C4水稻,我们的新挑战

自从上个世纪60年代末C4光合途径发现以来,人们对工程改造现有C3粮食作物使之具有C4光合能力进行了大量努力。目前,大量分子、生理和基因组水平研究的进展和证据表明,该目标将可能在10～15年之内实现。本综述结合目前国际C4研究的现状,详述了该领域目前所涉各项研究内容的理论依据。我们首先总结过去的经典杂交实验,然后论证新一代测序技术与C4光合研究模式系统狐尾草（Setaria viridis）的发展极大的促进了我们对C4光合特征遗传发育相关基因的发现与鉴定。最后,我们强调虽然C4光合工程改造的研究目前已在世界各国大规模展开,但其最终成功仍有赖于不同国家研究基金及私立慈善基金的大力和长期共同资助。     

The role of proteins in C(3) plants prior to their recruitment into the C(4) pathway

Our most productive crops and native vegetation use a modified version of photosynthesis known as the C(4) pathway. Leaves of C(4) crops have increased nitrogen and water use efficiencies compared with C(3) species. Although the modifications to leaves of C(4) plants are complex, their faster growth led to the proposal that C(4) photosynthesis should be installed in C(3) crops in order to increase yield potential. Typically, a limited set of proteins become restricted to mesophyll or bundle sheath cells, and this allows CO(2) to be concentrated around the primary carboxylase RuBisCO. The role that these proteins play in C(3) species prior to their recruitment into the C(4) pathway is addressed here. Understanding the role of these proteins in C(3) plants is likely to be of use in predicting how the metabolism of a C(3) leaf will alter as components of the C(4) pathway are introduced as part of efforts to install characteristics of C(4) photosynthesis in leaves of C(3) crops.     

植物高光效基因工程育种

C₄植物所具有的C₄光合途径赋予其较高的光合作用效率,而一些主要的农作物如水稻、小麦、大豆等均为C₃作物,光合效率低下。随着生物技术的发展,通过基因工程手段利用C₄光合特性来改善C₃植物的光合效率进而提高其生物产量逐渐成为植物高光效育种的一个研究热点。综述了目前这一领域的研究进展及存在问题,预测了这一领域的发展前景。     

C4 Rice-an Ideal Arena for Systems Biology Research

Engineering the C4 photosynthetic pathway into C3 crops has the potential to dramatically increase the yields of major C3 crops.The genetic control of features involved in C4 photosynthesis are still far from being understood; which partially explains why we have gained little success in C4 engineering thus far.Next generation sequencing techniques and other high throughput technologies are offering an unprecedented opportunity to elucidate the developmental and evolutionary processes of C4 photosynthesis.Two contrasting hypotheses about the evolution of C4 photosynthesis exist,i.e.the master switch hypothesis and the incremental gain hypothesis.These two hypotheses demand two different research strategies to proceed in parallel to maximize the success of C4 engineering.In either case,systems biology research will play pivotal roles in identifying key regulatory elements controlling development of C4 features,identifying essential biochemical and anatomical features required to achieve high photosynthetic efficiency,elucidating genetic mechanisms underlining C4 differentiation and ultimately identifying viable routes to engineer C4 rice.As a highly interdisciplinary project,the C4 rice project will have far-reaching impacts on both basic and applied research related to agriculture in the 21st century.     

Engineering C4 photosynthetic regulatory networks

C4 photosynthesis is a complex metabolic pathway responsible for carbon fixation in major feed, food and bioenergy crops. Although many enzymes driving this pathway have been identified, regulatory mechanisms underlying this system remain elusive. C4 photosynthesis contributes to photosynthetic efficiency in major bioenergy crops such as sugarcane, Miscanthus, switchgrass, maize and sorghum, and international efforts are underway to engineer C4 photosynthesis into C3 crops. A fundamental understanding of the C4 network is thus needed. New experimental and informatics methods can facilitate the accumulation and analysis of high-throughput data to define components of the C4 system. The use of new model plants, closely related to C4 crops, will also contribute to our understanding of the mechanisms that regulate this complex and important pathway.     

C(4) photosynthesis in terrestrial plants does not require Kranz anatomy

C(4) photosynthesis in terrestrial plants was thought to require Kranz anatomy because the cell wall between mesophyll and bundle sheath cells restricts leakage of CO(2). Recent work with the central Asian chenopods Borszczowia aralocaspica and Bienertia cycloptera show that C(4) photosynthesis functions efficiently in individual cells containing both the C(4) and C(3) cycles. These discoveries provide new inspiration for efforts to convert C(3) crops into C(4) plants because the anatomical changes required for C(4) photosynthesis might be less stringent than previously thought.     

Ectopic expression of C 4 photosynthetic pathway genes improves carbon assimilation and alleviate stress tolerance for future climate change

Alteration in atmospheric carbon dioxide concentration and other environmental factors are the significant cues of global climate change. Environmental factors affect the most fundamental biological process including photosynthesis and different metabolic pathways. The feeding of the rapidly growing world population is another challenge which imposes pressure to improve productivity and quality of the existing crops. C4 plants are considered the most productive, containing lower photorespiration, and higher water-use & N-assimilation efficiencies, compared to C3 plants. Besides, the C4-photosynthetic genes not only play an important role in carbon assimilation but also modulate abiotic stresses. In this review, fundamental three metabolic processes (C4, C3, and CAM) of carbon dioxide assimilation, the evolution of C4-photosynthetic genes, effect of elevated CO2 on photosynthesis, and overexpression of C4-photosynthetic genes for higher photosynthesis were discussed. Kranz-anatomy is considered an essential prerequisite for the terrestrial C4 carbon assimilation, but single-celled C4 plant species changed this well-established paradigm. C4 plants are insensitive to an elevated CO2 stress condition but performed better under stress conditions. Overexpression of essential C4-photosynthetic genes such as PEPC, PPDK, and NADP-ME in C3 plants like Arabidopsis, tobacco, rice, wheat, and potato not only improved photosynthesis but also provided tolerance to various environmental stresses, especially drought. The review provides useful information for sustainable productivity and yield under elevated CO2 environment, which to be explored further for CO2 assimilation and also abiotic stress tolerance. Additionally, it provides a better understanding to explore C4-photosynthetic gene(s) to cope with global warming and prospective adverse climatic changes.     

The future of C4 research--maize, Flaveria or Cleome?

C4 photosynthesis has evolved multiple times among the angiosperms: the spatial rearrangement of the photosynthetic apparatus, combined with alterations to the leaf structure, allows CO2 to be concentrated around Rubisco. Higher CO2 concentrations at Rubisco decrease the rate of oxygenation and therefore reduce the amount of energy lost through photorespiration. C4 plants are particularly prevalent in tropical and subtropical regions because they can sustain higher rates of net photosynthesis; they also represent some of our most productive crops. To date, most progress in identifying genes crucial for C4 photosynthesis has been made using maize and Flaveria. We propose that Cleome, the most closely related genus containing C4 species to the C3 model Arabidopsis, be used together with Arabidopsis resources to accelerate our progress in elucidating the genetic basis of C4 photosynthesis.     

The myth of plant transformation

Technology development is innovative to many aspects of basic and applied plant transgenic science. Plant genetic engineering has opened new avenues to modify crops, and provided new solutions to solve specific needs. Development of procedures in cell biology to regenerate plants from single cells or organized tissue, and the discovery of novel techniques to transfer genes to plant cells provided the prerequisite for the practical use of genetic engineering in crop modification and improvement. Plant transformation technology has become an adaptable platform for cultivar improvement as well as for studying gene function in plants. This success represents the climax of years of efforts in tissue culture improvement, in transformation techniques and in genetic engineering. Plant transformation vectors and methodologies have been improved to increase the efficiency of transformation and to achieve stable expression of transgenes in plants. This review provides a comprehensive discussion of important issues related to plant transformation as well as advances made in transformation techniques during three decades.     

Evolution of the C(4) photosynthetic mechanism: are there really three C(4) acid decarboxylation types?

Some of the most productive plants on the planet use a variant of photosynthesis known as the C(4) pathway. This photosynthetic mechanism uses a biochemical pump to concentrate CO(2) to levels up to 10-fold atmospheric in specialized cells of the leaf where Rubisco, the primary enzyme of C(3) photosynthesis, is located. The basic biochemical pathways underlying this process, discovered more than 40 years ago, have been extensively studied and, based on these pathways, C(4) plants have been subdivided into two broad groups according to the species of C(4) acid produced in the mesophyll cells and into three groups according to the enzyme used to decarboxylate C(4) acids in the bundle sheath to release CO(2). Recent molecular, biochemical, and physiological data indicate that these three decarboxylation types may not be rigidly genetically determined, that the possibility of flexibility between the pathways exists and that this may potentially be both developmentally and environmentally controlled. This evidence is synthesized here and the implications for C(4) engineering discussed.     

Towards a dynamic photosynthesis model to guide yield improvement in C4 crops

The most productive C4 food and biofuel crops, such as Saccharum officinarum (sugarcane), Sorghum bicolor (sorghum) and Zea mays (maize), all use NADP-ME-type C4 photosynthesis. Despite high productivities, these crops fall well short of the theoretical maximum solar conversion efficiency of 6%. Understanding the basis of these inefficiencies is key for bioengineering and breeding strategies to increase the sustainable productivity of these major C4 crops. Photosynthesis is studied predominantly at steady state in saturating light. In field stands of these crops light is continually changing, and often with rapid fluctuations. Although light may change in a second, the adjustment of photosynthesis may take many minutes, leading to inefficiencies. We measured the rates of CO₂ uptake and stomatal conductance of maize, sorghum and sugarcane under fluctuating light regimes. The gas exchange results were combined with a new dynamic photosynthesis model to infer the limiting factors under non-steady-state conditions. The dynamic photosynthesis model was developed from an existing C4 metabolic model for maize and extended to include: (i) post-translational regulation of key photosynthetic enzymes and their temperature responses; (ii) dynamic stomatal conductance; and (iii) leaf energy balance. Testing the model outputs against measured rates of leaf CO₂ uptake and stomatal conductance in the three C4 crops indicated that Rubisco activase, the pyruvate phosphate dikinase regulatory protein and stomatal conductance are the major limitations to the efficiency of NADP-ME-type C4 photosynthesis during dark-to-high light transitions. We propose that the level of influence of these limiting factors make them targets for bioengineering the improved photosynthetic efficiency of these key crops.     

Evolution of C4 photosynthetic genes and overexpression of maize C4 genes in rice

C3 plants including many agronomically important crops exhibit a lower photosynthetic efficiency due to inhibition of photosynthesis by O₂ and the associated photorespiration. C4 plants had evolved the C4 pathway to overcome low CO₂ and photorespiration. This review first focuses on the generation of a system for high level expression of the C4-specific gene for pyruvate, orthophosphate dikinase (Pdk), one of the key enzyme in C4 photosynthesis. Based on the results with transgenic rice plants, we have demonstrated that the regulatory system controlling thePdk expression in maize is not unique to C4 plants but rice (C3 plant) posses a similar system. Second, we discussed the possibility of the high level expression of maize C4-specific genes in transgenic rice plants. Introduction of the maize intact phosphoenolpyruvate carboxylase gene (Ppc) caused 30–100 fold higher PEPC activities than non-transgenic rice. These results demonstrated that intact C4-type genes are available for high level expression of C4 enzymes in rice plants. The extended abstract of a paper presented at the 13th International Symposium in Conjugation with Award of the International Prize for Biology “Frontier of Plant Biology”     

Biotechnology and the improvement of oil crops – genes,dreams and realities

During the past decade, there have been many optimistic claims concerning the potential of novel oil-based products from genetically engineered crops, particularly for the manufacture of a new generation of renewable, carbon-neutral, industrial materials. Such claims have been underpinned by an impressive series of scientific advances that have resulted in the isolation of genes encoding most of the enzymes directly involved in oil biosynthesis. In some cases, these enzymes have even been re-engineered by site-directed or random mutagenesis to allow production of new fatty acid profiles that are not present in any existing organism. This has opened up the prospect of engineering `designer oil crops' to produce novel fatty acids with chain lengths from C8 to C24 and with a wide range of industrially useful functionalities, including hydroxylation, epoxidation, and conjugated and non-conjugated double or triple bonds. However, there remain significant technical challenges before this promise of designer transgenic crops is likely to be translated into large-scale commercial reality. For example, it has proved surprisingly difficult to engineer high levels of novel fatty acids in genetically engineered transgenic plants, although many wild type seeds can readily accumulate 90–95% of a single fatty acid in their storage oil. Another complication is the recent discovery of multiple pathways of triacylglycerol biosynthesis and the difficulty in ensuring that novel fatty acids are only channelled towards storage triacylglycerols and not to membrane or signalling lipids in major target crops like rapeseed. New findings from our lab have suggested that there may also be problems with the tissue specificity of some of the `seed-specific' gene promoters that are commonly used in transgenic crops. There are also considerable and often underestimated challenges associated with the economics, management and public acceptability of all transgenic crops, even for non-food use. In most cases the projections of petroleum reserves over the next few decades make it unlikely that crop-derived commodity products that substitute for petroleum will be competitive. Also the scale of crop production required to generate millions of tonnes of commodity oils, e.g., for biodegradable plastics, is likely to seriously impinge on food production at a time of increasing global populations, and is therefore unlikely to be acceptable. An alternative strategy to transgenic oil crops is to use molecular breeding techniques in order to develop new crops that already synthesise high levels of novel fatty acids of interest. Finally, the most promising market sectors and product ranges for the future development of oil crop biotechnology will be discussed.     

Dissecting metabolic flux in C<Subscript>4</Subscript> plants: experimental and theoretical approaches

C₄ photosynthesis is the carbon fixation pathway in specific plant species, so called C₄ plants including maize, sorghum and sugarcane. It is characterized by the carboxylation reaction that forms four-carbon (C₄) molecules, which are then used to transport CO₂ to the proximity of RubisCO in the bundle sheath cells. Since C₄ photosynthesis confers high photosynthetic as well as water and nitrogen use efficiency on plants, worldwide efforts have been made to understand the mechanisms of C₄ photosynthesis and to properly introduce the pathway into C₃ crops. Metabolic flux analysis (MFA) is a research field trying to analyze the metabolic pathway structure and activity (i.e., flux) in vivo. Constraint-based reconstruction and analysis tools theoretically study the distribution of metabolic flux in genome-scale network-based models. Different types of MFA and model-based analyses have been contributing to the discovery of C₄ photosynthetic pathways and to analyze its operation in C₄ plant species. This article reviews the studies to dissect the operation of C₄ photosynthesis and adjacent pathways, from the pioneer studies using radioisotope-based MFA to the recent stable isotope-based MFA and the model-based approaches. These studies indicate complex interconnections among metabolic pathways and the importance of the integration of experimental and theoretical approaches. Perspectives on the integrative approach and major obstacles are also discussed.     

植物抗线虫基因的分子定位与克隆

植物线虫严重危害农业生产并造成产量损失。抗线虫基因的分子标记定位与克隆是抗线虫基因工程的基础。现就几种植物中的抗线虫基因的分子定位及克隆进行了综述 ,同时针对已克隆的抗线虫基因所编码的蛋白质的结构特征和克隆新方法进行评述 ,并对通过遗传途径发展抗线虫品种进行了展望     

Converging phenomics and genomics to study natural variation in plant photosynthetic efficiency

In recent years developments in plant phenomic approaches and facilities have gradually caught up with genomic approaches. An opportunity lies ahead to dissect complex, quantitative traits when both genotype and phenotype can be assessed at a high level of detail. This is especially true for the study of natural variation in photosynthetic efficiency, for which forward genetics studies have yielded only a little progress in our understanding of the genetic layout of the trait. High‐throughput phenotyping, primarily from chlorophyll fluorescence imaging, should help to dissect the genetics of photosynthesis at the different levels of both plant physiology and development. Specific emphasis should be directed towards understanding the acclimation of the photosynthetic machinery in fluctuating environments, which may be crucial for the identification of genetic variation for relevant traits in food crops. Facilities should preferably be designed to accommodate phenotyping of photosynthesis‐related traits in such environments. The use of forward genetics to study the genetic architecture of photosynthesis is likely to lead to the discovery of novel traits and/or genes that may be targeted in breeding or bio‐engineering approaches to improve crop photosynthetic efficiency. In the near future, big data approaches will play a pivotal role in data processing and streamlining the phenotype‐to‐gene identification pipeline.