Predicting C4 photosynthesis evolution: modular, individually adaptive steps on a Mount Fuji fitness landscape.

An ultimate goal of evolutionary biology is the prediction and experimental verification of adaptive trajectories on macroevolutionary timescales. This aim has rarely been achieved for complex biological systems, as models usually lack clear correlates of organismal fitness. Here, we simulate the fitness landscape connecting two carbon fixation systems: C3 photosynthesis, used by most plant species, and the C4 system, which is more efficient at ambient CO2 levels and elevated temperatures and which repeatedly evolved from C3. Despite extensive sign epistasis, C4 photosynthesis is evolutionarily accessible through individually adaptive steps from any intermediate state. Simulations show that biochemical subtraits evolve in modules; the order and constitution of modules confirm and extend previous hypotheses based on species comparisons. Plant-species-designated C3-C4 intermediates lie on predicted evolutionary trajectories, indicating that they indeed represent transitory states. Contrary to expectations, we find no slowdown of adaptation and no diminishing fitness gains along evolutionary trajectories.

Perspectives on improving photosynthesis to increase crop yield.

Improving photosynthesis, the fundamental process by which plants convert light energy into chemical energy, is a key area of research with great potential for enhancing sustainable agricultural productivity and addressing global food security challenges. This perspective delves into the latest advancements and approaches aimed at optimizing photosynthetic efficiency. Our discussion encompasses the entire process, beginning with light harvesting and its regulation and progressing through the bottleneck of electron transfer. We then delve into the carbon reactions of photosynthesis, focusing on strategies targeting the enzymes of the Calvin-Benson-Bassham (CBB) cycle. Additionally, we explore methods to increase carbon dioxide (CO2) concentration near the Rubisco, the enzyme responsible for the first step of CBB cycle, drawing inspiration from various photosynthetic organisms, and conclude this section by examining ways to enhance CO2 delivery into leaves. Moving beyond individual processes, we discuss two approaches to identifying key targets for photosynthesis improvement: systems modeling and the study of natural variation. Finally, we revisit some of the strategies mentioned above to provide a holistic view of the improvements, analyzing their impact on nitrogen use efficiency and on canopy photosynthesis.

The Gynandropsis gynandra genome provides insights into whole-genome duplications and the evolution of C4 photosynthesis in Cleomaceae.

Gynandropsis gynandra (Cleomaceae) is a cosmopolitan leafy vegetable and medicinal plant, which has also been used as a model to study C4 photosynthesis due to its evolutionary proximity to C3 Arabidopsis (Arabidopsis thaliana). Here, we present the genome sequence of G. gynandra, anchored onto 17 main pseudomolecules with a total length of 740 Mb, an N50 of 42 Mb and 30,933 well-supported gene models. The G. gynandra genome and previously released genomes of C3 relatives in the Cleomaceae and Brassicaceae make an excellent model for studying the role of genome evolution in the transition from C3 to C4 photosynthesis. Our analyses revealed that G. gynandra and its C3 relative Tarenaya hassleriana shared a whole-genome duplication event (Gg-α), then an addition of a third genome (Th-α, +1×) took place in T. hassleriana but not in G. gynandra. Analysis of syntenic copy number of C4 photosynthesis-related gene families indicates that G. gynandra generally retained more duplicated copies of these genes than C3T. hassleriana, and also that the G. gynandra C4 genes might have been under positive selection pressure. Both whole-genome and single-gene duplication were found to contribute to the expansion of the aforementioned gene families in G. gynandra. Collectively, this study enhances our understanding of the polyploidy history, gene duplication and retention, as well as their impact on the evolution of C4 photosynthesis in Cleomaceae.

Transcriptome dynamics in developing leaves from C3 and C4 Flaveria species.

C4 species have evolved more than 60 times independently from C3 ancestors. This multiple and parallel evolution of the complex C4 trait suggests common underlying evolutionary mechanisms, which could be identified by comparative analysis of closely related C3 and C4 species. Efficient C4 function depends on a distinctive leaf anatomy that is characterised by enlarged, chloroplast-rich bundle sheath cells and narrow vein spacing. To elucidate the molecular mechanisms that generate the Kranz anatomy, we analysed a developmental series of leaves from the C4 plant Flaveria bidentis and the closely related C3 species Flaveria robusta by comparing anatomies and transcriptomes. Vascular density measurements of all nine leaf developmental stages identified three leaf anatomical zones whose proportions vary with respect to the developmental stage. We then deconvoluted the transcriptome datasets using non-negative matrix factorisation, which identified four distinct transcriptome patterns in the growing leaves of both species. By integrating the leaf anatomy and transcriptome data, we were able to correlate the different transcriptional profiles with different developmental zones in the leaves. These comparisons revealed an important role for auxin metabolism, in particular auxin homeostasis (conjugation and deconjugation), in establishing the high vein density typical of C4 species.

Diurnal Rhythms in the Red Seaweed Gracilariopsis chorda are Characterized by Unique Regulatory Networks of Carbon Metabolism.

Cellular and physiological cycles are driven by endogenous pacemakers, the diurnal and circadian rhythms. Key functions such as cell cycle progression and cellular metabolism are under rhythmic regulation, thereby maintaining physiological homeostasis. The photoreceptors phytochrome and cryptochrome, in response to light cues, are central input pathways for physiological cycles in most photosynthetic organisms. However, among Archaeplastida, red algae are the only taxa that lack phytochromes. Current knowledge about oscillatory rhythms is primarily derived from model species such as Arabidopsis thaliana and Chlamydomonas reinhardtii in the Viridiplantae, whereas little is known about these processes in other clades of the Archaeplastida, such as the red algae (Rhodophyta). We used genome-wide expression profiling of the red seaweed Gracilariopsis chorda and identified 3,098 rhythmic genes. Here, we characterized possible cryptochrome-based regulation and photosynthetic/cytosolic carbon metabolism in this species. We found a large family of cryptochrome genes in G. chorda that display rhythmic expression over the diurnal cycle and may compensate for the lack of phytochromes in this species. The input pathway gates regulatory networks of carbon metabolism which results in a compact and efficient energy metabolism during daylight hours. The system in G. chorda is distinct from energy metabolism in most plants, which activates in the dark. The green lineage, in particular, land plants, balance water loss and CO2 capture in terrestrial environments. In contrast, red seaweeds maintain a reduced set of photoreceptors and a compact cytosolic carbon metabolism to thrive in the harsh abiotic conditions typical of intertidal zones.

MSIreg: an R package for unsupervised coregistration of mass spectrometry and H&E images.

SUMMARY: Joint analysis of mass spectrometry images (MS images) and microscopy images of hematoxylin and eosin (H&E) stained tissues assists pathologists in characterizing the morphological structure of the tissues, and in performing diagnosis. Unfortunately, the analysis is undermined by substantial differences between these modalities in terms of aspect ratios, spatial resolution, number of channels in each image, as well as by large global or small local elastic spatial deformations of one image with respect to the other. Therefore, accurate coregistration of the images is a critical pre-requisite for their joint interpretation. We introduce MSIreg, an open-source R package for coregistration of MSI and H&E images. MSIreg is designed for high-dimensional MSI experiments where each spatial location is represented by thousands of mass features. Unlike most existing coregistration methods, MSIreg implements a landmark free workflow, and quantitative metrics for performance evaluation. We evaluate the performance of MSIreg on six case studies, including coregistration of contiguous tissues with large deformations, as well as simultaneous coregistration of 29 tissue microarray cores. AVAILABILITY AND IMPLEMENTATION: The R package, installation instructions, and fully reproducible vignettes describing methods and Case Studies are available open-source under the GPL-3.0 license at https://github.com/sslakkimsetty/msireg/.

Leaf transcriptomes from C3, C3-C4 intermediate, and C4 Neurachne species give insights into C4 photosynthesis evolution.

The C4 photosynthetic pathway is hypothesized to have evolved from the ancestral C3 pathway through progressive changes in leaf anatomy and biochemistry with extant C3-C4 photosynthetic intermediate species representing phenotypes between species demonstrating full C3 and full C4 states. The Australian endemic genus Neurachne is the only known grass group that contains distinct, closely related species that carry out C3, C3-C4 intermediate, or C4 photosynthesis. To explore and understand the molecular mechanisms underlying C4 photosynthesis evolution in this genus, leaf transcriptomes were generated from two C3, three photosynthetic intermediate (proto-Kranz, C2-like, and C2), and two C4  Neurachne species. The data were used to reconstruct phylogenetic relationships in Neurachne, which confirmed two independent C4 origins in the genus. Relative transcript abundances substantiated the photosynthetic phenotypes of individual species and highlighted transcriptional investment differences between species, including between the two C4 species. The data also revealed proteins potentially involved in C4 cycle intermediate transport and identified molecular mechanisms responsible for the evolution of C4-associated proteins in the genus.

Natural alleles of the abscisic acid catabolism gene ZmAbh4 modulate water use efficiency and carbon isotope discrimination in maize.

Altering plant water use efficiency (WUE) is a promising approach for achieving sustainable crop production in changing climate scenarios. Here, we show that WUE can be tuned by alleles of a single gene discovered in elite maize (Zea mays) breeding material. Genetic dissection of a genomic region affecting WUE led to the identification of the gene ZmAbh4 as causative for the effect. CRISPR/Cas9-mediated ZmAbh4 inactivation increased WUE without growth reductions in well-watered conditions. ZmAbh4 encodes an enzyme that hydroxylates the phytohormone abscisic acid (ABA) and initiates its catabolism. Stomatal conductance is regulated by ABA and emerged as a major link between variation in WUE and discrimination against the heavy carbon isotope (Δ13C) during photosynthesis in the C4 crop maize. Changes in Δ13C persisted in kernel material, which offers an easy-to-screen proxy for WUE. Our results establish a direct physiological and genetic link between WUE and Δ13C through a single gene with potential applications in maize breeding.

Genomic Basis of Adaptation to a Novel Precipitation Regime.

Energy production and metabolism are intimately linked to ecological and environmental constraints across the tree of life. In plants, which depend on sunlight to produce energy, the link between primary metabolism and the environment is especially strong. By governing CO2 uptake for photosynthesis and transpiration, leaf pores, or stomata, couple energy metabolism to the environment and determine productivity and water-use efficiency (WUE). Although evolution is known to tune physiological traits to the local environment, we lack knowledge of the specific links between molecular and evolutionary mechanisms that shape this process in nature. Here, we investigate the evolution of stomatal conductance and WUE in an Arabidopsis population that colonized an island with a montane cloud scrubland ecosystem characterized by seasonal drought and fog-based precipitation. We find that stomatal conductance increases and WUE decreases in the colonizing population relative to its closest outgroup population from temperate North Africa. Genome-wide association mapping reveals a polygenic basis of trait variation, with a substantial contribution from a nonsynonymous single-nucleotide polymorphism in MAP KINASE 12 (MPK12 G53R), which explains 35% of the phenotypic variance in WUE in the island population. We reconstruct the spatially explicit evolutionary history of MPK12 53R on the island and find that this allele increased in frequency in the population due to positive selection as Arabidopsis expanded into the harsher regions of the island. Overall, these findings show how adaptation shaped quantitative eco-physiological traits in a new precipitation regime defined by low rainfall and high humidity.

PYRIDOX(AM)INE 5'-PHOSPHATE OXIDASE3 of Arabidopsis thaliana maintains carbon/nitrogen balance in distinct environmental conditions.

The identification of factors that regulate C/N utilization in plants can make a substantial contribution to optimization of plant health. Here, we explored the contribution of pyridox(am)ine 5'-phosphate oxidase3 (PDX3), which regulates vitamin B6 homeostasis, in Arabidopsis (Arabidopsis thaliana). Firstly, N fertilization regimes showed that ammonium application rescues the leaf morphological phenotype of pdx3 mutant lines but masks the metabolite perturbance resulting from impairment in utilizing soil nitrate as a source of N. Without fertilization, pdx3 lines suffered a C/N imbalance and accumulated nitrogenous compounds. Surprisingly, exploration of photorespiration as a source of endogenous N driving this metabolic imbalance, by incubation under high CO2, further exacerbated the pdx3 growth phenotype. Interestingly, the amino acid serine, critical for growth and N management, alleviated the growth phenotype of pdx3 plants under high CO2, likely due to the requirement of pyridoxal 5'-phosphate for the phosphorylated pathway of serine biosynthesis under this condition. Triggering of thermomorphogenesis by growth of plants at 28 °C (instead of 22 °C) did not appear to require PDX3 function, and we observed that the consequent drive toward C metabolism counters the C/N imbalance in pdx3. Further, pdx3 lines suffered a salicylic acid-induced defense response, probing of which unraveled that it is a protective strategy mediated by nonexpressor of pathogenesis related1 (NPR1) and improves fitness. Overall, the study demonstrates the importance of vitamin B6 homeostasis as managed by the salvage pathway enzyme PDX3 to growth in diverse environments with varying nutrient availability and insight into how plants reprogram their metabolism under such conditions.

The role of metabolomics in informing strategies for improving photosynthesis.

Photosynthesis plays a vital role in acclimating to and mitigating climate change, providing food and energy security for a population that is constantly growing, and achieving an economy with zero carbon emissions. A thorough comprehension of the dynamics of photosynthesis, including its molecular regulatory network and limitations, is essential for utilizing it as a tool to boost plant growth, enhance crop yields, and support the production of plant biomass for carbon storage. Photorespiration constrains photosynthetic efficiency and contributes significantly to carbon loss. Therefore, modulating or circumventing photorespiration presents opportunities to enhance photosynthetic efficiency. Over the past eight decades, substantial progress has been made in elucidating the molecular basis of photosynthesis, photorespiration, and the key regulatory mechanisms involved, beginning with the discovery of the canonical Calvin-Benson-Bassham cycle. Advanced chromatographic and mass spectrometric technologies have allowed a comprehensive analysis of the metabolite patterns associated with photosynthesis, contributing to a deeper understanding of its regulation. In this review, we summarize the results of metabolomics studies that shed light on the molecular intricacies of photosynthetic metabolism. We also discuss the methodological requirements essential for effective analysis of photosynthetic metabolism, highlighting the value of this technology in supporting strategies aimed at enhancing photosynthesis.

A synthetic C4 shuttle via the ß-hydroxyaspartate cycle in C3 plants.

Plants depend on the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) for CO2 fixation. However, especially in C3 plants, photosynthetic yield is reduced by formation of 2-phosphoglycolate, a toxic oxygenation product of Rubisco, which needs to be recycled in a high-flux-demanding metabolic process called photorespiration. Canonical photorespiration dissipates energy and causes carbon and nitrogen losses. Reducing photorespiration through carbon-concentrating mechanisms, such as C4 photosynthesis, or bypassing photorespiration through metabolic engineering is expected to improve plant growth and yield. The ß-hydroxyaspartate cycle (BHAC) is a recently described microbial pathway that converts glyoxylate, a metabolite of plant photorespiration, into oxaloacetate in a highly efficient carbon-, nitrogen-, and energy-conserving manner. Here, we engineered a functional BHAC in plant peroxisomes to create a photorespiratory bypass that is independent of 3-phosphoglycerate regeneration or decarboxylation of photorespiratory precursors. While efficient oxaloacetate conversion in Arabidopsis thaliana still masks the full potential of the BHAC, nitrogen conservation and accumulation of signature C4 metabolites demonstrate the proof of principle, opening the door to engineering a photorespiration-dependent synthetic carbon-concentrating mechanism in C3 plants.

Redox regulation of NADP-malate dehydrogenase is vital for land plants under fluctuating light environment.

Many enzymes involved in photosynthesis possess highly conserved cysteine residues that serve as redox switches in chloroplasts. These redox switches function to activate or deactivate enzymes during light-dark transitions and have the function of fine-tuning their activities according to the intensity of light. Accordingly, many studies on chloroplast redox regulation have been conducted under the hypothesis that "fine regulation of the activities of these enzymes is crucial for efficient photosynthesis." However, the impact of the regulatory system on plant metabolism is still unclear. To test this hypothesis, we here studied the impact of the ablation of a redox switch in chloroplast NADP-malate dehydrogenase (MDH). By genome editing, we generated a mutant plant whose MDH lacks one of its redox switches and is active even in dark conditions. Although NADPH consumption by MDH in the dark is expected to be harmful to plant growth, the mutant line did not show any phenotypic differences under standard long-day conditions. In contrast, the mutant line showed severe growth retardation under short-day or fluctuating light conditions. These results indicate that thiol-switch redox regulation of MDH activity is crucial for maintaining NADPH homeostasis in chloroplasts under these conditions.

Sub-optimal therapy of patients with primary biliary cholangitis (PBC) in the real-life stetting of the German PBC cohort.

Real-world data on the management of patients with primary biliary cholangitis (PBC) are so far scarce in Germany. Therefore, we aimed to establish a nationwide registry and describe the clinical characteristics and therapy of PBC patients.Three different cohorts defined as ursodeoxycholic acid (UDCA) responders, as inadequate responders according to Paris II criteria, and as newly diagnosed patients were prospectively recruited.This manuscript includes the baseline data of the project.In total, 33/77 (43%) contacted centres (58% of university hospitals, 38% of non-university hospitals, and 24% of private practices) recruited 515 patients including 204 UDCA responders, 221 inadequate responders to UDCA, and 90 newly diagnosed patients.All patients were treated with UDCA; however, a UDCA dosage below the recommended dosage of 13 mg/kg/d was observed in 38.5% of individuals after 12 months of treatment. UDCA dosages were lower in nonacademic compared to academic centres.Only 75/219 (38.5%) of inadequate responders to UDCA received a second-line therapy with obeticholic acid (OCA) and/or bezafibrate (BZF). OCA (13% vs. 4.5%) and BZF (14% vs. 6.5%) were significantly more often prescribed by academic vs. nonacademic centres.Pruritus (27% vs. 15.5%), fatigue (23% vs. 4.5%), and sicca syndrome (14% vs. 1%) were significantly more often reported by academic centres.The German PBC registry could be established, which indicates suboptimal therapy in a relevant proportion of patients and shows significant differences between academic and nonacademic centres. Results are fundamental to improving clinical management at different levels of care.

Basal osteotomy of the first metacarpal using patient-specific guides and instrumentation: biomechanical and 3D CT-based analysis.

INTRODUCTION: The aim of this study was to investigate the radiological outcomes of proximal closing metacarpal extension osteotomies using patient-specific guides and instruments (PSI) in early-stage trapeziometacarpal osteoarthritis to gain further insight into the joint loading surface and the benefits of the procedure. METHODS: In a prospective observational study, nine patients were included between 11/2020 and 12/2021, undergoing a total of ten proximal metacarpal extension osteotomies for basal thumb osteoarthritis. Computer-assisted surgical planning was performed using computed tomography (CT) and three-dimensional (3D) segmentation, allowing the fabrication of 3D-printed PSIs for surgical treatment. Inclusion criteria were a 1-year follow-up by CT to assess postoperative correction of the positional shift of the first metacarpal (MC1) and the location of peak loads compared with the preoperative situation. RESULTS: Radiographic analysis of the peak loading zone revealed a mean displacement on the articular surface of the trapezius of 0.4 mm ± 1.4 mm to radial and 0.1 mm ± 1.2 mm to palmar, and on the articular surface of the MC1 of 0.4 mm ± 1.4 mm to radial and 0.1 mm ± 1.2 mm to dorsal. CONCLUSION: There were trends indicating that a flatter pressure distribution and a dorsal shift of the peak loading zone may contribute to an improvement in subjective pain and patient satisfaction associated with this surgical procedure. The non-significant radiological results and the minor dorsal-radial shifts in our small study group limit a firm conclusion. LEVEL OF EVIDENCE: III.

Mitochondrial and peroxisomal NAD+ uptake are important for improved photosynthesis and seed yield under elevated CO2 concentrations.

As sessile organisms, plants must adapt their physiology and developmental processes to cope with challenging environmental circumstances, such as the ongoing elevation in atmospheric carbon dioxide (CO2 ) levels. Nicotinamide adenine dinucleotide (NAD+ ) is a cornerstone of plant metabolism and plays an essential role in redox homeostasis. Given that plants impaired in NAD metabolism and transport often display growth defects, low seed production and disturbed stomatal development/movement, we hypothesized that subcellular NAD distribution could be a candidate for plants to exploit the effects of CO2 fertilization. We report that an efficient subcellular NAD+ distribution is required for the fecundity-promoting effects of elevated CO2 levels. Plants with reduced expression of either mitochondrial (NDT1 or NDT2) or peroxisomal (PXN) NAD+ transporter genes grown under elevated CO2 exhibited reduced total leaf area compared with the wild-type while PXN mutants also displayed reduced leaf number. NDT2 and PXN lines grown under elevated CO2 conditions displayed reduced rosette dry weight and lower photosynthetic rates coupled with reduced stomatal conductance. Interestingly, high CO2 doubled seed production and seed weight in the wild-type, whereas the mutants were less responsive to increases in CO2 levels during reproduction, producing far fewer seeds than the wild-type under both CO2 conditions. These data highlight the importance of mitochondrial and peroxisomal NAD+ uptake mediated by distinct NAD transporter proteins to modulate photosynthesis and seed production under high CO2 levels.

Prospects for rice in 2050.

A key to achieve the goals put forward in the UN's 2030 Agenda for Sustainable Development, it will need transformative change to our agrifood systems. We must mount to the global challenge to achieve food security in a sustainable manner in the context of climate change, population growth, urbanization, and depletion of natural resources. Rice is one of the major staple cereal crops that has contributed, is contributing, and will still contribute to the global food security. To date, rice yield has held pace with increasing demands, due to advances in both fundamental and biological studies, as well as genomic and molecular breeding practices. However, future rice production depends largely on the planting of resilient cultivars that can acclimate and adapt to changing environmental conditions. This Special Issue highlight with reviews and original research articles the exciting and growing field of rice-environment interactions that could benefit future rice breeding. We also outline open questions and propose future directions of 2050 rice research, calling for more attentions to develop environment-resilient rice especially hybrid rice, upland rice and perennial rice.

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.

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.

Carbon monoxide exposure activates ULK1 via AMPK phosphorylation in murine embryonic fibroblasts.

Carbon monoxide (CO) is endogenously produced upon degradation of heme by heme oxygenases (HOs) and is suggested to act as a gaseous signaling molecule. The expression of HO-1 is triggered by the Nrf2-Keap1 signaling pathway which responds to exogenous stress signals and dietary constituents such as flavonoids and glucosinolates or reactive metabolic intermediates like 4-hydroxynonenal. Endogenous CO affects energy metabolism, regulates the utilization of glucose and addresses CYP450 enzymes. Using the CO releasing molecule-401 (CORM-401), we studied the effect of endogenous CO on ATP synthesis, AMP-signaling and activation of the AMPK pathway in cell culture. Upon exposure of cells to CORM-401, the mitochondrial ATP production rate was significantly decreased (P=0.007) to about 50%, while glycolytic ATP synthesis was unchanged (P=0.489). Total ATP levels were less affected as determined by mass spectrometry. Instead, levels of ADP and AMP were elevated following CORM-401 exposure by about two- (P=0.022) and four-fold (P=0.012) compared to control, respectively. Increased concentrations of AMP activate AMPK which was demonstrated by a 10 to 15-fold increased phosphorylation of Thr172 of the α-subunit of AMPK (P=0.025). A downstream target of AMPK is the kinase ULK1 which triggers autophagic and mitophagic processes. Activation of ULK1 after CO exposure was proven by a 3 to 5-fold elevated phosphorylation of ULK1 at Ser555 (P=0.004). The present data suggest that production of endogenous CO leads to increasing amounts of AMP which mediates AMPK-dependent downstream effects and likely triggers autophagic processes. Since dietary constituents and their metabolites induce the expression of the CO producing enzyme HO-1, CO signaling may also be involved in the cellular response to nutritional factors.