C3 Rice Research Articles

Revolutionizing Rice: The Synthesis of Nature's Evolution and Biotechnology in C4 Rice

Agriculture plays a fundamental role in meeting the essential needs of humanity, yet numerous factors continuously influence agricultural production and productivity. Over time, evolving agricultural practices have significantly increased yields of staple food crops, thereby enhancing global food security. However, as the world's population continues to grow, there is a pressing need to further augment crop yields. Historically, increases in yield were achieved by expanding cultivated land, but rapid industrialization and urbanization have diminished available agricultural land and depleted natural resources essential for farming. In response to these challenges, enhancing crop photosynthetic efficiency has emerged as a pivotal strategy. Photosynthesis, the process by which plants convert sunlight into energy, has been optimized in various crops through evolutionary and biotechnological approaches. One such innovation is the adaptation of the C4 photosynthetic pathway into C3 rice, a staple food for billions worldwide. The C4 pathway, found in several efficient crops like maize and sugarcane, enhances carbon dioxide uptake and reduces photorespiration, leading to improved water and nitrogen use efficiency. This review explores the integration of nature’s evolutionary principles with cutting-edge biotechnological advancements in the development of C4 rice. By introducing the C4 pathway into rice—a traditionally C3 plant—scientists aim to dramatically increase its productivity and resilience to environmental stressors. This transformative approach not only holds promise for meeting future food demands sustainably but also addresses the challenges posed by climate change and resource scarcity. Moreover, the review examines the scientific basis, technological innovations, and potential agricultural implications of implementing C4 rice. It highlights the collaborative efforts of researchers, agronomists, and biotechnologists worldwide in reimagining rice cultivation to secure global food supplies while minimizing environmental impact. Ultimately, the integration of C4 photosynthesis into rice represents a significant step forward in agricultural innovation, poised to redefine food production capabilities and enhance global food security in the face of escalating population growth and environmental change.

Convergent and divergent signaling pathways in C3 rice and C4 foxtail millet crops in response to salt stress

Salt stress is a global constraint on agricultural production. Therefore, the development of salt tolerant plants has become a current research hotspot. Salt tolerance evolves more frequently in C4 grass lineages. However, few studies have been carried out to explore the molecular bases underlying salt stress tolerance in C4 crop foxtail millet. In this study, we performed a multi-pronged approach spanning the omics analyses of transcriptomes and physiological analysis of C3 crop rice and C4 model crop foxtail millet in response to salt stress. Our results revealed specifically compared to C3 rice, C4 foxtail millet has upregulated ABA and notably reduced CK biosynthesis and signaling transduction under salt stress. Salt stress in C3 rice plants triggered rapid downregulation of photosynthesis related genes, which was coupled by severely decreased net photosynthetic rates. In the salt-threatened C3 rice and C4 foxtail millet, some stress responsive transcription factors (TFs), such as AP2/ERF, WRKY and MYB underwent strong and distinct transcriptional changes. Based on weighted gene co-expression network analysis (WGCNA), an AP2/ERF transcription factor Rice Starch Regulator1 SiRSR1 (Seita.3G044600) was identified as a key regulator of salt stress response. To confirmed its function, we generated OsRSR1-knockout lines with CRISPR/Cas9 genome editing in rice and its upstream repressor SiMIR172a-overexpressing (172a-OE) transgenic plants in foxtail millet, which increased salt tolerance. Overall, our study not only provided new insights into the convergent regulation of salt stress responses of foxtail millet and rice, but also shed light on the divergent signaling networks between them in response to salt stress.

A rapid and robust leaf ablation method to visualize bundle sheath cells and chloroplasts in C3 and C4 grasses

BackgroundIt has been proposed that engineering the C4 photosynthetic pathway into C3 crops could significantly increase yield. This goal requires an increase in the chloroplast compartment of bundle sheath cells in C3 species. To facilitate large-scale testing of candidate regulators of chloroplast development in the rice bundle sheath, a simple and robust method to phenotype this tissue in C3 species is required.ResultsWe established a leaf ablation method to accelerate phenotyping of rice bundle sheath cells. The bundle sheath cells and chloroplasts were visualized using light and confocal laser microscopy. Bundle sheath cell dimensions, chloroplast area and chloroplast number per cell were measured from the images obtained by confocal laser microscopy. Bundle sheath cell dimensions of maize were also measured and compared with rice. Our data show that bundle sheath width but not length significantly differed between C3 rice and C4 maize. Comparison of paradermal versus transverse bundle sheath cell width indicated that bundle sheath cells were intact after leaf ablation. Moreover, comparisons of planar chloroplast areas and chloroplast numbers per bundle sheath cell between wild-type and transgenic rice lines expressing the maize GOLDEN-2 (ZmG2) showed that the leaf ablation method allowed differences in chloroplast parameters to be detected.ConclusionsLeaf ablation is a simple approach to accessing bundle sheath cell files in C3 species. We show that this method is suitable for obtaining parameters associated with bundle sheath cell size, chloroplast area and chloroplast number per cell.

Bioengineering of Canopy Photosynthesis in Rice for Securing Global Food Security: A Critical Review

The emerging threat of climate change is posing a scientific conundrum for global food and nutritional security. As a primary staple food, half of the global human population is supported by rice crop. Thus, enhancing rice yield is highly critical to ensure food security. Photosynthesis is the defining physiological process of plants that determines maximum attainable yield. Efficiently capturing solar radiation and converting the carbon assimilates into rice grain is critical to achieve high yield. Genetic interventions to modify the plant architecture for enhanced light capture can improve rice yield significantly. Enhancement of cellular photosynthesis by synthetic biology approaches targeting important nodes of the light harvesting and carbon assimilation pathways are critical for breaking yield ceiling. The possible targets for improving photosynthesis include the light capture, chloroplast electron transport, Calvin cycle enzymes, sugar transport mechanisms, minimization of photorespiration, and improving source–sink relations. Conversion of C3 rice into a C4 type plant is also an option being vigorously pursued. Here, we review the determinants of canopy photosynthesis in rice with special reference to genetic factors and cellular photosynthetic capacity.

Overexpression of Setaria italica phosphoenolpyruvate carboxylase gene in rice positively impacts photosynthesis and agronomic traits

C4 plants have the inherent capacity to concentrate atmospheric CO2 in the vicinity of RuBisCo, thereby increasing carboxylation, and inhibiting photorespiration. Carbonic anhydrase (CA), the first enzyme of C4 photosynthesis, converts atmospheric CO2 to HCO3−, which is utilized by PEPC to produce C4 acids. Bioengineering of C4 traits into C3 crops is an attractive strategy to increase photosynthesis and water use efficiency. In the present study, we isolated the PEPC gene from the C4 plant Setaria italica and transferred it to C3 rice. Overexpression of SiPEPC resulted in a 2-6-fold increment in PEPC enzyme activity in transgenic lines with respect to non-transformed control. Photosynthetic efficiency was enhanced in transformed plants, which was associated with increased ФPSII, ETR, lower NPQ, and higher chlorophyll accumulation. Water use efficiency was increased by 16–22% in PEPC transgenic rice lines. Increased PEPC activity enhanced quantum yield and carboxylation efficiency of PEPC transgenic lines. Transgenic plants exhibited higher light saturation photosynthesis rate and lower CO2 compensation point, as compared to non-transformed control. An increase in net photosynthesis increased the yield by (23–28.9%) and biomass by (24.1–29%) in transgenic PEPC lines. Altogether, our findings indicate that overexpression of C4-specific SiPEPC enzyme is able to enhance photosynthesis and related parameters in transgenic rice.

Unique features of regulation of sulfate assimilation in monocots.

Sulfate assimilation is an essential pathway of plant primary metabolism, regulated by the demand for reduced sulfur (S). The S-containing tripeptide glutathione (GSH) is the key signal for such regulation in Arabidopsis, but little is known about the conservation of these regulatory mechanisms beyond this model species. Using two model monocot species, C3 rice (Oryza sativa) and C4Setaria viridis, and feeding of cysteine or GSH, we aimed to find out how conserved are the regulatory mechanisms described for Arabidopsis in these species. We showed that while in principle the regulation is similar, there are many species-specific differences. For example, thiols supplied by the roots are translocated to the shoots in rice but remain in the roots of Setaria. Cysteine and GSH concentrations are highly correlated in Setaria, but not in rice. In both rice and Setaria, GSH seems to be the signal for demand-driven regulation of sulfate assimilation. Unexpectedly, we observed cysteine oxidation to sulfate in both species, a reaction that does not occur in Arabidopsis. This reaction is dependent on sulfite oxidase, but the enzyme(s) releasing sulfite from cysteine still need to be identified. Altogether our data reveal a number of unique features in the regulation of S metabolism in the monocot species and indicate the need for using multiple taxonomically distinct models to better understand the control of nutrient homeostasis, which is important for generating low-input crop varieties.

Estimating photosynthetic parameter values of rice, wheat, maize and sorghum to enable smart crop cultivation

Crop models can support the design of smart crop management practices. The Farquhar-von Caemmerer-Berry (FvCB) model is increasingly being used in these models for quantifying leaf photosynthesis. Nitrogen (N) is required for many functional machineries of photosynthesis, thus relationships between FvCB-model parameters and leaf N content (LNC) should be established. We conducted combined gas exchange and chlorophyll fluorescence measurements on fully expanded leaves of two C3 crops, rice (Oryza sativa) and wheat (Triticum aestivum), and two C4 crops, maize (Zea mays) and sorghum (Sorghum bicolor), grown under three N levels. Photosynthetic parameters were estimated and linear relationships between these parameters and LNC were quantified in both C3 and C4 crop types. The efficiency of converting incident light into linear electron transport for C3 crops or into ATP production for C4 crops showed a weak increase with LNC. The maximum electron transport rate (Jmax) for C3 crops or the maximum ATP production rate (Jmax,atp) for C4 crops significantly increased with LNC. The increase in Rubisco carboxylation capacity (Vcmax) with LNC was significantly higher in C3 than in C4 crops. Triose phosphate utilization for C3 crops and PEP carboxylation capacity (Vpmax) for C4 crops increased significantly with LNC as well. Except for Jmax at 21% O2 and Vcmax of C3 crops, there was no significant difference among crops in the relationship between estimated photosynthetic parameters and LNC. The tight associations of photosynthesis parameters with LNC were discussed in view of decision making on N management in the context of smart farming.

Revisiting Why Plants Become N Deficient Under Elevated CO2: Importance to Meet N Demand Regardless of the Fed-Form.

An increase in plant biomass under elevated CO2 (eCO2) is usually lower than expected. N-deficiency induced by eCO2 is often considered to be a reason for this. Several hypotheses explain the induced N-deficiency: (1) eCO2 inhibits nitrate assimilation, (2) eCO2 lowers nitrate acquisition due to reduced transpiration, or (3) eCO2 reduces plant N concentration with increased biomass. We tested them using C3 (wheat, rice, and potato) and C4 plants (guinea grass, and Amaranthus) grown in chambers at 400 (ambient CO2, aCO2) or 800 (eCO2) μL L−1 CO2. In most species, we could not confirm hypothesis (1) with the measurements of plant nitrate accumulation in each organ. The exception was rice showing a slight inhibition of nitrate assimilation at eCO2, but the biomass was similar between the nitrate and urea-fed plants. Contrary to hypothesis (2), eCO2 did not decrease plant nitrate acquisition despite reduced transpiration because of enhanced nitrate acquisition per unit transpiration in all species. Comparing to aCO2, eCO2 remarkably enhanced water-use efficiency, especially in C3 plants, decreasing water demand for CO2 acquisition. As our results supported hypothesis (3) without any exception, we then examined if lowered N concentration at eCO2 indeed limits the growth using C3 wheat and C4 guinea grass under various levels of nitrate-N supply. While eCO2 significantly increased relative growth rate (RGR) in wheat but not in guinea grass, each species increased RGR with higher N supply and then reached a maximum as no longer N was limited. To achieve the maximum RGR, wheat required a 1.3-fold N supply at eCO2 than aCO2 with 2.2-fold biomass. However, the N requirement by guinea grass was less affected by the eCO2 treatment. The results reveal that accelerated RGR by eCO2 could create a demand for more N, especially in the leaf sheath rather than the leaf blade in wheat, causing N-limitation unless the additional N was supplied. We concluded that eCO2 amplifies N-limitation due to accelerated growth rate rather than inhibited nitrate assimilation or acquisition. Our results suggest that plant growth under higher CO2 will become more dependent on N but less dependent on water to acquire both CO2 and N.

Retraction: Jiang et al. Formation of Proto-Kranz in C3 Rice Induced by Spike-Stalk Injection Method. Int. J. Mol. Sci. 2021, 22, 4305.

The journal and the authors retract the article "Formation of Proto-Kranz in C3 Rice Induced by Spike-Stalk Injection Method" [...].

Formation of Proto-Kranz in C3 Rice Induced by Spike-Stalk Injection Method.

Introduction of C4 photosynthetic traits into C3 crops is an important strategy for improving photosynthetic capacity and productivity. Here, we report the research results of a variant line of sorghum–rice (SR) plant with big panicle and high spikelet density by introducing sorghum genome DNA into rice by spike-stalk injection. The whole-genome resequencing showed that a few sorghum genes could be integrated into the rice genome. Gene expression was confirmed for two C4 photosynthetic enzymes containing pyruvate, orthophosphate dikinase and phosphoenolpyruvate carboxykinase. Exogenous sorghum DNA integration induced a series of key traits associated with the C4 pathway called “proto-Kranz” anatomy, including leaf thickness, bundle sheath number and size, and chloroplast size in bundle sheath cells. Significantly, transgenic plants exhibited enhanced photosynthetic capacity resulting from both photosynthetic CO2-concentrating effect and improved energy balance, which led to an increase in carbohydrate levels and productivity. Furthermore, such rice plant exhibited delayed leaf senescence. In summary, this study provides a proof for the feasibility of inducing the transition from C3 leaf anatomy to proto-Kranz by spike-stalk injection to achieve efficient photosynthesis and increase productivity.

Dietary shifts and diversities of individual life histories reveal cultural dynamics and interplay of millets and rice in the Chengdu Plain, China during the Late Neolithic (2500–2000 cal. BC)

We undertook a more comprehensive analyses than our previous study (Yi et al., International Journal of Osteoarchaeology, 2018, 28, 636-644) at the Gaoshan site (~2500-2000 cal. BC) to expand our understanding of the different roles of rice and millets to human subsistence strategies, diachronic shift of human diets, diversity of human life histories and cultural influence (dental ablation) to human population. Carbon and nitrogen isotopic analyses of human (ribs and long bones, n = 68) and animal (n = 15) bones as well as carbonized seeds from rice and millets (n = 8) were undertaken. Human bones were directly AMS-14 C dated. In addition, sequential sampling of 16 individuals with varied age and sex and cultural contexts for isotopic analysis was also conducted. The calibrated dating results (n = 27) indicate that there existed two periods of occupation at the site, ~2500-2200 BC and ~2200-1900 BC. Moderate high δ15 N values were found in the crops. Isotopic data from bone collagen suggest that the humans mainly relied on C3 -based animal protein and were supplemented by C4 -based foods. This isotopic pattern is also seen in pigs. Significant difference of human δ13 C values (Mann-Whitney U test, p < 0.05) between the two periods was observed. Compared to the bones, the isotopic data of human dentine serial sections show a much wider variability and higher δ13 C values. The crops (millets and rice) were probably manured. Human individuals subsisted on rice/millet agriculture and pigs in general. In combination with the radiocarbon dates, the increase of C3 (rice) consumption by the humans was found from the early (2500-2200 cal. BC) to late (2200-2000 cal. BC) periods, indicating the intensification of rice agriculture through time. However, four categories of human life histories during childhood are identified given different isotopic profiles of dentine sections, demonstrating that C3 (rice) and C4 (millets)-based foods played different roles in human lives. Even though, there were similar weaning practices among the human populations. It is surprising that human individuals with dental ablation, cultural characteristic in the Middle and Lower Yangtze River Valley, consumed more millets during childhood in the early period than those without dental ablation in the late period. Our study here provides novel insights into cultural dynamics and the interplay between rice and millets in rice-millet agricultural system during the Late Neolithic in Southwest China.

A Partial C4 Photosynthetic Biochemical Pathway in Rice

Introduction of a C4 photosynthetic pathway into C3 rice (Oryza sativa) requires installation of a biochemical pump that concentrates CO2 at the site of carboxylation in modified bundle sheath cells. To investigate the feasibility of this, we generated a quadruple line that simultaneously accumulates four of the core C4 photosynthetic enzymes from the NADP-malic enzyme subtype, phosphoenolpyruvate carboxylase (ZmPEPC), NADP-malate dehydrogenase (ZmNADP-MDH), NADP-malic enzyme (ZmNADP-ME), and pyruvate phosphate dikinase (ZmPPDK). This led to enhanced enzyme activity and mild phenotypic perturbations but was largely neutral in its effects on photosynthetic rate. Measurements of the flux of 13CO2 through photosynthetic metabolism revealed a significant increase in the incorporation of 13C into malate, consistent with increased fixation of 13CO2 via PEP carboxylase in lines expressing the maize PEPC enzyme. However, there was no significant differences in labeling of 3-phosphoglycerate (3PGA) indicating that there was no carbon flux through NADP-ME into the Calvin-Benson cycle. There was also no significant difference in labeling of phosphoenolpyruvate (PEP) indicating that there was no carbon flux through PPDK. Crossing the quadruple line with a line with reduced glycine decarboxylase H-protein (OsGDCH) abundance led to a photosynthetic phenotype characteristic of the reduced OsGDCH line and higher labeling of malate, aspartate and citrate than in the quintuple line. There was evidence of 13C labeling of aspartate indicating 13CO2 fixation into oxaloacetate by PEPC and conversion to aspartate by the endogenous aspartate aminotransferase activity. While Kranz anatomy or other anatomical modifications have not yet been installed in these plants to enable a fully functional C4 cycle, these results demonstrate for the first-time a partial flux through the carboxylation phase of NADP-ME C4 metabolism in transgenic rice containing two of the key metabolic steps in the C4 pathway.

Food Matrix Reference Materials for Hydrogen, Carbon, Nitrogen, Oxygen, and Sulfur Stable Isotope-Ratio Measurements: Collagens, Flours, Honeys, and Vegetable Oils.

An international project developed, quality-tested, and measured isotope-delta values of 10 new food matrix reference materials (RMs) for hydrogen, carbon, nitrogen, oxygen, and sulfur stable isotope-ratio measurements to support food authenticity testing and food provenance verification. These new RMs, USGS82 to USGS91, will enable users to normalize measurements of samples to isotope-delta scales. The RMs include (i) two honeys from Canada and tropical Vietnam, (ii) two flours from C3 (rice) and C4 (millet) plants, (iii) four vegetable oils from C3 (olive, peanut) and C4 (corn) plants, and (iv) two collagen powders from marine fish and terrestrial mammal origins. An errors-in-variables regression model included the uncertainty associated with the measured and assigned values of the RMs, and it was applied centrally to normalize results and obtain consensus values and measurement uncertainties. Utilization of these new RMs should facilitate mutual compatibility of stable isotope data if accepted normalization procedures are applied and documented.

A leaf-level biochemical model simulating the introduction of C2 and C4 photosynthesis in C3 rice: gains, losses and metabolite fluxes.

This work aims at developing an adequate theoretical basis for comparing assimilation of the ancestral C3 pathway with CO2 concentrating mechanisms (CCM) that have evolved to reduce photorespiratory yield losses. We present a novel model for C3 , C2 , C2 +C4 and C4 photosynthesis simulating assimilatory metabolism, energetics and metabolite traffic at the leaf level. It integrates a mechanistic description of light reactions to simulate ATP and NADPH production, and a variable engagement of cyclic electron flow. The analytical solutions are compact and thus suitable for larger scale simulations. Inputs were derived with a comprehensive gas-exchange experiment. We show trade-offs in the operation of C4 that are in line with ecophysiological data. C4 has the potential to increase assimilation over C3 at high temperatures and light intensities, but this benefit is reversed under low temperatures and light. We apply the model to simulate the introduction of progressively complex levels of CCM into C3 rice, which feeds >3.5 billion people. Increasing assimilation will require considerable modifications such as expressing the NAD(P)H Dehydrogenase-like complex and upregulating cyclic electron flow, enlarging the bundle sheath, and expressing suitable transporters to allow adequate metabolite traffic. The simpler C2 rice may be a desirable alternative.

Carbon and nitrogen isotopic composition of commercial dog food in Brazil.

BackgroundBrazil is a low- to medium-income country and has the second largest pet food market in the world with 8% of world pet food consumption. The lowest-income social class spends around 17% of their domestic budget on pet food and other items related to pets. Consumers are frequently misled by advertising as there is no precise information about the main sources of protein, carbohydrates and fat in the labels, and the Brazilian pet food industry can legally claim that their products contain certain items like salmon or beef even if they use just a flavoring compound.MethodsThe stable isotope methodology compares the stable isotope ratios of carbon (13C/12C) and nitrogen (15N/14N) between source and product. The 13C/12C ratio of a specific product (e.g., dog food) reveals the proportions of C4 (maize) and C3 (soybean, rice and wheat) plants in that product and the 15N/14N ratio reveals the proportion of the compounds derived from animals. With this isotopic data, we used MixSIAR, a Bayesian stable isotope-mixing model, to estimate the proportion of maize, grains, poultry and beef in dog food.ResultsThe δ13C values of dry dog food ranged from −24.2‰ to −12.8‰, with an average (± standard-deviation) of −17.1‰ ± 2.8‰. The δ13C values of wet pet food ranged from −25.4‰ to −16.9‰, with an average (± standard-deviation) of −21.2‰ ± 2.4‰, which was significantly lower (p < 0.01). The δ15N values of the dry and wet food ranged from 1.7‰ to 4.2‰, and from 0.5‰ to 5.5‰, respectively. The average δ15N values of dry food (2.9‰ ± 0.5‰) was not higher than the wet food (2.6‰ ± 1.3‰) (p > 0.01). The output of the MixSIAR showed a low proportion of bovine products in dry dog food samples. On the other hand, poultry was obviously the dominant ingredient present in most of the samples. Maize was the second dominant ingredient. Wet and dry dog food showed similar isotopic analysis results. The only difference was a lower proportion of maize and higher proportion of grains in wet dog food.DiscussionThe main finding is that dog food in Brazil is mostly made of approximately 60% (ranging from 32% to 86%) animal-based and 40% (ranging from 14% to 67%) plant-based products. Poultry and maize are the main ingredients. Poultry is added as a by-product or meal, which avoids competition between dogs and humans for meat products, while they can compete for maize. On the other hand, a large proportion of plant-based products in dog food decreases the energy and environmental footprint, since plant-based food products tend to be less harmful compared to animal-based products. Labels can mislead consumers by showing pictures of items that are not necessarily part of the product composition and by not showing the detailed information on the proportion of each ingredient. This information would allow customers to make their own choices considering their pet’s nutrition, the competition between animals and humans for resources and environmental sustainability.

Estimating global agricultural effects of geoengineering using volcanic eruptions.

Solar radiation management is increasingly considered to be an option for managing global temperatures1,2, yet the economic effects of ameliorating climatic changes by scattering sunlight back to space remain largely unknown3. Although solar radiation management may increase crop yields by reducing heat stress4, the effects of concomitant changes in available sunlight have never been empirically estimated. Here we use the volcanic eruptions that inspired modern solar radiation management proposals as natural experiments to provide the first estimates, to our knowledge, of how the stratospheric sulfate aerosols created by the eruptions of El Chichón and Mount Pinatubo altered the quantity and quality of global sunlight, and how these changes in sunlight affected global crop yields. We find that the sunlight-mediated effect of stratospheric sulfate aerosols on yields is negative for both C4 (maize) and C3 (soy, rice and wheat) crops. Applying our yield model to a solar radiation management scenario based on stratospheric sulfate aerosols, we find that projected mid-twenty-first century damages due to scattering sunlight caused by solar radiation management are roughly equal in magnitude to benefits from cooling. This suggests that solar radiation management-if deployed using stratospheric sulfate aerosols similar to those emitted by the volcanic eruptions it seeks to mimic-would, on net, attenuate little of the global agricultural damage from climate change. Our approach could be extended to study the effects of solar radiation management on other global systems, such as human health or ecosystem function.

SiSTL2 Is Required for Cell Cycle, Leaf Organ Development, Chloroplast Biogenesis, and Has Effects on C4 Photosynthesis in Setaria italica (L.) P. Beauv.

Deoxycytidine monophosphate deaminase (DCD) is a key enzyme in the de novo dTTP biosynthesis pathway. Previous studies have indicated that DCD plays key roles in the maintenance of the balance of dNTP pools, cell cycle progression, and plant development. However, few studies have elucidated the functions of the DCD gene in Panicoideae plants. Setaria has been proposed as an ideal model of Panicoideae grasses, especially for C4 photosynthesis research. Here, a Setaria italica stripe leaf mutant (sistl2) was isolated from EMS-induced lines of “Yugu1,” the wild-type parent. The sistl2 mutant exhibited semi-dwarf, striped leaves, abnormal chloroplast ultrastructure, and delayed cell cycle progression compared with Yugu1. High-throughput sequencing and map-based cloning identified the causal gene SiSTL2, which encodes a DCD protein. The occurrence of a single-base G to A substitution in the fifth intron introduced alternative splicing, which led to the early termination of translation. Further physiological and transcriptomic investigation indicated that SiSTL2 plays an essential role in the regulation of chloroplast biogenesis, cell cycle, and DNA replication, which suggested that the gene has conserved functions in both foxtail millet and rice. Remarkably, in contrast to DCD mutants in C3 rice, sistl2 showed a significant reduction in leaf cell size and affected C4 photosynthetic capacity in foxtail millet. qPCR showed that SiSTL2 had a similar expression pattern to typical C4 genes in response to a low CO2 environment. Moreover, the loss of function of SiSTL2 resulted in a reduction of leaf 13C content and the enrichment of DEGs in photosynthetic carbon fixation. Our research provides in-depth knowledge of the role of DCD in the C4 photosynthesis model S. italica and proposed new directions for further study of the function of DCD.

Physiological analysis of C3 rice [Oryza sativa (L.)] and C4 millet [Setaria italica (L.)] to identify photosynthetically efficient plants

The study was undertaken with the aim of developing a reliable and rapid screening protocol to identify crop plants with higher photosynthetic efficiencies. The vascular architecture and physiological functions of the leaves of C3 and C4 photosynthetic genetic model systems were compared. Vein densities, leaf chlorophyll index, Chlorophyll a fluorescence and gas exchange parameters were correlated with photosynthetic efficiency to develop an efficient protocol. The photosynthetic rates of Setaria italica were almost double when compared with the C3 plants. The higher photosynthetic efficiency of Setaria italica was supported by high photosynthetic rates coupled with moderate to high stomatal conductance and low transpiration rates. Setaria italica showed a significant increase in PSII efficiency as the growth progressed, while no significant changes in Fv/Fm values were observed in Oryza sativa genotypes. The distribution of SPAD index values among the rice genotypes during the entire growth period did not exceed 34.35. Setaria italica genotypes exhibited a denser arrangement of SLV’s, almost three times higher than Oryza sativa, with an interveinal distance of 0.317–0.329 mm. Denser networks of small longitudinal veins, higher SPAD indices and PSII efficiency were key traits to look for photosynthetic efficiency in crop plants.

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.

Breastfeeding, weaning, and dietary practices during the Western Zhou Dynasty (1122-771 BC) at Boyangcheng, Anhui Province, China.

Here we investigate breastfeeding and weaning practices and adult dietary habits at the Western Zhou Dynasty (1122-771 BC) site of Boyangcheng () located in Anhui Province, China. In addition, we utilize the differences in bone collagen turnover rates between rib and long bones from the same individual to examine past life histories, such as changes in diet or residence. Bone collagen from both the rib and long bones (either femora or humeri) of 42 individuals was measured for stable isotope ratios of carbon (δ13 C) and nitrogen (δ15 N). In addition, δ13 C and δ15 N values are reported for 35 animals (dogs, cows, horses, pigs, and deer). The human δ13 C values range from -20.7‰ to -12.0‰ with a mean value of -18.8 ± 1.6‰. The human δ15 N values range from 9.1‰ to 13.4‰ with a mean value of 10.9 ± 1.0‰. The animals display a wide range of δ13 C (-21.5‰ to -8.2‰; -15.8 ± 4.5‰) and δ15 N values (4.0‰ to 9.5‰; 6.5 ± 1.8‰). The adult δ13 C and δ15 N results indicate that mixed C3 (rice) and C4 (millet) terrestrial diets with varying levels of animal protein (mostly pigs and deer) were consumed. The elevated subadult δ15 N results return to adult levels by approximately 3-4 years of age, indicating that the weaning process was completed during this period. Individuals between 2 and 10 years old, with lower δ13 C and δ15 N results than the adult mean, possibly consumed more plant-based diets, and this is consistent with Chinese medical teachings ∼1500 years later during the Tang Dynasty (AD 618-907). The isotopic offsets between the ribs and long bones revealed that five adults experienced dramatic dietary shifts in their later lives, switching from predominately C3 /C4 to C3 diets. This research provides the first isotopic information about ancient Chinese breastfeeding and weaning practices and establishes a foundation for future studies to examine diachronic trends.