Tribulus (Zygophyllaceae) as a case study for the evolution of C2 and C4 photosynthesis.

C2 photosynthesis is a photosynthetic pathway in which photorespiratory CO2 release and refixation are enhanced in leaf bundle sheath (BS) tissues. The evolution of C2 photosynthesis has been hypothesized to be a major step in the origin of C4 photosynthesis, highlighting the importance of studying C2 evolution. In this study, physiological, anatomical, ultrastructural, and immunohistochemical properties of leaf photosynthetic tissues were investigated in six non-C4 Tribulus species and four C4 Tribulus species. At 42°C, T. cristatus exhibited a photosynthetic CO2 compensation point in the absence of respiration (C*) of 21 µmol mol-1, below the C3 mean C* of 73 µmol mol-1. Tribulus astrocarpus had a C* value at 42°C of 55 µmol mol-1, intermediate between the C3 species and the C2 T. cristatus. Glycine decarboxylase (GDC) allocation to BS tissues was associated with lower C*. Tribulus cristatus and T. astrocarpus allocated 86% and 30% of their GDC to the BS tissues, respectively, well above the C3 mean of 11%. Tribulus astrocarpus thus exhibits a weaker C2 (termed sub-C2) phenotype. Increased allocation of mitochondria to the BS and decreased length-to-width ratios of BS cells, were present in non-C4 species, indicating a potential role in C2 and C4 evolution.

Microbial exposure during early life regulates development of bile duct inflammation.

OBJECTIVES: The early life microbiome has been linked to inflammatory diseases in adulthood and a role for the microbiome in bile duct inflammation is supported by both human and murine studies. We utilized the NOD.c3c4 mouse model that develops a spontaneous immune-driven biliary disease with a known contribution of the microbiome to evaluate the temporal effects of the early life microbiome. MATERIALS AND METHODS: Germ-free (GF) NOD.c3c4 mice were conventionalized into a specific pathogen free environment at birth (conventionally raised, CONV-R) or at weaning (germ-free raised, GF-R) and compared with age and gender-matched GF and conventional (CONV) NOD.c3c4 mice. At 9 weeks of age, liver pathology was assessed by conventional histology and flow cytometry immunophenotyping. RESULTS: Neonatal exposure to microbes (CONV-R) increased biliary inflammation to similar levels as regular conventional NOD.c3c4 mice, while delayed exposure to microbes (GF-R) restrained the biliary inflammation. Neutrophil infiltration was increased in all conventionalized mice compared to GF. An immunophenotype in the liver similar to CONV was restored in both CONV-R and GF-R compared to GF mice displaying a proportional increase of B cells and reduction of T cells in the liver. CONCLUSIONS: Microbial exposure during early life has a temporal impact on biliary tract inflammation in the NOD.c3c4 mouse model suggesting that age-sensitive interaction with commensal microbes have long-lasting effects on biliary immunity that can be of importance for human cholangiopathies.

Scalp acupuncture guidance for identifying the optimal site for transcranial electrical stimulation of the hand.

Transcranial electrical stimulation (tES) often targets the EEG-guided C3/C4 area that may not accurately represent M1 for hand muscles. This study aimed to determine if the neuroanatomy-based scalp acupuncture-guided site (AC) was a more effective spot than the C3 site for neuromodulation. Fifteen healthy subjects received one 20-minute session of high-definition transcranial alternating current stimulation (HD-tACS) intervention (20 Hz at 2 mA) at the AC or C3 sites randomly with a 1-week washout period. Subjects performed ball-squeezing exercises with the dominant hand during the HD-tACS intervention. The AC site was indiscernible from the finger flexor hotspot detected by TMS. At the baseline, the MEP amplitude from finger flexors was greater with less variability at the AC site than at the C3 site. HD-tACS intervention at the AC site significantly increased the MEP amplitude. However, no significant changes were observed after tACS was applied to the C3 site. Our results provide evidence that HD-tACS at the AC site produces better neuromodulation effects on the flexor digitorum superficialis (FDS) muscle compared to the C3 site. The AC localization approach can be used for future tES studies.

A genomic panel for studying C3-C4 intermediate photosynthesis in the Brassiceae tribe.

Research on C4 and C3-C4 photosynthesis has attracted significant attention because the understanding of the genetic underpinnings of these traits will support the introduction of its characteristics into commercially relevant crop species. We used a panel of 19 taxa of 18 Brassiceae species with different photosynthesis characteristics (C3 and C3-C4) with the following objectives: (i) create draft genome assemblies and annotations, (ii) quantify orthology levels using synteny maps between all pairs of taxa, (iii) ⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠⁠describe the phylogenetic relatedness across all the species, and (iv) track the evolution of C3-C4 intermediate photosynthesis in the Brassiceae tribe. Our results indicate that the draft de novo genome assemblies are of high quality and cover at least 90% of the gene space. Therewith we more than doubled the sampling depth of genomes of the Brassiceae tribe that comprises commercially important as well as biologically interesting species. The gene annotation generated high-quality gene models, and for most genes extensive upstream sequences are available for all taxa, yielding potential to explore variants in regulatory sequences. The genome-based phylogenetic tree of the Brassiceae contained two main clades and indicated that the C3-C4 intermediate photosynthesis has evolved five times independently. Furthermore, our study provides the first genomic support of the hypothesis that Diplotaxis muralis is a natural hybrid of D. tenuifolia and D. viminea. Altogether, the de novo genome assemblies and the annotations reported in this study are a valuable resource for research on the evolution of C3-C4 intermediate photosynthesis.

Leaf anatomy explains the strength of C4 activity within the grass species Alloteropsis semialata.

C4 photosynthesis results from anatomical and biochemical characteristics that together concentrate CO2 around ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), increasing productivity in warm conditions. This complex trait evolved through the gradual accumulation of components, and particular species possess only some of these, resulting in weak C4 activity. The consequences of adding C4 components have been modelled and investigated through comparative approaches, but the intraspecific dynamics responsible for strengthening the C4 pathway remain largely unexplored. Here, we evaluate the link between anatomical variation and C4 activity, focusing on populations of the photosynthetically diverse grass Alloteropsis semialata that fix various proportions of carbon via the C4 cycle. The carbon isotope ratios in these populations range from values typical of C3 to those typical of C4 plants. This variation is statistically explained by a combination of leaf anatomical traits linked to the preponderance of bundle sheath tissue. We hypothesize that increased investment in bundle sheath boosts the strength of the intercellular C4 pump and shifts the balance of carbon acquisition towards the C4 cycle. Carbon isotope ratios indicating a stronger C4 pathway are associated with warmer, drier environments, suggesting that incremental anatomical alterations can lead to the emergence of C4 physiology during local adaptation within metapopulations.

Brassicaceae display variation in efficiency of photorespiratory carbon-recapturing mechanisms.

Carbon-concentrating mechanisms enhance the carboxylase efficiency of Rubisco by providing supra-atmospheric concentrations of CO2 in its surroundings. Beside the C4 photosynthesis pathway, carbon concentration can also be achieved by the photorespiratory glycine shuttle which requires fewer and less complex modifications. Plants displaying CO2 compensation points between 10 ppm and 40 ppm are often considered to utilize such a photorespiratory shuttle and are termed 'C3-C4 intermediates'. In the present study, we perform a physiological, biochemical, and anatomical survey of a large number of Brassicaceae species to better understand the C3-C4 intermediate phenotype, including its basic components and its plasticity. Our phylogenetic analysis suggested that C3-C4 metabolism evolved up to five times independently in the Brassicaceae. The efficiency of the pathway showed considerable variation. Centripetal accumulation of organelles in the bundle sheath was consistently observed in all C3-C4-classified taxa, indicating a crucial role for anatomical features in CO2-concentrating pathways. Leaf metabolite patterns were strongly influenced by the individual species, but accumulation of photorespiratory shuttle metabolites glycine and serine was generally observed. Analysis of phosphoenolpyruvate carboxylase activities suggested that C4-like shuttles have not evolved in the investigated Brassicaceae. Convergent evolution of the photorespiratory shuttle indicates that it represents a distinct photosynthesis type that is beneficial in some environments.

Evolutionary implications of C2 photosynthesis: how complex biochemical trade-offs may limit C4 evolution.

The C2 carbon-concentrating mechanism increases net CO2 assimilation by shuttling photorespiratory CO2 in the form of glycine from mesophyll to bundle sheath cells, where CO2 concentrates and can be re-assimilated. This glycine shuttle also releases NH3 and serine into the bundle sheath, and modelling studies suggest that this influx of NH3 may cause a nitrogen imbalance between the two cell types that selects for the C4 carbon-concentrating mechanism. Here we provide an alternative hypothesis outlining mechanisms by which bundle sheath NH3 and serine play vital roles to not only influence the status of C2 plants along the C3 to C4 evolutionary trajectory, but to also convey stress tolerance to these unique plants. Our hypothesis explains how an optimized bundle sheath nitrogen hub interacts with sulfur and carbon metabolism to mitigate the effects of high photorespiratory conditions. While C2 photosynthesis is typically cited for its intermediary role in C4 photosynthesis evolution, our alternative hypothesis provides a mechanism to explain why some C2 lineages have not made this transition. We propose that stress resilience, coupled with open flux tricarboxylic acid and photorespiration pathways, conveys an advantage to C2 plants in fluctuating environments.

Metabolic Background, Not Photosynthetic Physiology, Determines Drought and Drought Recovery Responses in C3 and C2 Moricandias.

Distinct photosynthetic physiologies are found within the Moricandia genus, both C3-type and C2-type representatives being known. As C2-physiology is an adaptation to drier environments, a study of physiology, biochemistry and transcriptomics was conducted to investigate whether plants with C2-physiology are more tolerant of low water availability and recover better from drought. Our data on Moricandia moricandioides (Mmo, C3), M. arvensis (Mav, C2) and M. suffruticosa (Msu, C2) show that C3 and C2-type Moricandias are metabolically distinct under all conditions tested (well-watered, severe drought, early drought recovery). Photosynthetic activity was found to be largely dependent upon the stomatal opening. The C2-type M. arvensis was able to secure 25-50% of photosynthesis under severe drought as compared to the C3-type M. moricandioides. Nevertheless, the C2-physiology does not seem to play a central role in M. arvensis drought responses and drought recovery. Instead, our biochemical data indicated metabolic differences in carbon and redox-related metabolism under the examined conditions. The cell wall dynamics and glucosinolate metabolism regulations were found to be major discriminators between M. arvensis and M. moricandioides at the transcription level.

A scheme for C4 evolution derived from a comparative analysis of the closely related C3, C3-C4 intermediate, C4-like, and C4 species in the genus Flaveria.

KEY MESSAGE: A comparative analysis of the genus Flaveria showed a C4 evolutionary process in which the anatomical and metabolic features of C4 photosynthesis were gradually acquired through C3-C4 intermediate stages. C4 photosynthesis has been acquired in multiple lineages of angiosperms during evolution to suppress photorespiration. Crops that perform C4 photosynthesis exhibit high rates of CO2 assimilation and high grain production even under high-temperature in semiarid environments; therefore, engineering C4 photosynthesis in C3 plants is of great importance in the application field. The genus Flaveria contains a large number of C3, C3-C4 intermediate, C4-like, and C4 species, making it a good model genus to study the evolution of C4 photosynthesis, and these studies indicate the direction for C4 engineering. C4 photosynthesis was acquired gradually through the C3-C4 intermediate stage. First, a two-celled C2 cycle called C2 photosynthesis was acquired by localizing glycine decarboxylase activity in the mitochondria of bundle sheath cells. With the development of two-cell metabolism, anatomical features also changed. Next, the replacement of the two-celled C2 cycle by the two-celled C4 cycle was induced by the acquisition of cell-selective expression in addition to the upregulation of enzymes in the C4 cycle during the C3-C4 intermediate stage. This was supported by an increase in cyclic electron transport activity in response to an increase in the ATP/NADPH demand for metabolism. Suppression of the C3 cycle in mesophyll cells was induced after the functional establishment of the C4 cycle, and optimization of electron transport by suppressing the activity of photosystem II also occurred during the final phase of C4 evolution.

Structural diversity in salt excreting glands and salinity tolerance in Oryza coarctata, Sporobolus anglicus and Urochondra setulosa.

MAIN CONCLUSION: Unlike the bicellular glands characteristic of all known excreting grasses, unique single-celled salt glands were discovered in the only salt tolerant species of the genus Oryza, Oryza coarctata. Salt tolerance has evolved frequently in a large number of grass lineages with distinct difference in mechanisms. Mechanisms of salt tolerance were studied in three species of grasses characterized by salt excretion: C3 wild rice species Oryza coarctata, and C4 species Sporobolus anglicus and Urochondra setulosa. The leaf anatomy and ultrastructure of salt glands, pattern of salt excretion, gas exchange, accumulation of key photosynthetic enzymes, leaf water content and osmolality, and levels of some osmolytes, were compared when grown without salt, with 200 mM NaCl versus 200 mM KCl. Under salt treatments, there was little effect on the capacity for CO2 assimilation, while stomatal conductance decreased with a reduction in water loss by transpiration and an increase in water use efficiency. All three species accumulate compatible solutes but with drastic differences in osmolyte composition. Having high capacity for salt excretion, they have distinct structural differences in the salt excreting machinery. S. anglicus and U. setulosa have bicellular glands while O. coarctata has unique single-celled salt glands with a partitioning membrane system that are responsible for salt excretion rather than multiple hairs as previously suggested. The features of physiological responses and salt excretion indicate similar mechanisms are involved in providing tolerance and excretion of Na+ and K+.

Upregulation of C4 characteristics does not consistently improve photosynthetic performance in intraspecific hybrids of a grass.

C4 photosynthesis is thought to have evolved via intermediate stages, with changes towards the C4 phenotype gradually enhancing photosynthetic performance. This hypothesis is widely supported by modelling studies, but experimental tests are missing. Mixing of C4 components to generate artificial intermediates can be achieved via crossing, and the grass Alloteropsis semialata represents an outstanding study system since it includes C4 and non-C4 populations. Here, we analyse F1 hybrids between C3 and C4 , and C3 +C4 and C4 genotypes to determine whether the acquisition of C4 characteristics increases photosynthetic performance. The hybrids have leaf anatomical characters and C4 gene expression profiles that are largely intermediate between those of their parents. Carbon isotope ratios are similarly intermediate, which suggests that a partial C4 cycle coexists with C3 carbon fixation in the hybrids. This partial C4 phenotype is associated with C4 -like photosynthetic efficiency in C3 +C4 × C4 , but not in C3 × C4 hybrids, which are overall less efficient than both parents. Our results support the hypothesis that the photosynthetic gains from the upregulation of C4 characteristics depend on coordinated changes in anatomy and biochemistry. The order of acquisition of C4 components is thus constrained, with C3 +C4 species providing an essential step for C4 evolution.

Stress-induced higher vein density in the C3-C4 intermediate Moricandia suffruticosa under drought and heat stress.

The evolution of C4 photosynthesis involved multiple anatomical and physiological modifications, yet our knowledge of the genetic regulation involved remains elusive. In this study, systematic analyses were conducted comparing the C3-C4 intermediate Moricandia suffruticosa and its C3 relative Brassica napus (rapeseed). We found that the leaves of M. suffruticosa had significantly higher vein density than those of B. napus, and the vein density was further increased in M. suffruticosa under drought and heat stress. Moreover, the bundle sheath distance, as the mean distance from the outer wall of one bundle sheath to the outer wall of an adjacent one, decreased and the number of centripetal chloroplasts in bundle sheath cells was found to be altered in M. suffruticosa leaves under drought and heat treatments. These results suggest that abiotic stress can induce a change in an intermediate C3-C4 anatomy towards a C4-like anatomy in land plants. By integrating drought and heat factors, co-expression network and comparative transcriptome analyses between M. suffruticosa and B. napus revealed that inducible auxin signaling regulated vascular development, and autophagy-related vesicle trafficking processes were associated with this stress-induced anatomical change. Overexpressing three candidate genes, MsERF02, MsSCL01, and MsDOF01, increased leaf vein density and/or enhanced photosynthetic assimilation and drought adaptability in the transgenic lines. The findings of this study may improve our understanding of the genetic regulation and evolution of C4 anatomy.

Effects of growth temperature and nitrogen nutrition on expression of C3-C4 intermediate traits in Chenopodium album.

Proto-Kranz plants represent an initial phase in the evolution from C3 to C3-C4 intermediate to C4 plants. The ecological and adaptive aspects of C3-C4 plants would provide an important clue to understand the evolution of C3-C4 plants. We investigated whether growth temperature and nitrogen (N) nutrition influence the expression of C3-C4 traits in Chenopodium album (proto-Kranz) in comparison with Chenopodium quinoa (C3). Plants were grown during 5 weeks at 20 or 30 °C under standard or low N supply levels (referred to as 20SN, 20LN, 30SN, and 30LN). Net photosynthetic rate and leaf N content were higher in 20SN and 30SN plants than in 20LN and 30LN plants of C. album but did not differ among growth conditions in C. quinoa. The CO2 compensation point (Γ) of C. album was lowest in 30LN plants (36 µmol mol-1), highest in 20SN plants (51 µmol mol-1), and intermediate in 20LN and 30SN plants, whereas Γ of C. quinoa did not differ among the growth conditions (51-52 µmol mol-1). The anatomical structure of leaves was not considerably affected by growth conditions in either species. However, ultrastructural observations in C. album showed that the number of mitochondria per mesophyll or bundle sheath (BS) cell was lower in 20LN and 30LN plants than in 20SN and 30SN plants. Immunohistochemical observations revealed that lower accumulation level of P-protein of glycine decarboxylase (GDC-P) in mesophyll mitochondria than in BS mitochondria is the major factor causing the decrease in Γ values in C. album plants grown under low N supply and high temperature. These results suggest that high growth temperature and low N supply lead to the expression of C3-C4 traits (the reduction of Γ) in the proto-Kranz plants of C. album through the regulation of GDC-P expression.

C2 photosynthesis: a promising route towards crop improvement?

C2 photosynthesis is a carbon concentrating mechanism that can increase net CO2 assimilation by capturing, concentrating and re-assimilating CO2 released by photorespiration. Empirical and modelling studies indicate that C2 plants assimilate more carbon than C3 plants under high temperature, bright light, and low CO2 conditions. I argue that engineering C2 photosynthesis into C3 crops is a promising approach to improve photosynthetic performance under these - and temporally heterogeneous - environments, and review the modifications that may re-create a C2 phenotype in C3 plants. Although a C2 engineering program would encounter many of the same challenges faced by C4 engineering programmes, the simpler leaf anatomical requirements make C2 engineering a feasible approach to improve crops in the medium term.

Photosynthesis research: a model to bridge fundamental science, translational products, and socio-economic considerations in agriculture.

Despite impressive success in molecular physiological understanding of photosynthesis, and preliminary evidence on its potential for quantum shifts in agricultural productivity, the question remains of whether increased photosynthesis, without parallel fine-tuning of the associated processes, is enough. There is a distinct lack of formal socio-economic impact studies that address the critical questions of product profiling, cost-benefit analysis, environmental trade-offs, and technological and market forces in product acceptability. When a relatively well understood process gains enough traction for translational value, its broader scientific and technical gap assessment, in conjunction with its socio-economic impact assessment for success, should be a prerequisite. The successes in the upstream basic understanding of photosynthesis should be integrated with a gap analysis for downstream translational applications to impact the farmers' and customers' lifestyles and livelihoods. The purpose of this review is to assess how the laboratory, the field, and the societal demands from photosynthesis could generate a transformative product. Two crucial recommendations from the analysis of the state of knowledge and potential ways forward are (i) the formulation of integrative mega-projects, which span the multistakeholder spectrum, to ensure rapid success in harnessing the transformative power of photosynthesis; and (ii) stipulating spatiotemporal, labour, and economic criteria to stage-gate deliverables.

Trade-offs in soil carbon protection mechanisms under aerobic and anaerobic conditions.

Oxygen (O2 ) limitation is generally understood to suppress oil carbon (C) decomposition and is a key mechanism impacting terrestrial C stocks under global change. Yet, O2 limitation may differentially impact kinetic or thermodynamic versus physicochemical C protection mechanisms, challenging our understanding of how soil C may respond to climate-mediated changes in O2 dynamics. Although O2 limitation may suppress decomposition of new litter C inputs, release of physicochemically protected C due to iron (Fe) reduction could potentially sustain soil C losses. To test this trade-off, we incubated two disparate upland soils that experience periodic O2 limitation-a tropical rainforest Oxisol and a temperate cropland Mollisol-with added litter under either aerobic (control) or anaerobic conditions for 1 year. Anoxia suppressed total C loss by 27% in the Oxisol and by 41% in the Mollisol relative to the control, mainly due to the decrease in litter-C decomposition. However, anoxia sustained or even increased decomposition of native soil-C (11.0% vs. 12.4% in the control for the Oxisol and 12.5% vs. 5.3% in the control for the Mollisol, in terms of initial soil C mass), and it stimulated losses of metal- or mineral-associated C. Solid-state 13 C nuclear magnetic resonance spectroscopy demonstrated that anaerobic conditions decreased protein-derived C but increased lignin- and carbohydrate-C relative to the control. Our results indicate a trade-off between physicochemical and kinetic/thermodynamic C protection mechanisms under anaerobic conditions, whereby decreased decomposition of litter C was compensated by more extensive loss of mineral-associated soil C in both soils. This challenges the common assumption that anoxia inherently protects soil C and illustrates the vulnerability of mineral-associated C under anaerobic events characteristic of a warmer and wetter future climate.

Land transformation in tropical savannas preferentially decomposes newly added biomass, whether C3 or C4 derived.

As tropical savannas are undergoing rapid conversion to other land uses, native C3 -C4 vegetation mixtures are often transformed to C3 - or C4 -dominant systems, resulting in poorly understood changes to the soil carbon (C) cycle. Conventional models of the soil C cycle are based on assumptions that more labile components of the heterogenous soil organic C (SOC) pool decompose at faster rates. Meanwhile, previous work has suggested that the C4 -derived component of SOC is more labile than C3 -derived SOC. Here we report on long-term (18 months) soil incubations from native and transformed tropical savannas of northern Australia. We test the hypothesis that, regardless of the type of land conversion, the C4 component of SOC will be preferentially decomposed. We measured changes in the SOC and pyrogenic carbon (PyC) pools, as well as the carbon isotope composition of SOC, PyC and respired CO2 , from 63 soil cores collected intact from different land use change scenarios. Our results show that land use change had no consistent effect on the size of the SOC pool, but strong effects on SOC decomposition rates, with slower decomposition rates at C4 -invaded sites. While we confirm that native savanna soils preferentially decomposed C4 -derived SOC, we also show that transformed savanna soils preferentially decomposed the newly added pool of labile SOC, regardless of whether it was C4 -derived (grass) or C3 -derived (forestry) biomass. Furthermore, we provide evidence that in these fire-prone landscapes, the nature of the PyC pool can shed light on past vegetation composition: while the PyC pool in C4 -dominant sites was mainly derived from C3 biomass, PyC in C3-dominant sites and native savannas was mainly derived from C4 biomass. We develop a framework to systematically assess the effects of recent land use change vs. prior vegetation composition.

C4 anatomy can evolve via a single developmental change.

C4 photosynthesis is a complex trait that boosts productivity in warm environments. Paradoxically, it evolved independently in numerous plant lineages, despite requiring specialised leaf anatomy. The anatomical modifications underlying C4 evolution have previously been evaluated through interspecific comparisons, which capture numerous changes besides those needed for C4 functionality. Here, we quantify the anatomical changes accompanying the transition between non-C4 and C4 phenotypes by sampling widely across the continuum of leaf anatomical traits in the grass Alloteropsis semialata. Within this species, the only trait that is shared among and specific to C4 individuals is an increase in vein density, driven specifically by minor vein development that yields multiple secondary effects facilitating C4 function. For species with the necessary anatomical preconditions, developmental proliferation of veins can therefore be sufficient to produce a functional C4 leaf anatomy, creating an evolutionary entry point to complex C4 syndromes that can become more specialised.

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.

Transition from C3 to proto-Kranz to C3-C4 intermediate type in the genus Chenopodium (Chenopodiaceae).

The Chenopodiaceae is one of the families including C4 species among eudicots. In this family, the genus Chenopodium is considered to include only C3 species. However, we report here a transition from C3 photosynthesis to proto-Kranz to C3-C4 intermediate type in Chenopodium. We investigated leaf anatomical and photosynthetic traits of 15 species, of which 8 species showed non-Kranz anatomy and a CO2 compensation point (Γ) typical of C3 plants. However, 5 species showed proto-Kranz anatomy and a C3-like Γ, whereas C. strictum showed leaf anatomy and a Γ typical of C3-C4 intermediates. Chenopodium album accessions examined included both proto-Kranz and C3-C4 intermediate types, depending on locality. Glycine decarboxylase, a key photorespiratory enzyme that is involved in the decarboxylation of glycine, was located predominantly in the mesophyll (M) cells of C3 species, in both M and bundle-sheath (BS) cells in proto-Kranz species, and exclusively in BS cells in C3-C4 intermediate species. The M/BS tissue area ratio, number of chloroplasts and mitochondria per BS cell, distribution of these organelles to the centripetal region of BS cells, the degree of inner positioning (vacuolar side of chloroplasts) of mitochondria in M cells, and the size of BS mitochondria also changed with the change in glycine decarboxylase localization. All Chenopodium species examined were C3-like regarding activities and amounts of C3 and C4 photosynthetic enzymes and δ13C values, suggesting that these species perform photosynthesis without contribution of the C4 cycle. This study demonstrates that Chenopodium is not a C3 genus and is valuable for studying evolution of C3-C4 intermediates.