the cytoplasm. OMT1 further plays a prominent role in C4 photosynthesis: OAA resulting from phosphoenolpyruvate carboxylation is imported into the chloroplast, reduced to malate by plastidic NADP-malate dehydrogenase, and then exported for transport to bundle sheath cells. Both transport steps are catalyzed by OMT1, at the rate of net carbon assimilation. To engineer C4 photosynthesis into C3 crops, OMT1 must be expressed in high amounts on top of core C4 metabolic enzymes. We report here high-level expression of ZmOMT1 from maize in rice (Oryza sativa ssp. indica IR64). Increased activity of the transporter in transgenic rice was confirmed by reconstitution of transporter activity into proteoliposomes. Unexpectedly, overexpression of ZmOMT1 in rice negatively affected growth, CO2 assimilation rate, total free amino acid content, tricarboxylic acid cycle metabolites, as well as sucrose and starch contents. Accumulation of high amounts of aspartate and the impaired growth phenotype of OMT1 rice lines could be suppressed by simultaneous overexpression of ZmDiT2. Implications for engineering C4 rice are discussed.

Variation in photosynthetic induction between rice accessions and its potential for improving productivity.

Photosynthetic induction describes the transient increase in leaf CO2 uptake with an increase in light. During induction, efficiency is lower than at steady state. Under field conditions of fluctuating light, this lower efficiency during induction may cost > 20% of potential crop assimilation. Accelerating induction would boost photosynthetic and resource-use efficiencies. Variation between rice accessions and potential for accelerating induction was analysed by gas exchange. Induction during shade to sun transitions of 14 accessions representing five subpopulations from the 3000 Rice Genome Project Panel (3K RGP) was analysed. Differences of 109% occurred in the CO2 fixed during the first 300 s of induction, 117% in the half-time to completion of induction, and 65% in intrinsic water-use efficiency during induction, between the highest and lowest performing accessions. Induction in three accessions with contrasting responses (AUS 278, NCS 771 A and IR64-21) was compared for a range of [CO2 ] to analyse limitations. This showed in vivo capacity for carboxylation at Rubisco (Vc,max ), and not stomata, as the primary limitation to induction, with significant differences between accessions. Variation in nonsteady-state efficiency greatly exceeded that at steady state, suggesting a new and more promising opportunity for selection of greater crop photosynthetic efficiency in this key food crop.

Rice with reduced stomatal density conserves water and has improved drought tolerance under future climate conditions.

Much of humanity relies on rice (Oryza sativa) as a food source, but cultivation is water intensive and the crop is vulnerable to drought and high temperatures. Under climate change, periods of reduced water availability and high temperature are expected to become more frequent, leading to detrimental effects on rice yields. We engineered the high-yielding rice cultivar 'IR64' to produce fewer stomata by manipulating the level of a developmental signal. We overexpressed the rice epidermal patterning factor OsEPF1, creating plants with substantially reduced stomatal density and correspondingly low stomatal conductance. Low stomatal density rice lines were more able to conserve water, using c. 60% of the normal amount between weeks 4 and 5 post germination. When grown at elevated atmospheric CO2 , rice plants with low stomatal density were able to maintain their stomatal conductance and survive drought and high temperature (40°C) for longer than control plants. Low stomatal density rice gave equivalent or even improved yields, despite a reduced rate of photosynthesis in some conditions. Rice plants with fewer stomata are drought tolerant and more conservative in their water use, and they should perform better in the future when climate change is expected to threaten food security.

Transgenic maize phosphoenolpyruvate carboxylase alters leaf-atmosphere CO2 and 13CO2 exchanges in Oryza sativa.

The engineering process of C4 photosynthesis into C3 plants requires an increased activity of phosphoenolpyruvate carboxylase (PEPC) in the cytosol of leaf mesophyll cells. The literature varies on the physiological effect of transgenic maize (Zea mays) PEPC (ZmPEPC) leaf expression in Oryza sativa (rice). Therefore, to address this issue, leaf-atmosphere CO2 and 13CO2 exchanges were measured, both in the light (at atmospheric O2 partial pressure of 1.84 kPa and at different CO2 levels) and in the dark, in transgenic rice expressing ZmPEPC and wild-type (WT) plants. The in vitro PEPC activity was 25 times higher in the PEPC overexpressing (PEPC-OE) plants (~20% of maize) compared to the negligible activity in WT. In the PEPC-OE plants, the estimated fraction of carboxylation by PEPC (ß) was ~6% and leaf net biochemical discrimination against 13CO2[Formula: see text] was ~ 2‰ lower than in WT. However, there were no differences in leaf net CO2 assimilation rates (A) between genotypes, while the leaf dark respiration rates (Rd) over three hours after light-dark transition were enhanced (~ 30%) and with a higher 13C composition [Formula: see text] in the PEPC-OE plants compared to WT. These data indicate that ZmPEPC in the PEPC-OE rice plants contributes to leaf carbon metabolism in both the light and in the dark. However, there are some factors, potentially posttranslational regulation and PEP availability, which reduce ZmPEPC activity in vivo.

Knockdown of glycine decarboxylase complex alters photorespiratory carbon isotope fractionation in Oryza sativa leaves.

The influence of reduced glycine decarboxylase complex (GDC) activity on leaf atmosphere CO2 and 13CO2 exchange was tested in transgenic Oryza sativa with the GDC H-subunit knocked down in leaf mesophyll cells. Leaf measurements on transgenic gdch knockdown and wild-type plants were carried out in the light under photorespiratory and low photorespiratory conditions (i.e. 18.4 kPa and 1.84 kPa atmospheric O2 partial pressure, respectively), and in the dark. Under approximately current ambient O2 partial pressure (18.4 kPa pO2), the gdch knockdown plants showed an expected photorespiratory-deficient phenotype, with lower leaf net CO2 assimilation rates (A) than the wild-type. Additionally, under these conditions, the gdch knockdown plants had greater leaf net discrimination against 13CO2 (Δo) than the wild-type. This difference in Δo was in part due to lower 13C photorespiratory fractionation (f) ascribed to alternative decarboxylation of photorespiratory intermediates. Furthermore, the leaf dark respiration rate (Rd) was enhanced and the 13CO2 composition of respired CO2 (δ13CRd) showed a tendency to be more depleted in the gdch knockdown plants. These changes in Rd and δ13CRd were due to the amount and carbon isotopic composition of substrates available for dark respiration. These results demonstrate that impairment of the photorespiratory pathway affects leaf 13CO2 exchange, particularly the 13C decarboxylation fractionation associated with photorespiration.

Key changes in gene expression identified for different stages of C4 evolution in Alloteropsis semialata.

C4 photosynthesis is a complex trait that boosts productivity in tropical conditions. Compared with C3 species, the C4 state seems to require numerous novelties, but species comparisons can be confounded by long divergence times. Here, we exploit the photosynthetic diversity that exists within a single species, the grass Alloteropsis semialata, to detect changes in gene expression associated with different photosynthetic phenotypes. Phylogenetically informed comparative transcriptomics show that intermediates with a weak C4 cycle are separated from the C3 phenotype by increases in the expression of 58 genes (0.22% of genes expressed in the leaves), including those encoding just three core C4 enzymes: aspartate aminotransferase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxylase. The subsequent transition to full C4 physiology was accompanied by increases in another 15 genes (0.06%), including only the core C4 enzyme pyruvate orthophosphate dikinase. These changes probably created a rudimentary C4 physiology, and isolated populations subsequently improved this emerging C4 physiology, resulting in a patchwork of expression for some C4 accessory genes. Our work shows how C4 assembly in A. semialata happened in incremental steps, each requiring few alterations over the previous step. These create short bridges across adaptive landscapes that probably facilitated the recurrent origins of C4 photosynthesis through a gradual process of evolution.

Natural variation in tolerance to sub-zero temperatures among populations of Arabidopsis lyrata ssp. petraea.

BACKGROUND: Temperature is one of the most important abiotic factors limiting plant growth and productivity. Many plants exhibit cold acclimation to prepare for the likelihood of freezing as temperatures decrease towards 0 °C. The physiological mechanisms associated with enabling increased tolerance to sub-zero temperatures vary between species and genotypes. Geographically and climatically diverse populations of Arabidopsis lyrata ssp. petraea were examined for their ability to survive, maintain functional photosynthetic parameters and cellular electrolyte leakage integrity after being exposed to sub-zero temperatures. The duration of cold acclimation prior to sub-zero temperatures was also manipulated (2 and 14 days). RESULTS: We found that there was significant natural variation in tolerances to sub-zero temperatures among populations of A. petraea. The origin of the population affected the acclimation response and survival after exposure to sub-zero temperatures. Cold acclimation of plants prior to sub-zero temperatures affected the maximum quantum efficiency of photosystem II (PSII) (Fv/Fm) in that plants that were cold acclimated for longer periods had higher values of Fv/Fm as a result of sub-zero temperatures. The inner immature leaves were better able to recover Fv/Fm from sub-zero temperatures than mature outer leaves. The Irish population (Leitrim) acclimated faster, in terms of survival and electrolyte leakage than the Norwegian population (Helin). CONCLUSION: The ability to survive, recover photosynthetic processes and cellular electrolyte leakage after exposure to sub-zero temperatures is highly dependent on the duration of cold acclimation.

Diurnal Solar Energy Conversion and Photoprotection in Rice Canopies.

Genetic improvement of photosynthetic performance of cereal crops and increasing the efficiency with which solar radiation is converted into biomass has recently become a major focus for crop physiologists and breeders. The pulse amplitude modulated chlorophyll fluorescence technique (PAM) allows quantitative leaf level monitoring of the utilization of energy for photochemical light conversion and photoprotection in natural environments, potentially over the entire crop lifecycle. Here, the diurnal relationship between electron transport rate (ETR) and irradiance was measured in five cultivars of rice (Oryza sativa) in canopy conditions with PAM fluorescence under natural solar radiation. This relationship differed substantially from that observed for conventional short term light response curves measured under controlled actinic light with the same leaves. This difference was characterized by a reduced curvature factor when curve fitting was used to model this diurnal response. The engagement of photoprotective processes in chloroplast electron transport in leaves under canopy solar radiation was shown to be a major contributor to this difference. Genotypic variation in the irradiance at which energy flux into photoprotective dissipation became greater than ETR was observed. Cultivars capable of higher ETR at midrange light intensities were shown to produce greater leaf area over time, estimated by noninvasive imaging.

Combined Chlorophyll Fluorescence and Transcriptomic Analysis Identifies the P3/P4 Transition as a Key Stage in Rice Leaf Photosynthetic Development.

Leaves are derived from heterotrophic meristem tissue that, at some point, must make the transition to autotrophy via the initiation of photosynthesis. However, the timing and spatial coordination of the molecular and cellular processes underpinning this switch are poorly characterized. Here, we report on the identification of a specific stage in rice (Oryza sativa) leaf development (P3/P4 transition) when photosynthetic competence is first established. Using a combined physiological and molecular approach, we show that elements of stomatal and vascular differentiation are coordinated with the onset of measurable light absorption for photosynthesis. Moreover, by exploring the response of the system to environmental perturbation, we show that the earliest stages of rice leaf development have significant plasticity with respect to elements of cellular differentiation of relevance for mature leaf photosynthetic performance. Finally, by performing an RNA sequencing analysis targeted at the early stages of rice leaf development, we uncover a palette of genes whose expression likely underpins the acquisition of photosynthetic capability. Our results identify the P3/P4 transition as a highly dynamic stage in rice leaf development when several processes for the initiation of photosynthetic competence are coordinated. As well as identifying gene targets for future manipulation of rice leaf structure/function, our data highlight a developmental window during which such manipulations are likely to be most effective.

Targeted Knockdown of GDCH in Rice Leads to a Photorespiratory-Deficient Phenotype Useful as a Building Block for C4 Rice.

The glycine decarboxylase complex (GDC) plays a critical role in the photorespiratory C2 cycle of C3 species by recovering carbon following the oxygenation reaction of ribulose-1,5-bisphosphate carboxylase/oxygenase. Loss of GDC from mesophyll cells (MCs) is considered a key early step in the evolution of C4 photosynthesis. To assess the impact of preferentially reducing GDC in rice MCs, we decreased the abundance of OsGDCH (Os10g37180) using an artificial microRNA (amiRNA) driven by a promoter that preferentially drives expression in MCs. GDC H- and P-proteins were undetectable in leaves of gdch lines. Plants exhibited a photorespiratory-deficient phenotype with stunted growth, accelerated leaf senescence, reduced chlorophyll, soluble protein and sugars, and increased glycine accumulation in leaves. Gas exchange measurements indicated an impaired ability to regenerate ribulose 1,5-bisphosphate in photorespiratory conditions. In addition, MCs of gdch lines exhibited a significant reduction in chloroplast area and coverage of the cell wall when grown in air, traits that occur during the later stages of C4 evolution. The presence of these two traits important for C4 photosynthesis and the non-lethal, down-regulation of the photorespiratory C2 cycle positively contribute to efforts to produce a C4 rice prototype.

Standards for plant synthetic biology: a common syntax for exchange of DNA parts.

Inventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.

Evolution of GOLDEN2-LIKE gene function in C(3) and C (4) plants.

A pair of GOLDEN2-LIKE transcription factors is required for normal chloroplast development in land plant species that encompass the range from bryophytes to angiosperms. In the C(4) plant maize, compartmentalized function of the two GLK genes in bundle sheath and mesophyll cells regulates dimorphic chloroplast differentiation, whereas in the C(3) plants Physcomitrella patens and Arabidopsis thaliana the genes act redundantly in all photosynthetic cells. To assess whether the cell-specific function of GLK genes is unique to maize, we analyzed gene expression patterns in the C(4) monocot Sorghum bicolor and C(4) eudicot Cleome gynandra. Compartmentalized expression was observed in S. bicolor, consistent with the development of dimorphic chloroplasts in this species, but not in C. gynandra where bundle sheath and mesophyll chloroplasts are morphologically similar. The generation of single and double mutants demonstrated that GLK genes function redundantly in rice, as in other C(3) plants, despite the fact that GLK gene duplication in monocots preceded the speciation of rice, maize and sorghum. Together with phylogenetic analyses of GLK gene sequences, these data have allowed speculation on the evolutionary trajectory of GLK function. Based on current evidence, most species that retain single GLK genes belong to orders that contain only C(3) species. We therefore propose that the ancestral state is a single GLK gene, and hypothesize that GLK gene duplication enabled sub-functionalization, which in turn enabled cell-specific function in C(4) plants with dimorphic chloroplasts. In this scenario, GLK gene duplication preconditioned the evolution of C(4) physiology that is associated with chloroplast dimorphism.

Antisense suppression of the small chloroplast protein CP12 in tobacco alters carbon partitioning and severely restricts growth.

The thioredoxin-regulated chloroplast protein CP12 forms a multienzyme complex with the Calvin-Benson cycle enzymes phosphoribulokinase (PRK) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PRK and GAPDH are inactivated when present in this complex, a process shown in vitro to be dependent upon oxidized CP12. The importance of CP12 in vivo in higher plants, however, has not been investigated. Here, antisense suppression of CP12 in tobacco (Nicotiana tabacum) was observed to impact on NAD-induced PRK and GAPDH complex formation but had little effect on enzyme activity. Additionally, only minor changes in photosynthetic carbon fixation were observed. Despite this, antisense plants displayed changes in growth rates and morphology, including dwarfism and reduced apical dominance. The hypothesis that CP12 is essential to separate oxidative pentose phosphate pathway activity from Calvin-Benson cycle activity, as proposed in cyanobacteria, was tested. No evidence was found to support this role in tobacco. Evidence was seen, however, for a restriction to malate valve capacity, with decreases in NADP-malate dehydrogenase activity (but not protein levels) and pyridine nucleotide content. Antisense repression of CP12 also led to significant changes in carbon partitioning, with increased carbon allocation to the cell wall and the organic acids malate and fumarate and decreased allocation to starch and soluble carbohydrates. Severe decreases were also seen in 2-oxoglutarate content, a key indicator of cellular carbon sufficiency. The data presented here indicate that in tobacco, CP12 has a role in redox-mediated regulation of carbon partitioning from the chloroplast and provides strong in vivo evidence that CP12 is required for normal growth and development in plants.

C acid decarboxylases required for C photosynthesis are active in the mid-vein of the C species Arabidopsis thaliana, and are important in sugar and amino acid metabolism.

Cells associated with veins of petioles of C(3) tobacco possess high activities of the decarboxylase enzymes required in C(4) photosynthesis. It is not clear whether this is the case in other C(3) species, nor whether these enzymes provide precursors for specific biosynthetic pathways. Here, we investigate the activity of C(4) acid decarboxylases in the mid-vein of Arabidopsis, identify regulatory regions sufficient for this activity, and determine the impact of removing individual isoforms of each protein on mid-vein metabolite profiles. This showed that radiolabelled malate and bicarbonate fed to the xylem stream were incorporated into soluble and insoluble material in the mid-vein of Arabidopsis leaves. Compared with the leaf lamina, mid-veins possessed high activities of NADP-dependent malic enzyme (NADP-ME), NAD-dependent malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PEPCK). Transcripts derived from both NAD-ME, one PCK and two of the four NADP-ME genes were detectable in these veinal cells. The promoters of each decarboxylase gene were sufficient for expression in mid-veins. Analysis of insertional mutants revealed that cytosolic NADP-ME2 is responsible for 80% of NADP-ME activity in mid-veins. Removing individual decarboxylases affected the abundance of amino acids derived from pyruvate and phosphoenolpyruvate. Reducing cytosolic NADP-ME activity preferentially affected the sugar content, whereas abolishing NAD-ME affected both the amino acid and the glucosamine content of mid-veins.

Systemic low temperature signaling in Arabidopsis.

When leaves are exposed to low temperature, sugars accumulate and transcription factors in the C-repeat binding factor (CBF) family are expressed, which, together with CBF-independent pathways, are known to contribute to the cold acclimation process and an increase in freezing tolerance. What is not known, however, is whether expression of these cold-regulated genes can be induced systemically in response to a localized cold treatment. To address this, pre-existing, mature leaves of warm-grown Arabidopsis thaliana were exposed to a localized cold treatment (near 10 °C) whilst conjoined newly developing leaves continued only to experience warmer temperatures. In initial experiments on wild-type A. thaliana (Col-0) using real-time reverse transcription--PCR (RT-PCR) we observed that some genes--including CBF genes, certain downstream cold-responsive (COR) targets and CBF-independent transcription factors--respond to a direct 9 °C treatment of whole plants. In subsequent experiments, we found that the treatment of expanded leaves with temperatures near 10 °C can induce cold-associated genes in conjoined warm-maintained tissues. CBF1 showed a particularly strong systemic response, although CBF-independent transcription factors also responded. Moreover, the localized cold treatment of A. thaliana (C24) plants with a luciferase reporter fused to the promoter region of KIN2 indicated that in warm-maintained leaves, KIN2 might respond to a systemic signal from remote, directly cold-treated leaves. Collectively, our study provides strong evidence that the processes involved in cold acclimation are partially mediated by a signal that acts systemically. This has the potential to act as an early-warning system to enable developing leaves to cope better with the cold environment in which they are growing.

High-throughput chlorophyll fluorescence screening of Setaria viridis for mutants with altered CO2 compensation points.

To assist with efforts to engineer a C4 photosynthetic pathway into rice, forward-genetic approaches are being used to identify the genes modulating key C4 traits. Currently, a major challenge is how to screen for a variety of different traits in a high-throughput manner. Here we describe a method for identifying C4 mutant plants with increased CO2 compensation points. This is used as a signature for decreased photosynthetic efficiency associated with a loss of C4 function. By exposing plants to a CO2 concentration close to the CO2 compensation point of a wild-type plant, individuals can be identified from measurements of chlorophyll a fluorescence. We use this method to screen a mutant population of the C4 monocot Setaria viridis (L.)P.Beauv. generated using N-nitroso-N-methylurea (NMU). Mutants were identified at a frequency of 1 per 157 lines screened. Forty-six candidate lines were identified and one line with a heritable homozygous phenotype selected for further characterisation. The CO2 compensation point of this mutant was increased to a value similar to that of C3 rice. Photosynthesis and growth was significantly reduced under ambient conditions. These data indicate that the screen was capable of identifying mutants with decreased photosynthetic efficiency. Characterisation and next-generation sequencing of all the mutants identified in this screen may lead to the discovery of novel genes underpinning C4 photosynthesis. These can be used to engineer a C4 photosynthetic pathway into rice.

Re-creation of a Key Step in the Evolutionary Switch from C3 to C4 Leaf Anatomy.

The C4 photosynthetic pathway accounts for ∼25% of primary productivity on the planet despite being used by only 3% of species. Because C4 plants are higher yielding than C3 plants, efforts are underway to introduce the C4 pathway into the C3 crop rice. This is an ambitious endeavor; however, the C4 pathway evolved from C3 on multiple independent occasions over the last 30 million years, and steps along the trajectory are evident in extant species. One approach toward engineering C4 rice is to recapitulate this trajectory, one of the first steps of which was a change in leaf anatomy. The transition from C3 to so-called "proto-Kranz" anatomy requires an increase in organelle volume in sheath cells surrounding leaf veins. Here we induced chloroplast and mitochondrial development in rice vascular sheath cells through constitutive expression of maize GOLDEN2-LIKE genes. Increased organelle volume was accompanied by the accumulation of photosynthetic enzymes and by increased intercellular connections. This suite of traits reflects that seen in "proto-Kranz" species, and, as such, a key step toward engineering C4 rice has been achieved.