Molecular biology of C4 phosphoenolpyruvate carboxylase: Structure,regulation and genetic engineering

Three to four families of nuclear genes encode different isoforms of phosphoenolpyruvate (PEP) carboxylase (PEPC): C₄-specific, C₃ or etiolated, CAM and root forms. C₄ leaf PEPC is encoded by a single gene (ppc) in sorghum and maize, but multiple genes in the C₄-dicot Flaveria trinervia. Selective expression of ppc in only C₄-mesophyll cells is proposed to be due to nuclear factors, DNA methylation and a distinct gene promoter. Deduced amino acid sequences of C₄-PEPC pinpoint the phosphorylatable serine near the N-terminus, C₄-specific valine and serine residues near the C-terminus, conserved cysteine, lysine and histidine residues and PEP binding/catalytic sites. During the PEPC reaction, PEP and bicarbonate are first converted into carboxyphosphate and the enolate of pyruvate. Carboxyphosphate decomposes within the active site into Pi and CO₂, the latter combining with the enolate to form oxalacetate. Besides carboxylation, PEPC catalyzes a HCO₃ ^--dependent hydrolysis of PEP to yield pyruvate and Pi. Post-translational regulation of PEPC occurs by a phosphorylation/dephosphorylation cascade in vivo and by reversible enzyme oligomerization in vitro. The interrelation between phosphorylation and oligomerization of the enzyme is not clear. PEPC-protein kinase (PEPC-PK), the enzyme responsible for phosphorylation of PEPC, has been studied extensively while only limited information is available on the protein phosphatase 2A capable of dephosphorylating PEPC. The C₄ ppc was cloned and expressed in Escherichia coli as well as tobacco. The transformed E. coli produced a functional/phosphorylatable C₄ PEPC and the transgenic tobacco plants expressed both C₃ and C₄ isoforms. Site-directed mutagenesis of ppc indicates the importance of His¹³⁸, His⁵⁷⁹ and Arg⁵⁸⁷ in catalysis and/or substrate-binding by the E. coli enzyme, Ser⁸ in the regulation of sorghum PEPC. Important areas for further research on C₄ PEPC are: mechanism of transduction of light signal during photoactivation of PEPC-PK and PEPC in leaves, extensive use of site-directed mutagenesis to precisely identify other key amino acid residues, changes in quarternary structure of PEPC in vivo, a high-resolution crystal structure, and hormonal regulation of PEPC expression.Abbreviations OAA oxalacetate - PEP phosphoenolpyruvate - PEPC PEP carboxylase - PEPC-PK PEPC-protein kinase - PPDK pyruvate, orthophosphate dikinase - Rubisco ribulose 1,5-bis-phosphate carboxylase/oxygenase - CAM Crassulacean acid metabolism     

Leaf anatomy and C4 photosynthetic enzymes in three reed ecotypes

Differences in leaf interveinal distances, chloroplasts distribution in bundle sheath cells (BSC) and activities of C₄ photosynthetic enzymes in the leaves of three ecotypes of Phragmites communis Trinius, namely swamp reed (SR), heavy salt meadow reed (HSMR) and dune reed (DR), occurring in the desert region of northwest China were investigated. The two terrestrial ecotypes, DR and HSMR, had denser vascular system, more and longer BSC chloroplasts and higher capacity of CO₂ concentrating mechanism of NAD-ME subtype as compared with the SR ecotype. The enhanced NADP-ME pathway in the HSMR might contribute to its adaptation to the salinity habitat.     

Molecular evolution: first enzymes, gases as substrates and genetic templates.

A fundamental problem in biology is the self-assembly of the first cells capable of growth and division under anoxic conditions on the Earth. Evolution proceeded by self-assembling and self-replicating cells that reproduced their own genetic information and also changed their genetic code over time. Was it also possible that some of the first proteins were catalytic and used gases as substrates and also acted as genetic templates? This paper explores the possibility that primitive protein enzymes used gases as their substrates, and reverse translation may have been a feature in the self-assembly of the first cell(s).     

Comparative genomic analysis of C4 photosynthetic pathway evolution in grasses

Background  

Sorghum is the first C4 plant and the second grass with a full genome sequence available. This makes it possible to perform a whole-genome-level exploration of C4 pathway evolution by comparing key photosynthetic enzyme genes in sorghum, maize (C4) and rice (C3), and to investigate a long-standing hypothesis that a reservoir of duplicated genes is a prerequisite for the evolution of C4 photosynthesis from a C3 progenitor.     

Overproduction of C4 photosynthetic enzymes in transgenic rice plants: an approach to introduce the C4-like photosynthetic pathway into rice

Four enzymes, namely, the maize C(4)-specific phosphoenolpyruvate carboxylase (PEPC), the maize C(4)-specific pyruvate, orthophosphate dikinase (PPDK), the sorghum NADP-malate dehydrogenase (MDH), and the rice C(3)-specific NADP-malic enzyme (ME), were overproduced in the mesophyll cells of rice plants independently or in combination. Overproduction individually of PPDK, MDH or ME did not affect the rate of photosynthetic CO(2) assimilation, while in the case of PEPC it was slightly reduced. The reduction in CO(2) assimilation in PEPC overproduction lines remained unaffected by overproduction of PPDK, ME or a combination of both, however it was significantly restored by the combined overproduction of PPDK, ME, and MDH to reach levels comparable to or slightly higher than that of non-transgenic rice. The extent of the restoration of CO(2) assimilation, however, was more marked at higher CO(2) concentrations, an indication that overproduction of the four enzymes in combination did not act to concentrate CO(2) inside the chloroplast. Transgenic rice plants overproducing the four enzymes showed slight stunting. Comparison of transformants overproducing different combinations of enzymes indicated that overproduction of PEPC together with ME was responsible for stunting, and that overproduction of MDH had some mitigating effects. Possible mechanisms underlying these phenotypic effects, as well as possibilities and limitations of introducing the C(4)-like photosynthetic pathway into C(3) plants, are discussed.     

Natural genetic engineering in evolution

The results of molecular genetics have frequently been difficult to explain by conventional evolutionary theory. New findings about the genetic conservation of protein structure and function across very broad taxonomic boundaries, the mosaic structure of genomes and genetic loci, and the molecular mechanisms of genetic change all point to a view of evolution as involving the rearrangement of basic genetic motifs. A more detailed examination of how living cells restructure their genomes reveals a wide variety of sophisticated biochemical systems responsive to elaborate regulatory networks. In some cases, we know that cells are able to accomplish extensive genome reorganization within one or a few cell generations. The emergence of bacterial antibiotic resistance is a contemporary example of evolutionary change; molecular analysis of this phenomenon has shown that it occurs by the addition and rearrangement of resistance determinants and genetic mobility systems rather than by gradual modification of pre-existing cellular genomes. In addition, bacteria and other organisms have intricate repair systems to prevent genetic change by sporadic physicochemical damage or errors of the replication machinery. In their ensemble, these results show that living cells have (and use) the biochemical apparatus to evolve by a genetic engineering process. Future research will reveal how well the regulatory systems integrate genomic change into basic life processes during evolution.     

Molecular evolution of hydrogen peroxide degrading enzymes

For efficient removal of intra- and/or extracellular hydrogen peroxide by dismutation to harmless dioxygen and water (2H(2)O(2) → O(2) + 2H(2)O), nature designed three metalloenzyme families that differ in oligomeric organization, monomer architecture as well as active site geometry and catalytic residues. Here we report on the updated reconstruction of the molecular phylogeny of these three gene families. Ubiquitous typical (monofunctional) heme catalases are found in all domains of life showing a high structural conservation. Their evolution was directed from large subunit towards small subunit proteins and further to fused proteins where the catalase fold was retained but lost its original functionality. Bifunctional catalase-peroxidases were at the origin of one of the two main heme peroxidase superfamilies (i.e. peroxidase-catalase superfamily) and constitute a protein family predominantly present among eubacteria and archaea, but two evolutionary branches are also found in the eukaryotic world. Non-heme manganese catalases are a relatively small protein family with very old roots only present among bacteria and archaea. Phylogenetic analyses of the three protein families reveal features typical (i) for the evolution of whole genomes as well as (ii) for specific evolutionary events including horizontal gene transfer, paralog formation and gene fusion. As catalases have reached a striking diversity among prokaryotic and eukaryotic pathogens, understanding their phylogenetic and molecular relationship and function will contribute to drug design for prevention of diseases of humans, animals and plants.     

Strategies for engineering a two-celled C(4) photosynthetic pathway into rice

Every day almost one billion people suffer from chronic hunger, and the situation is expected to deteriorate with a projected population growth to 9 billion worldwide by 2050. In order to provide adequate nutrition into the future, rice yields in Asia need to increase by 60%, a change that may be achieved by introduction of the C(4) photosynthetic cycle into rice. The international C(4) Rice Consortium was founded in order to test the feasibility of installing the C(4) engine into rice. This review provides an update on two of the many approaches employed by the C(4) Rice Consortium: namely, metabolic C(4) engineering and identification of determinants of leaf anatomy by mutant screens. The aim of the metabolic C(4) engineering approach is to generate a two-celled C(4) shuttle in rice by expressing the classical enzymes of the NADP-ME C(4) cycle in a cell-appropriate manner. The aim is also to restrict RuBisCO and glycine decarboxylase expression to the bundle sheath (BS) cells of rice in a C(4)-like fashion by specifically down-regulating their expression in rice mesophyll (M) cells. In addition to the changes in biochemistry, two-celled C(4) species show a convergence in leaf anatomy that include increased vein density and reduced numbers of M cells between veins. By screening rice activation-tagged lines and loss-of-function sorghum mutants we endeavour to identify genes controlling these key traits.     

Lessons from engineering a single-cell C(4) photosynthetic pathway into rice

The transfer of C(4) plant traits into C(3) plants has long been a strategy for improving the photosynthetic performance of C(3) plants. The introduction of a pathway mimicking the C(4) photosynthetic pathway into the mesophyll cells of C(3) plants was only a realistic approach when transgenic technology was sufficiently well developed and widely adopted. Here an attempt to introduce a single-cell C(4)-like pathway in which CO(2) capture and release occur in the mesophyll cell, such as the one found in the aquatic plant Hydrilla verticillata (L.f.) Royle, into rice (Oryza sativa L.) is described. Four enzymes involved in this pathway were successfully overproduced in the transgenic rice leaves, and 12 different sets of transgenic rice that overproduce these enzymes independently or in combination were produced and analysed. Although none of these transformants has yet shown dramatic improvements in photosynthesis, these studies nonetheless have important implications for the evolution of C(4) photosynthetic genes and their metabolic regulation, and have shed light on the unique aspects of rice physiology and metabolism. This article summarizes the lessons learned during these attempts to engineer single-cell C(4) rice.     

A possible role for C4 photosynthetic enzymes in tolerance of Zea mays to NaCl

Treatment of 14-day-old maize cultivars (Hybrid351 and Giza2) with 250 mM NaCl significantly reduced shoot fresh and dry weights and protein content during the subsequent 12 days. The magnitude of reduction was more pronounced in Giza than Hybrid. Both cultivars contained converging levels of protein for the enzymes phosphoenolpyruvate carboxylase (PEPC), malate dehydrogenase (MDH), pyruvate phosphate dikinase (PPDK) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) under normal conditions; however, NaCl led to increase these levels in Hybrid and decrease them in Giza. Moreover, NaCl significantly inhibited the activities of PEPC, MDH and PPDK in both cultivars during the first 2 days, thereafter the inhibition nullified only in Hybrid; nonetheless, Rubisco was the least affected enzyme in both cultivars. In addition, NaCl slightly increased V _max of PEPC, MDH and PPDK in Hybrid with no change in K _m; nevertheless V _max dropped in Giza with an increase in K _m of only PEPC and MDH. Also K _cat, K _cat/K _m and V _max/K _m of all enzymes were lower in treated Giza than in treated Hybrid. The increased V _max of all enzymes in only Hybrid by NaCl confirms that they were synthesised more in Hybrid than in Giza. However, the decreased V _max in Giza concomitant with the increased K _m points to an interference of salinity with synthesis of enzymes and their structural integrity. This would lead to a noncompetitive inhibition for the enzymes. These findings declare that maize tolerance to NaCl was larger in Hybrid compared to Giza due to a role for C4 enzymes.     

A critical review on the improvement of photosynthetic carbon assimilation in C3 plants using genetic engineering

Global warming is one of the most serious challenges facing us today. It may be linked to the increase in atmospheric CO2 and other greenhouse gases (GHGs), leading to a rise in sea level, notable shifts in ecosystems, and in the frequency and intensity of wild fires. There is a strong interest in stabilizing the atmospheric concentration of CO2 and other GHGs by decreasing carbon emission and/or increasing carbon sequestration. Biotic sequestration is an important and effective strategy to mitigate the effects of rising atmospheric CO2 concentrations by increasing carbon sequestration and storage capacity of ecosystems using plant photosynthesis and by decreasing carbon emission using biofuel rather than fossil fuel. Improvement of photosynthetic carbon assimilation, using transgenic engineering, potentially provides a set of available and effective tools for enhancing plant carbon sequestration. In this review, firstly different biological methods of CO2 assimilation in C3, C4 and CAM plants are introduced and three types of C4 pathways which have high photosynthetic performance and have evolved as CO2 pumps are briefly summarized. Then (i) the improvement of photosynthetic carbon assimilation of C3 plants by transgenic engineering using non-C4 genes, and (ii) the overexpression of individual or multiple C4 cycle photosynthetic genes (PEPC, PPDK, PCK, NADP-ME and NADP-MDH) in transgenic C3 plants (e.g. tobacco, potato, rice and Arabidopsis) are highlighted. Some transgenic C3 plants (e.g. tobacco, rice and Arabidopsis) overexpressing the FBP/SBPase, ictB and cytochrome c6 genes showed positive effects on photosynthetic efficiency and growth characteristics. However, over the last 28 years, efforts to overexpress individual, double or multiple C4 enzymes in C3 plants like tobacco, potato, rice, and Arabidopsis have produced mixed results that do not confirm or eliminate the possibility of improving photosynthesis of C3 plants by this approach. Finally, a prospect is provided on the challenges of enhancing carbon assimilation of C3 plants using transgenic engineering in the face of global warming, and the trends of the most promising approaches to improving the photosynthetic performance of C3 plants.     

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.     

Salt stress induces Kranz anatomy and expression of C4 photosynthetic enzymes in the amphibious sedge Eleocharis vivipara

Photosynthesis Research - Eleocharis vivipara Link is a unique amphibious leafless plant of the Cyperaceae. The terrestrial form develops culms with Kranz anatomy and C4-like traits, while the...     

Evolutionary switch and genetic convergence on rbcL following the evolution of C4 photosynthesis

Rubisco is responsible for the fixation of CO2 into organic compounds through photosynthesis and thus has a great agronomic importance. It is well established that this enzyme suffers from a slow catalysis, and its low specificity results into photorespiration, which is considered as an energy waste for the plant. However, natural variations exist, and some Rubisco lineages, such as in C4 plants, exhibit higher catalytic efficiencies coupled to lower specificities. These C4 kinetics could have evolved as an adaptation to the higher CO2 concentration present in C4 photosynthetic cells. In this study, using phylogenetic analyses on a large data set of C3 and C4 monocots, we showed that the rbcL gene, which encodes the large subunit of Rubisco, evolved under positive selection in independent C4 lineages. This confirms that selective pressures on Rubisco have been switched in C4 plants by the high CO2 environment prevailing in their photosynthetic cells. Eight rbcL codons evolving under positive selection in C4 clades were involved in parallel changes among the 23 independent monocot C4 lineages included in this study. These amino acids are potentially responsible for the C4 kinetics, and their identification opens new roads for human-directed Rubisco engineering. The introgression of C4-like high-efficiency Rubisco would strongly enhance C3 crop yields in the future CO2-enriched atmosphere.     

Molecular engineering of industrial enzymes: recent advances and future prospects

Many enzymes are efficiently produced by microbes. However, the use of natural enzymes as biocatalysts has limitations such as low catalytic efficiency, low activity, and low stability, especially under industrial conditions. Many protein engineering technologies have been developed to modify natural enzymes and eliminate these limitations. Commonly used protein engineering strategies include directed evolution, site-directed mutagenesis, truncation, and terminal fusion. This review summarizes recent advances in the molecular engineering of industrial enzymes and discusses future prospects in this field. We expect this review to increase interest in and advance the molecular engineering of industrial enzymes.     

High photosynthetic capacity of Sahelian C3 and C4 plants

Photosynthesis Research - The semi-arid ecosystems of the African Sahel play an important role in the global carbon cycle and are among the most sensitive ecosystems to future environmental...     

实验室适应性进化技术在光合蓝细菌底盘工程中的研究进展

蓝细菌是唯一可进行放氧光合作用的原核微生物,基于光合蓝细菌构建“自养型细胞工厂”具有广阔前景。但以蓝细菌作为底盘进行生物燃料及化学品的合成仍存在细胞耐受能力差、产量低等问题,导致实现工业化生产的经济可行性还比较低,亟需通过合成生物学等技术手段构建新的藻株。近年来,实验室适应性进化(adaptive laboratory evolution,ALE)已被用于底盘工程中,实现了优化生长速度、增加耐受性、加强底物利用和提高产品产量等目标。ALE在提高蓝细菌鲁棒性方面取得了一定进展,已获得了耐受高光、重金属离子、高盐和高浓度有机溶剂胁迫的进化藻株。但是,蓝细菌中的ALE策略效率相对较低,耐受各胁迫的分子机制并未阐释完全。本文综述了ALE相关技术策略及其在蓝细菌底盘工程中的应用,讨论了如何借鉴其他微生物中ALE手段,构建更大ALE突变文库、增加菌株的突变频率、缩短进化时间、探索多重胁迫耐受工程菌构建原则及研究策略等,高效解析进化菌株的突变体库,构建高产量、鲁棒性强的工程菌株等,以期未来促进蓝细菌底盘的改造及其工程菌的规模化应用。