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Many tropical and subtropical plant lineages have independently evolved C4 photosynthesis. The convergent evolution of this complex functional trait from different ancestors is reflected in variations in the structural and biochemical characteristics of C4 components such as enzymes and cellular specializations. The mechanism of C4 carbon concentration mostly involves coordinated function of mesophyll and bundle sheath cells. Important adaptations of the C4 syndrome include increased vein density and the development of photosynthetic bundle sheath cells with low gas conductance. In addition, the enzymes and transporters of the C4 pathway evolved via the co-option of multiple genes, each derived from a specific lineage of isoforms present in nonC4-ancestors. In particular, the adaptation of C4 enzymes resulted in a variety of structural and biochemical modifications, generally leading to increased catalytic efficiency and regulation by metabolites and post-translational modifications. Differences in these adaptations are particularly evident in the C4-acid decarboxylation step, which can be catalyzed by three decarboxylases that define the C4 subtypes. Associated with the biochemical subtypes, there are also differences in the extend of grana staking and localization of bundle sheath cells chloroplasts. The presence of a suberin layer and symplastic connections also likely vary among the different C4-subtypes. This review examines the current understanding of the diversity of structural and functional changes in key components of the C4 carbon concentration mechanism. This knowledge is necessary not only to identify divergent solutions for convergent optimization of C4 components in different C4 lineages, but also to guide their creation for rational synthetic biology approaches.

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BACKGROUND: C4 photosynthesis is a mechanism that plants have evolved to reduce the rate of photorespiration during the carbon fixation process. The C4 pathway allows plants to adapt to high temperatures and light while more efficiently using resources, such as water and nitrogen. Despite decades of studies, the evolution of the C4 pathway from a C3 ancestor remains a biological enigma. Interestingly, species with C3-C4 intermediates photosynthesis are usually found closely related to the C4 lineages. Indeed, current models indicate that the assembly of C4 photosynthesis was a gradual process that included the relocalization of photorespiratory enzymes, and the establishment of intermediate photosynthesis subtypes. More than a third of the C4 origins occurred within the grass family (Poaceae). In particular, the Otachyriinae subtribe (Paspaleae tribe) includes 35 American species from C3, C4, and intermediates taxa making it an interesting lineage to answer questions about the evolution of photosynthesis. RESULTS: To explore the molecular mechanisms that underpin the evolution of C4 photosynthesis, the transcriptomic dynamics along four different leaf segments, that capture different stages of development, were compared among Otachyriinae non-model species. For this, leaf transcriptomes were sequenced, de novo assembled, and annotated. Gene expression patterns of key pathways along the leaf segments showed distinct differences between photosynthetic subtypes. In addition, genes associated with photorespiration and the C4 cycle were differentially expressed between C4 and C3 species, but their expression patterns were well preserved throughout leaf development. CONCLUSIONS: New, high-confidence, protein-coding leaf transcriptomes were generated using high-throughput short-read sequencing. These transcriptomes expand what is currently known about gene expression in leaves of non-model grass species. We found conserved expression patterns of C4 cycle and photorespiratory genes among C3, intermediate, and C4 species, suggesting a prerequisite for the evolution of C4 photosynthesis. This dataset represents a valuable contribution to the existing genomic resources and provides new tools for future investigation of photosynthesis evolution.

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Trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is an essential signal metabolite in plants, linking growth and development to carbon status. Our current understanding of Tre6P metabolism and signaling pathways in plants is based almost entirely on studies performed with Arabidopsis thaliana, a model plant that performs C3 photosynthesis. Conversely, our knowledge on the molecular mechanisms involved in Tre6P regulation of carbon partitioning and metabolism in C4 plants is scarce. This topic is especially relevant due to the agronomic importance of crops performing C4 photosynthesis, such as maize, sorghum and sugarcane. In this review, we focused our attention on recent developments related to Tre6P metabolism in C4 species and raised some open questions that should be addressed in the near future to improve the yield of economically important crops.

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Portulaca species can switch between C4 and crassulacean acid metabolism (CAM) depending on environmental conditions. However, the regulatory mechanisms behind this rare photosynthetic adaptation remain elusive. Using Portulaca oleracea as a model system, here we investigated the involvement of the circadian clock, plant hormones, and transcription factors in coordinating C4 and CAM gene expression. Free-running experiments in constant conditions suggested that C4 and CAM gene expression are intrinsically connected to the circadian clock. Detailed time-course, drought, and rewatering experiments revealed distinct time frames for CAM induction and reversion (days versus hours, respectively), which were accompanied by changes in abscisic acid (ABA) and cytokinin metabolism and signaling. Exogenous ABA and cytokinins were shown to promote and repress CAM expression in P. oleracea, respectively. Moreover, the drought-induced decline in C4 transcript levels was completely recovered upon cytokinin treatment. The ABA-regulated transcription factor genes HB7, NFYA7, NFYC9, TT8, and ARR12 were identified as likely candidate regulators of CAM induction following this approach, whereas NFYC4 and ARR9 were connected to C4 expression patterns. Therefore, we provide insights into the signaling events controlling C4-CAM transitions in response to water availability and over the day/night cycle, highlighting candidate genes for future functional studies in the context of facultative C4-CAM photosynthesis.

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C4 photosynthesis is typically characterized by the spatial compartmentalization of the photosynthetic reactions into mesophyll (M) and bundle sheath (BS) cells. Initial carbon fixation within M cells gives rise to C4 acids, which are transported to the BS cells. There, C4 acids are decarboxylated so that the resulting CO2 is incorporated into the Calvin cycle. This work is focused on the study of Setaria viridis, a C4 model plant, closely related to several major feed and bioenergy grasses. First, we performed the heterologous expression and biochemical characterization of Setaria isoforms for chloroplastic NADP-malic enzyme (NADP-ME) and mitochondrial NAD-malic enzyme (NAD-ME). The kinetic parameters obtained agree with a major role for NADP-ME in the decarboxylation of the C4 acid malate in the chloroplasts of BS cells. In addition, mitochondria-located NAD-ME showed regulatory properties that could be important in the context of the operation of the C4 carbon shuttle. Secondly, we compared the proteomes of M and BS compartments and found 825 differentially accumulated proteins that could support different metabolic scenarios. Most interestingly, we found evidence of metabolic strategies to insulate the C4 core avoiding the leakage of intermediates by either up-regulation or down-regulation of chloroplastic, mitochondrial, and peroxisomal proteins. Overall, the results presented in this work provide novel data concerning the complexity of C4 metabolism, uncovering future lines of research that will undoubtedly contribute to the expansion of knowledge on this topic.

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ABSTRACT Leaf anatomy characteristics provide important evidences about the transition between C3 and C4 pathways. The C4 photosynthesis pathway allowed to reduce the C3 photorespiratory rate, concentrating CO2 around the Rubisco site and using structures and machinery already presented in C3 plants. In monocots, it is observed a high number of C4 lineages, most of them phylogenetically related to C3 groups. The genus Apochloa (C3), subtribe Arthropogoninae, is related to two C4 genera Coleataenia and Cyphonanthus. The aim of this study was to evaluate four Apochloa species in order to establish anatomical characteristics related to the evolution of C4 pathway in this group. By means of transverse sections fully expanded leaves of A. euprepes, A. lorea, A. molinioides, and A. poliophylla were collected and the characteristics of the mesophyll (M) and bundle sheath (BS) cells were determined. These species showed a rustic Kranz anatomy with enlarged and radial arranged BS cells, which have few organelles organized in a centrifugal position. Although the modifications of BS cells are probably related to the maintenance of plant water status, we also discuss the evolution for the establishment of C4 photosynthesis in the related C4 genera.

RESUMEN Las características de la anatomía de la hoja proporcionan evidencias importantes sobre la transición entre las vías C3 y C4. La fotosíntesis C4 surgió para reducir la tasa de fotorrespiración C3, concentrando el CO2 alrededor del sitio de la Rubisco y utilizando estructuras y maquinaria ya presentes en las plantas C3. En monocotiledóneas, se observa un alto número de linajes C4, la mayoría de ellas filogenéticamente relacionadas con grupos C3. El género C3 Apochloa, que pertenece a la subtribu Arthropogoninae, está relacionado con dos géneros C4 Coleataenia and Cyphonanthus. En este contexto, el objetivo fue evaluar cuatro especies de Apochloa para establecer las características anatómicas relacionadas con la evolución de la via C4 en este grupo. Se colectaron hojas completamente expandidas de A. euprepes, A. lorea, A. molinioides y A. poliophylla y se determinaron las características de las células del mesófilo (M) y del haz de la vaina (HV) a partir de secciones transversales de la hoja. Las especies presentaron una anatomía rústica de Kranz con células HV agrandadas y de distribución radial, con pocas organelas organizadas en posición centrífuga. Aunque las modificaciones de las células HV están probablemente relacionadas con el mantenimiento del estado hídrico de la planta, se puede inferir que facilitan el establecimiento de la fotosíntesis en los géneros C4 relacionados.

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In different lineages of C4 plants, the release of CO2 by decarboxylation of a C4 acid near rubisco is catalyzed by NADP-malic enzyme (ME) or NAD-ME, and the facultative use of phosphoenolpyruvate carboxykinase. The co-option of gene lineages during the evolution of C4-NADP-ME has been thoroughly investigated, whereas that of C4-NAD-ME has received less attention. In this work, we aimed at elucidating the mechanism of recruitment of NAD-ME for its function in the C4 pathway by focusing on the eudicot family Cleomaceae. We identified a duplication of NAD-ME in vascular plants that generated the two paralogs lineages: α- and ß-NAD-ME. Both gene lineages were retained across seed plants, and their fixation was likely driven by a degenerative process of sub-functionalization, which resulted in a NAD-ME operating primarily as a heteromer of α- and ß-subunits. We found most angiosperm genomes maintain a 1:1 ß-NAD-ME/α-NAD-ME (ß/α) relative gene dosage, but with some notable exceptions mainly due to additional duplications of ß-NAD-ME subunits. For example, a significantly high proportion of species with C4-NAD-ME-type photosynthesis have a non-1:1 ratio of ß/α. In the Brassicales, we found C4 species with a 2:1 ratio due to a ß-NAD-ME duplication (ß1 and ß2); this was also observed in the C3 Tarenaya hassleriana and Brassica crops. In the independently evolved C4 species, Gynandropsis gynandra and Cleome angustifolia, all three genes were affected by C4 evolution with α- and ß1-NAD-ME driven by adaptive selection. In particular, the ß1-NAD-MEs possess many differentially substituted amino acids compared with other species and the ß2-NAD-MEs of the same species. Five of these amino acids are identically substituted in ß1-NAD-ME of G. gynandra and C. angustifolia, two of them were identified as positively selected. Using synteny analysis, we established that ß-NAD-ME duplications were derived from ancient polyploidy events and that α-NAD-ME is in a unique syntenic context in both Cleomaceae and Brassicaceae. We discuss our hypotheses for the evolution of NAD-ME and its recruitment for C4 photosynthesis. We propose that gene duplications provided the basis for the recruitment of NAD-ME in C4 Cleomaceae and that all members of the NAD-ME gene family have been adapted to fit the C4-biochemistry. Also, one of the ß-NAD-ME gene copies was independently co-opted for its function in the C4 pathway.

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BACKGROUND: C4 plants have been classified into three subtypes based on the enzymes used to decarboxylate C4 acids in the bundle sheath cells (NADP-ME, NAD-ME and PEPCK pathways). Evidences indicate that, depending on environmental factors, C4 plants may exhibit a certain degree of flexibility in the use of the decarboxylation mechanisms. In this context, the objective was to extend the knowledge on the degree of flexibility between the pathways of decarboxylation in sugarcane, a NADP-ME species, at different levels of water deficit. RESULTS: An experiment was carried out with two cultivars - RB92579 (tolerant to water deficit) and SP80-3280 (susceptible to water deficit) subjected to moderate level (- 1.5 to - 1.8 MPa), severe level (below - 2.0 MPa) and recovery (48 h after rehydration) and changes in the activities of the enzymes involved in the three C4 mechanisms and in gene expression were investigated. Our results showed that sugarcane uses the PEPCK pathway as a decarboxylation mechanism in addition to the NADP-ME, which was more evident under water deficit conditions for both cultivars. CONCLUSIONS: The results obtained here, show that sugarcane increases the use of the PEPCK pathway as a decarboxylation mechanism, in addition to the NADP-ME pathway, under conditions of water deficit, particularly in the tolerant cultivar.

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The Portulacaceae enable the study of the evolutionary relationship between C4 and crassulacean acid metabolism (CAM) photosynthesis. Shoots of well-watered plants of the C3-C4 intermediate species Portulaca cryptopetala Speg. exhibit net uptake of CO2 solely during the light. CO2 fixation is primarily via the C3 pathway as indicated by a strong stimulation of CO2 uptake when shoots were provided with air containing 2% O2. When plants were subjected to water stress, daytime CO2 uptake was reduced and CAM-type net CO2 uptake in the dark occurred. This was accompanied by nocturnal accumulation of acid in both leaves and stems, also a defining characteristic of CAM. Following rewatering, net CO2 uptake in the dark ceased in shoots, as did nocturnal acidification of the leaves and stems. With this unequivocal demonstration of stress-related reversible, i.e. facultative, induction of CAM, P. cryptopetala becomes the first C3-C4 intermediate species reported to exhibit CAM. Portulaca molokiniensis Hobdy, a C4 species, also exhibited CAM only when subjected to water stress. Facultative CAM has now been demonstrated in all investigated species of Portulaca, which are well sampled from across the phylogeny. This strongly suggests that in Portulaca, a lineage in which species engage predominately in C4 photosynthesis, facultative CAM is ancestral to C4. In a broader context, it has now been demonstrated that CAM can co-exist in leaves that exhibit any of the other types of photosynthesis known in terrestrial plants: C3, C4 and C3-C4 intermediate.

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Demonstration of crassulacean acid metabolism (CAM) in species with low usage of this system relative to C3-photosynthetic CO2 assimilation can be challenging experimentally but provides crucial information on the early steps of CAM evolution. Here, weakly expressed CAM was detected in the well-known pantropical coastal, leaf-succulent herb Sesuvium portulacastrum, demonstrating that CAM is present in the Sesuvioideae, the only sub-family of the Aizoaceae in which it had not yet been shown conclusively. In outdoor plots in Panama, leaves and stems of S. portulacastrum consistently exhibited a small degree of nocturnal acidification which, in leaves, increased during the dry season. In potted plants, nocturnal acidification was mainly facultative, as levels of acidification increased in a reversible manner following the imposition of short-term water-stress. In drought-stressed plants, nocturnal net CO2 exchange approached the CO2-compensation point, consistent with low rates of CO2 dark fixation sufficient to eliminate respiratory carbon loss. Detection of low-level CAM in S. portulacastrum adds to the growing number of species that cannot be considered C3 plants sensu stricto, although they obtain CO2 principally via the C3 pathway. Knowledge about the presence/absence of low-level CAM is critical when assessing trajectories of CAM evolution in lineages. The genus Sesuvium is of particular interest because it also contains C4 species.

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The C4 grass Echinochloa polystachya, which forms dense and extensive monotypic stands on the Varzea floodplains of the Amazon region, provides the most productive natural higher plant communities known. The seasonal cycle of growth of this plant is closely linked to the annual rise and fall of water level over the floodplain surface. Diurnal cycles of leaf photosynthesis and transpiration were measured at monthly intervals, in parallel with measurements of leaf area index, canopy light interception and biomass. By artificial manipulation of the light flux incident on leaves in the field light-response curves of photosynthesis at the top and near to the base of the canopy were generated. Fitted light-response curves of CO2 uptake were combined with information of leaf area index, incident light and light penetration of the canopy to estimate canopy rates of photosynthesis. Throughout the period in which the floodplains were submerged photosynthetic rates of CO2 uptake (A) for the emergent leaves were high with a mean of c. 30 µmol m-2 s-1 at mid-day and occasional values of 40 µmol m-2 s-1. During the brief dry phase, when the floodplain surface is uncovered, there was a significant depression of A, with mid-day mean values of c. 17 µmol m-2 s-1. This corresponded with a c. 50% decrease in stomatal conductance, and a c. 35% depression in the ratio of the leaf inter-cellular to external CO2 concentration (c i/c a). During the dry phase, a midday depression of rates of CO2 assimilation was observed. The lowest leaf area index (F) was c. 2 in November-December, when the flood plain was dry, and again in May, when the rising floodwaters were submerging leaves faster than they were replaced. The maximum F of c. 5 was in August when the floodwaters were receding rapidly. Canopy light interception efficiency varied from 0.90 to 0.98. Calculated rates of canopy photosynthesis exceeded 18 mol C m-2 mo-1 throughout the period of flooding, with a peak of 37 mol C m-2 mo-1 in August, but declined to 13 mol C m-2 mo-1 in November during the dry phase. Estimated uptake of carbon by the canopy from the atmosphere, over 12 months, was 3.57 kg C m-2. This was insufficient to account for the 3.99 kg C m-2 of net primary production, measured simultaneously by destructive harvesting. It is postulated that this discrepancy might be accounted for by internal diffusion of CO2 from the CO2-rich waters and sediments via the roots and stems to the sites of assimilation in the leaves.