Recent progress in the single-cell C4 photosynthesis in terrestrial plants

Currently, single-cell C4 photosynthesis has been reported in four terrestrial plant species, Bienertia cycloptera, B. sinuspersici, B. kavirense and Suaeda aralocaspica, of family Chenopodiaceae. These species possess novel mechanisms of C4 photosynthesis through spatial partitioning of organelles and key enzymes in distinct cytoplasmic domains within single chlorenchyma cells. Anatomical and biochemical studies have shown that the three Bienertia species and S. aralocaspica utilize biochemical and organellar compartmentation to achieve the equivalent spatial separation of Kranz anatomy but within a single photosynthetic cell. These discoveries have challenged the paradigm for C4 photosynthesis in terrestrial plants which had suggested for more than 40 years that the Kranz feature was indispensably required for its C4 function. In this review, we focus on the recent progress in understanding the cellular and molecular mechanisms that control the spatial relationship of organelles in these unique single-cell C4 systems. The demonstrated interaction of dimorphic chloroplasts with microtubules and actin filaments has shed light on the importance of these cytoskeleton components in the intracellular partitioning of organelles. Future perspectives on the potential function of the cytoskeleton in targeting gene products to specific subcellular compartments are discussed.

Enhanced tolerance to drought in transgenic rice plants overexpressing C_4 photosynthesis enzymes

Maize-specific pyruvate orthophosphate dikinase(PPDK) was overexpressed in rice independently or in combination with the maize C4-specific phosphoenolpyruvate carboxylase(PCK). The wild-type(WT) cultivar Kitaake and transgenic plants were evaluated in independent field and tank experiments. Three soil moisture treatments,well-watered(WW), moderate drought(MD) and severe drought(SD), were imposed from 9d post-anthesis till maturity. Leaf physiological and biochemical traits, root activities,biomass, grain yield, and yield components in the untransformed WT and two transgenic rice lines(PPDK and PCK) were systematically studied. Compared with the WT, both transgenic rice lines showed increased leaf photosynthetic rate: by 20%–40% under WW, by45%–60% under MD, and by 80%–120% under SD. The transgenic plants produced 16.1%,20.2% and 20.0% higher grain yields than WT under the WW, MD and SD treatments,respectively. Under the same soil moisture treatments, activities of phosphoenolpyruvate carboxylase(PEPC) and carbonic anhydrase(CA) in transgenic plants were 3–5-fold higher than those in WT plants. Compared with ribulose-1,5-bisphosphate carboxylase, activities of PEPC and CA were less reduced under both MD and SD treatments. The transgenic plants also showed higher leaf water content, stomatal conductance, transpiration efficiency, and root oxidation activity and a stronger active oxygen scavenging system than the WT under all soil moisture treatments, especially MD and SD. The results suggest that drought tolerance is greatly enhanced in transgenic rice plants overexpressing C4photosynthesis enzymes. This study was performed under natural conditions and normal planting density to evaluate yield advantages on a field basis. It may open a new avenue to droughttolerance breeding via overexpression of C4enzymes in rice.

Species- and Habitat-variability of Photosynthesis, Transpiration and Water Use Efficiency of Different Plant Species in Maowusu Sand Area

Photosynthesis ( P n ), transpiration ( E ) and water use efficiency ( WUE ) of more than 66 arid sand species from different environmental habitats, shifting sand dune, fixed sand dune, lowland and wetland in the Maowusu Sand Area were analyzed and the relation among these characteristics and the resource utilization efficiency, taxonomic categories and growth forms of the species were assessed. The results showed that species from Chenopodiaceae, Gramineae, Leguminosae which possessed the C 4 photosynthesis pathway, or C 3 pathway and also with nitrogen_fixation capacities had higher or the highest P n values, i.e., 20～30 μmol CO 2·m -2 ·s -1 , while that of evergreen shrub of Pinaceae had the lowest P n values, i.e., 0～5 μmol CO 2·m -2 ·s -1 . Those species from Compositae, Scrophulariaceae, and Gramineae with C 3 pathway but no N_fixation capacity had the highest E rates, i.e., 20～30 mmol H 2O·m -2 ·s -1 and again the evergreen shrub together with some species from Salicaceae and Compositae had the lowest E rates, i.e., 0～5 mmol H 2O·m -2 ·s -1 . Species from Leguminosae, Gramineae and Chenopodiaceae with C 4 pathway or C 3 pathway with N_fixation capacity, both shrubs and grasses, generally had higher WUE . However, even the physiological traits of the same species were habitat_ and season_specific. The values of both P n and E in late summer were much higher than those in early summer, with average increases of 26%, 40% respectively in the four habitats. WUE in late summer was, however, 12% lower. Generally, when the environments became drier as a result of habitats changed, i.e., in the order of wetland, lowland, fixed sand dune and shifting sand dune, P n and E decreased but WUE increased.

转C_4光合基因水稻特征特性及其在两系杂交稻育种中的应用

对pepc、ppdk和pepc +ppdk三种转基因水稻农艺性状观察表明 ,与原种Kitaake相比单株有效穗有不同程度的增多 ,单株产量相应提高 ,特别是pepc和ppdk基因聚合后 ,单株有效穗和单株产量分别比受体亲本Kitaake提高 2 9.1%和2 7.0 %。三种转基因材料作基因供体分别与受体光敏核不育系培矮 6 4S、2 30 4S和 2 30 6S杂交后 ,这些基因在新的遗传背景下不仅稳定遗传和高水平表达 ,而且表现增穗增产 ,特别当pepc和ppdk基因聚合时 ,与受体相比 ,F1的PEPC活性提高 5 .8～ 18.6倍 ,PPDK活性提高 0 .5～ 1.3倍 ,植株饱和光合速率提高 5 0 %左右。转育的转基因材料结实率有所降低 ,是值得进一步研究的问题。

中国C_4植物的地理分布与生态学研究 Ⅰ.中国C_4植物及其与气候环境的关系

通过调查记录了中国５３３种４０变种和３亚种具有Ｃ４光合作用的植物和８种Ｃ３～Ｃ４中间植物。它们隶属于１６０属２４科，其中４６属９７种、８变种和１亚种隶属于双子叶植物，１１４属４３６种、３２变种和２亚种隶属于单子叶植物。Ｃ４植物主要属于禾本科（９６属３２４种、３２变种和２亚种），莎草科（１４属１０８种），藜科（１３属３７种、７变种和１亚种）和苋科（３属１６种１亚种）。根据中国的温度气候（寒温带，冷温带，中温带，暖温带，亚热带和边缘热带）和大气水分状况（极干旱，干旱，半干旱，半湿润，季节性干旱和湿润）将中国除了南海诸岛以外的地理区域划为７个区和１２个亚区，并总结出中国Ｃ４种的地理与气候分布区。Ｃ４植物种数（尤其是禾本科和莎草科）随着大气温度与水分增高而增加。然而，藜科的Ｃ４植物种数随着大气温度与水分增高而减少。调查结果表明中国Ｃ４植物具有广阔的地理分布特点，在寒温带也有一定数量的分布。

Simulating leaf net CO_2 assimilation rate of C_3 & C_4 plants and its response to environmental factors

Basic structure and algorithm of leaf mechanism photosynthesis model were described in first part of this study based on former researcher results. Then, considering some environmental factors influencing on leaf photosynthesis, three numerical sensitivity experiments were carried out. We simulated the sing le leaf net CO2 assimilation, which acts as a function of different light, carbo n dioxide and temperature conditions. The relationships between leaf net photosy nthetic rate of C3 and C4 plant with CO2 concentration intercellular, leaf tempe rature, and photosynthetic active radiation (PAR) were presented, respectively. The results show the numerical experiment may indicate the main characteristic o f plant photosynthesis in C3 and C4 plant, and further can be used to integrate with the regional climate model and act as land surface process scheme, and bett er understand the interaction between vegetation and atmosphere.

Simulating leaf net CO2 assimilation rate of C3 & C4 plants and its response to environmental factors

Basic structure and algorithm of leaf mechanism photosynthesis model were described in first part of this study based on former researcher results. Then, considering some environmental factors influencing on leaf photosynthesis, three numerical sensitivity experiments were carried out. We simulated the sing le leaf net CO2 assimilation, which acts as a function of different light, carbo n dioxide and temperature conditions. The relationships between leaf net photosy nthetic rate of C3 and C4 plant with CO2 concentration intercellular, leaf tempe rature, and photosynthetic active radiation (PAR) were presented, respectively. The results show the numerical experiment may indicate the main characteristic o f plant photosynthesis in C3 and C4 plant, and further can be used to integrate with the regional climate model and act as land surface process scheme, and bett er understand the interaction between vegetation and atmosphere.

水稻田稗草叶片光合作用对开放式空气CO_2浓度增高(FACE)的适应

于分蘖、拔节和抽穗 3个时期在空气CO2 浓度 (380 μmol·mol-1)下测定稻田中稗草叶片的净光合速率 (Pn) ,发现在开放式CO2 浓度增高 (FACE)条件下生长的稗草叶片后 2个时期的Pn显著低于普通空气中生长的对照 ,比对照下降约 2 0 % ,说明FACE条件下稗草叶片光合作用对高CO2 浓度发生了明显的适应 .同时 ,叶片的气孔导度 (Gs)和胞间CO2 浓度 (Ci)的下降更为明显 .与对照相比 ,叶片可溶性蛋白含量明显降低 ,拔节期只有对照的 6 2 .4 % ;高CO2 浓度下生长的稗草叶片Rubisco含量也降低 ,分蘖期和拔节期分别为对照的 87%和 84 % ,但其差异未达到显著水平 .可以认为 ,长期生长在高CO2 浓度下的C4植物稗草叶片光合作用的适应是叶片气孔部分关闭和可溶性蛋白含量下降的结果 .

Characterization of photosynthesis,photoinhibition and the activities of C_4 pathway enzymes in a superhigh-yield rice,Liangyoupeijiu

Characteristics of photosynthetic gas exchange, photoinhibition and C4 pathway enzyme activities in both flag leaves and lemma were compared between a superhigh-yield rice (Oryza sativa L.) hybrid, Liangyoupeijiu and a traditional rice hybrid, Shanyou63. Liangyoupeijiu had a similar light saturated assimilation rate (Asat) to Shanyou63, but a much higher apparent quantum yield (AQY), carboxylation efficiency (CE) and quantum yield of CO2 fixation (φCO2). Liangyoupeijiu also showed a higher resistance to photoinhibition and higher non-radiative energy dissipation associated with the xanthophyll cycle than Shanyou63 when subjected to strong light. In addition, Liangyoupeijiu had higher activities of the C4 pathway enzymes in both flag leaves and lemmas than Shanyou63. These results indicate that higher light and CO2 use efficiency, higher resistance to photoinhibition and C4 pathway in both flag leaf and lemma may contribute to the higher yield of the superhigh-yield rice hybrid, Liangyoupeijiu.

A well-supported nuclear phylogeny of Poaceae and implications for the evolution of C4 photosynthesis

Poaceae(the grasses)includes rice,maize,wheat,and other crops,and is the most economically important angiosperm family.Poaceae is also one of the largest plant families,consisting of over 11000 species with a global distribution that contributes to diverse ecosystems.Poaceae species are classified into 12 subfamilies,with generally strong phylogenetic support for their monophyly.However,many relationships within subfamilies,among tribes and/or subtribes,remain uncertain.To better resolve the Poaceae phylogeny,we generated 342 transcriptomic and seven genomic datasets;these were combined with other genomic and transcriptomic datasets to provide sequences for 357 Poaceae species in 231 genera,representing 45 tribes and all 12 subfamilies.Over 1200 low-copy nuclear genes were retrieved from these datasets,with several subsets obtained using additional criteria,and used for coalescent analyses to reconstruct a Poaceae phylogeny.Our results strongly support the monophyly of 11 subfamilies;however,the subfamily Puelioideae was separated into two non-sister clades,one for each of the two previously defined tribes,supporting a hypothesis that places each tribe in a separate subfamily.Molecular clock analyses estimated the crown age of Poaceae to be101 million years old.Ancestral character reconstruction of C3/C4 photosynthesis supports the hypothesis of multiple independent origins of C4 photosynthesis.These origins are further supported by phylogenetic analysis of the ppc gene family that encodes the phosphoenolpyruvate carboxylase,which suggests that members of three paralogous subclades(ppc-aL1a,ppc-aL1b,and ppcB2)were recruited as functional C4 ppc genes.This study provides valuable resources and a robust phylogenetic framework for evolutionary analyses of the grass family.

Dynamics of soil organic carbon following land-use change:insights from stable C-isotope analysis in black soil of Northeast China

Intensive soil tillage is a significant factor in soil organic matter decline in cultivated soils. Both cultivation abandonment and foregoing tillage have been encouraged in the past 30 years to reduce greenhouse gas emissions and soil erosion. However, the dynamic processes of soil organic carbon (SOC) in areas of either continuous cultivation or abandonment remain unclear and inconsistent.Our aims were to assess and model the dynamic processes of SOC under continuous tillage and after cultivation abandonment in the black soil of Northeast China. Soil profiles were collected of cultivated or abandoned land with cultivation history of 0–100 years. An isotope mass balance equation was used to calculate the proportion of SOC derived from corn debris (C_4) and from natural vegetation (C_3) to deduce the dynamic process. Approximately 40% of SOC in the natural surface soil (0–10 cm) was eroded in the first 5 years of cultivation, increasing to about 75% within 40 years, before a slow recovery. C_4 above 30 cm soil depth increased by 4.5%–5% or 0.11–0.12 g·kg^(-1) on average per year under continuous cultivation, while it decreased by approximately 0.34% annually in the surface soil after cultivation abandonment.The increase in the percentage of C_4 was fitted to a linear equation with given intercepts in the upper 30 cm of soil in cultivated land. A significant relationship between the change of C_4 and time was found only in the surface soil after abandonment of cultivation. These results demonstrate the loss and accumulation of corn-derived SOC in surface black soil of Northeast China under continuous tillage or cultivation abandonment.

Expression Profiles of <i>psbA, ALS, EPSPS</i>, and Other Chloroplastic Genes in Response to PSII-, ALS-, and EPSPS-Inhibitor Treatments in <i>Kochia scoparia</i>

Kochia (Kochia scoparia L. Schrad.), also known as tumbleweed, is an economically important annual C4 broadleaf weed found throughout the US Great Plains. Several herbicides with different modes of action are used in the management of kochia. The effect of commonly used herbicides on the expression of their target site(s) and photosynthetic/chloroplastic genes is poorly understood in weed species, including kochia. The objective of this research was to characterize the expression profiles of herbicide target-site genes, KspsbA, KsALS, and KsEPSPS upon treatment with PSII- (e.g. atrazine), ALS- (e.g. chlorsulfuron), and EPSPS- (e.g. glyphosate)-inhibitors, respectively, in kochia. Furthermore, the expression of genes involved in photosynthesis (e.g. KsRubisco, KsCAB, and KsPPDK) was also determined in response to these herbicide treatments. KspsbA was strongly upregulated (>200-fold) 24 h after atrazine treatment. Transcript levels of the KsALS or KsEPSPS genes were 7 and 3-fold higher 24 h after chlorsulfuron or glyphosate treatment, respectively. KsRubisco, a Calvin cycle gene important for CO2 fixation, was upregulated 7 and 2.6-fold 8 and 24 h after glyphosate and chlorsulfuron treatments, whereas it downregulated 8 and 24 h after atrazine treatment. The transcript levels of KsPPDK remained unchanged after glyphosate treatment but increased 1.8-fold and decreased 2-fold at 24 h after chlorsulfuron and atrazine treatments, respectively. KsCAB remained unchanged after chlorsulfuron treatment, but was downregulated after glyphosate and atrazine treatments. The results show that herbicide treatments not only affect the respective target-site gene expression, but also influence the genes involved in the critical photosynthetic pathway.

Effects of Media Composition and Auxins on Adventitious Rooting of Bienertia sinuspersici Cuttings

An efficient in vitro method for rapid vegetative propagation of Bienertia sinuspersici, one of four terrestrial species of family Chenopodiaceae capable of performing C4 photosynthesis within a single cell, was developed. Cuttings of B. sinuspersici were used to examine the effects of Murashige and Skoog (MS) media strength and auxins on adventitious root formation. Half-strength MS medium was determined to be ideal for adventitious root formation in Bienertia cuttings. Although cuttings cultured in medium containing 5.0 mg/L α-naphthalene acetic acid (NAA) promoted the highest number of adventitious roots, cuttings cultured in medium supplemented with 1.0 mg/L indole-3-butyric acid (IBA) produced the longest adventitious roots and had the highest survival rate upon transplanting to soil. Histological analysis revealed variations in the root anatomy generated by the various auxins which may affect adventitious root formation and subsequent establishment of cuttings in soil. Overall, the established procedure provides a simple and cost-effective means for the rapid propagation of the single-cell C4 species B. sinuspersici.

Photoinhibition of Leaves with Different Photosynthetic Carbon Assimilation Characteristics in Maize (<i>Zea mays</i>)

Strong light decreases the rate of photosynthesis and assimilates production of crop plants. Plants with different carbon reduction cycles respond differently to strong light stress. However, variation in photoinhibition in leaves with different photosynthetic characteristics in maize is not clear. In this experiment, we used the first leaves (with an incomplete C4 cycle) and fifth leaves (with a complete C4 cycle) of maize plants as well as the fifth leaves (C3 cycle) of tobacco plants as a reference to measure the photosynthetic rate (PN) and chlorophyll a parameters under strong light stress. During treatment, PN, the maximal fluorescence (Fm), the maximal quantum yield of PSII photochemistry (Fv/Fm), and the number of active photosystem II (PSII) reaction centers per excited cross-section (RC/CSm) declined dramatically in all three types of leaves but to different degrees. PN, Fm, Fv/Fm, and RC/CSm were less inhibited by strong light in C4 leaves. The results showed that maize C4 leaves with higher rates of photosynthesis are more tolerant to strong light stress than incomplete C4 leaves, and the carbon reduction cycle is more important to photoprotection in C4 leaves, while state transition is critical in incomplete C4 leaves.

内蒙古浑善达克沙地97种植物的光合生理特征(英文)

报告了内蒙古浑善达克沙地不同生境下 97种不同科、属植物的光合速率、蒸腾速率和水分利用效率特征。结果表明 :猪毛菜 (Salsolacollina)、沙米 (Agriophyllumpungens)、黑沙蒿 (Artemisiaordosica)、狗尾草 (Setariaviridis)、柠条 (Caraganamicrophylla)等具有C4 光合碳同化途径或具固氮能力的植物种具有较高的光合能力 ,其净光合速率大于 30 μmolCO2 ·m- 2 ·s- 1 ,而大部分具C3途径和无固氮能力的植物种的净光合速率较低 ,为 1 .2 9～ 1 0 μmolCO2 ·m- 2 ·s- 1 ;71 %的植物种蒸腾速率集中在 2～ 1 0mmolH2 O·m- 2 ·s- 1 。所选出的高光效植物种在当地植被恢复与重建过程中应有很高利用价值。C4 植物种占所测植物种的 1 / 5左右 ,主要分布于固定沙丘上 ,且随着生境土壤湿度的增大其与C3植物种的光合速率差异逐渐缩小。在 3种生境条件下 ,灌木和草本植物的光合速率和蒸腾速率顺序为 :低湿地 >滩地 >固定沙丘 (p <0 .0 1 ) ,而乔木为固定沙丘 >滩地 (p <0 .0 1 )。不同功能型植物的气体交换特征随生境的不同而异 ,在固定沙丘上 ,草本的蒸腾速率最高 ,乔木的水分利用率最高 。

Hybrid Rubisco with Complete Replacement of Rice Rubisco Small Subunits by Sorghum Counterparts Confers C4 Plant-like High Catalytic Activity

Photosynthetic rate at the present atmospheric condition is limited by the CO2-fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco)because of its extremely low catalytic rate(kcat)and poor affinity for C02(Kc)and specificity for CO2(Sc/O).Rubisco in C4 plants generally shows higher k cat than that in C3 plants.Rubisco consists of eight large subunits and eight small subunits(RbcS).Previously,the chimeric incorporation of sorghum C4-type RbcS significantly increased the k cat of Rubisco in a C3 plant,rice.In this study,we knocked out rice RbcS multigene family using the CRISPR-Cas9 technology and completely replaced rice RbcS with sorghum RbcS in rice Rubisco.Obtained hybrid Rubisco showed almost C4 plant-like catalytic properties,i.e.,higher kcai,higher Kc,and lower Sc/O.Transgenic lines expressing the hybrid Rubisco accumulated reduced levels of Rubisco,whereas they showed slightly but significantly higher photosynthetic capacity and similar biomass production under high C 02 condition compared with wild-type rice.High-resolution crystal structural analysis of the wild-type Rubisco and hybrid Rubisco revealed the structural differences around the central pore of Rubisco and the pC-pD hairpin in RbcS.We propose that such differences,particularly in the pC-pD hairpin,may impact the flexibility of Rubisco catalytic site and change its catalytic properties.

Machine Learning Techniques for Predicting Crop Photosynthetic Capacity from Leaf Reflectance Spectra

Harnessing natural variation in photosynthetic capacity is a promising route toward yield increases, but physiological phenotyping is still too laborious for large-scale genetic screens. Here, we evaluate the potential of leaf reflectance spectroscopy to predict parameters of photosynthetic capacity in Brassica oleracea and Zea mays, a C3 and a C4 crop, respectively. To this end, we systematically evaluated properties of reflectance spectra and found that they are surprisingly similar over a wide range of species. We assessed the performance of a wide range of machine learning methods and selected recursive feature elimination on untransformed spectra followed by partial least squares regression as the preferred algorithm that yielded the highest predictive power. Learning curves of this algorithm suggest optimal species-specific sample sizes. Using the Brassica relative Moricandia, we evaluated the model transferability between spe- cies and found that cross-species performance cannot be predicted from phylogenetic proximity. The final intra-species models predict crop photosynthetic capacity with high accuracy. Based on the estimated model accuracy, we simulated the use of the models in selective breeding experiments, and showed that high-throughput photosynthetic phenotyping using our method has the potential to greatly improve breeding success. Our results indicate that leaf reflectance phenotyping is an efficient method for improving crop photosynthetic capacity.

Interactive response of photosynthetic characteristics in Haloxylon ammodendron and Hedysarum scoparium exposed to soil water and air vapor pressure deficits

C4 plants possess better drought tolerance than C3 plants. However, Hedysarum scoparium, a C3 species, is dominant and widely distributed in the desert areas of northwestern China due to its strong drought tolerance. This study compared it with Haloxylon ammodendron, a C4 species, regarding the interactive effects of drought stress and different leaf–air vapor pressure deficits. Variables of interest included gas exchange, the activity levels of key C4 photosynthetic enzymes, and cellular anatomy. In both species, gas exchange parameters were more sensitive to high vapor pressure deficit than to strong water stress, and the net CO2 assimilation rate（A n） was enhanced as vapor pressure deficits increased. A close relationship between A n and stomatal conductance（g s） suggested that the species shared a similar response mechanism. In H. ammodendron, the activity levels of key C4 enzymes were higher, including those of phosphoenolpyruvate carboxylase（PEPC） and nicotinamide adenine dinucleotide phosphate-malate enzyme（NADP-ME）, whereas in H. scoparium, the activity level of nicotinamide adenine dinucleotide-malate enzyme（NAD-ME） was higher.Meanwhile, H. scoparium utilized adaptive structural features, including a larger relative vessel area and a shorter distance from vein to stomata, which facilitated the movement of water. These findings implied that some C4 biochemical pathways were present in H. scoparium to respond to environmental challenges.

Initiation of Setaria as a model plant

Model organisms such as Arabidopsis(Arabidopsis thaliana)and rice(Oryza sativa)have proven essential for efficient scientific discovery and development of new methods.With the diversity of plant lineages,some important processes such as C4 photosynthesis are not found in either Arabidopsis or rice,so new model species are needed.Due to their small diploid genomes,short life cycles,self-pollination,small adult statures and prolific seed production,domesticated foxtail millet(Setaria italica)and its wild ancestor,green foxtail(S.viridis),have recently been proposed as novel model species for functional genomics of the Panicoideae,especially for study of C4 photosynthesis.This review outlines the development of these species as model organisms,and discusses current challenges and future potential of a Setaria model.