Cytokinin-promoted secondary growth and nutrient storage in the perennial stem zone of Arabis alpina.

Perennial plants maintain their lifespan through several growth seasons. Arabis alpina serves as a model Brassicaceae species to study perennial traits. Lateral stems of A. alpina have a proximal vegetative zone with a dormant bud zone and a distal senescing seed-producing inflorescence zone. We addressed how this zonation is distinguished at the anatomical level, whether it is related to nutrient storage and which signals affect the zonation. We found that the vegetative zone exhibits secondary growth, which we termed the perennial growth zone (PZ). High-molecular-weight carbon compounds accumulate there in cambium and cambium derivatives. Neither vernalization nor flowering were requirements for secondary growth and the sequestration of storage compounds. The inflorescence zone with only primary growth, termed the annual growth zone (AZ), or roots exhibited different storage characteristics. Following cytokinin application cambium activity was enhanced and secondary phloem parenchyma was formed in the PZ and also in the AZ. In transcriptome analysis, cytokinin-related genes represented enriched gene ontology terms and were expressed at a higher level in the PZ than in the AZ. Thus, A. alpina primarily uses the vegetative PZ for nutrient storage, coupled to cytokinin-promoted secondary growth. This finding lays a foundation for future studies addressing signals for perennial growth.

Multiomics resolution of molecular events during a day in the life of Chlamydomonas.

The unicellular green alga Chlamydomonas reinhardtii displays metabolic flexibility in response to a changing environment. We analyzed expression patterns of its three genomes in cells grown under light-dark cycles. Nearly 85% of transcribed genes show differential expression, with different sets of transcripts being up-regulated over the course of the day to coordinate cellular growth before undergoing cell division. Parallel measurements of select metabolites and pigments, physiological parameters, and a subset of proteins allow us to infer metabolic events and to evaluate the impact of the transcriptome on the proteome. Among the findings are the observations that Chlamydomonas exhibits lower respiratory activity at night compared with the day; multiple fermentation pathways, some oxygen-sensitive, are expressed at night in aerated cultures; we propose that the ferredoxin, FDX9, is potentially the electron donor to hydrogenases. The light stress-responsive genes PSBS, LHCSR1, and LHCSR3 show an acute response to lights-on at dawn under abrupt dark-to-light transitions, while LHCSR3 genes also exhibit a later, second burst in expression in the middle of the day dependent on light intensity. Each response to light (acute and sustained) can be selectively activated under specific conditions. Our expression dataset, complemented with coexpression networks and metabolite profiling, should constitute an excellent resource for the algal and plant communities.

Overexpression of the chloroplastic 2-oxoglutarate/malate transporter disturbs carbon and nitrogen homeostasis in rice.

The chloroplastic 2-oxaloacetate (OAA)/malate transporter (OMT1 or DiT1) takes part in the malate valve that protects chloroplasts from excessive redox poise through export of malate and import of OAA. Together with the glutamate/malate transporter (DCT1 or DiT2), it connects carbon with nitrogen assimilation, by providing 2-oxoglutarate for the GS/GOGAT (glutamine synthetase/glutamate synthase) reaction and exporting glutamate to the cytoplasm. OMT1 further plays a prominent role in C4 photosynthesis: OAA resulting from phosphoenolpyruvate carboxylation is imported into the chloroplast, reduced to malate by plastidic NADP-malate dehydrogenase, and then exported for transport to bundle sheath cells. Both transport steps are catalyzed by OMT1, at the rate of net carbon assimilation. To engineer C4 photosynthesis into C3 crops, OMT1 must be expressed in high amounts on top of core C4 metabolic enzymes. We report here high-level expression of ZmOMT1 from maize in rice (Oryza sativa ssp. indica IR64). Increased activity of the transporter in transgenic rice was confirmed by reconstitution of transporter activity into proteoliposomes. Unexpectedly, overexpression of ZmOMT1 in rice negatively affected growth, CO2 assimilation rate, total free amino acid content, tricarboxylic acid cycle metabolites, as well as sucrose and starch contents. Accumulation of high amounts of aspartate and the impaired growth phenotype of OMT1 rice lines could be suppressed by simultaneous overexpression of ZmDiT2. Implications for engineering C4 rice are discussed.

The mitochondrial NAD+ transporter (NDT1) plays important roles in cellular NAD+ homeostasis in Arabidopsis thaliana.

Nicotinamide adenine dinucleotide (NAD+ ) is an essential coenzyme required for all living organisms. In eukaryotic cells, the final step of NAD+ biosynthesis is exclusively cytosolic. Hence, NAD+ must be imported into organelles to support their metabolic functions. Three NAD+ transporters belonging to the mitochondrial carrier family (MCF) have been biochemically characterized in plants. AtNDT1 (At2g47490), focus of the current study, AtNDT2 (At1g25380), targeted to the inner mitochondrial membrane, and AtPXN (At2g39970), located in the peroxisomal membrane. Although AtNDT1 was presumed to reside in the chloroplast membrane, subcellular localization experiments with green fluorescent protein (GFP) fusions revealed that AtNDT1 locates exclusively in the mitochondrial membrane in stably transformed Arabidopsis plants. To understand the biological function of AtNDT1 in Arabidopsis, three transgenic lines containing an antisense construct of AtNDT1 under the control of the 35S promoter alongside a T-DNA insertional line were evaluated. Plants with reduced AtNDT1 expression displayed lower pollen viability, silique length, and higher rate of seed abortion. Furthermore, these plants also exhibited an increased leaf number and leaf area concomitant with higher photosynthetic rates and higher levels of sucrose and starch. Therefore, lower expression of AtNDT1 was associated with enhanced vegetative growth but severe impairment of the reproductive stage. These results are discussed in the context of the mitochondrial localization of AtNDT1 and its important role in the cellular NAD+ homeostasis for both metabolic and developmental processes in plants.

Downregulation of a Mitochondrial NAD+ Transporter (NDT2) Alters Seed Production and Germination in Arabidopsis.

Despite the fundamental importance of nicotinamide adenine dinucleotide (NAD+) for metabolism, the physiological roles of NAD+ carriers in plants remain unclear. We previously characterized the Arabidopsis thaliana gene (At1g25380), named AtNDT2, encoding a protein located in the mitochondrial inner membrane, which imports NAD+ from the cytosol using ADP and AMP as counter-exchange substrates for NAD+. Here, we further investigated the physiological roles of NDT2, by isolating a T-DNA insertion line, generating an antisense line and characterizing these genotypes in detail. Reduced NDT2 expression affected reproductive phase by reducing total seed yield. In addition, reduced seed germination and retardation in seedling establishment were observed in the mutant lines. Moreover, remarkable changes in primary metabolism were observed in dry and germinated seeds and an increase in fatty acid levels was verified during seedling establishment. Furthermore, flowers and seedlings of NDT2 mutants displayed upregulation of de novo and salvage pathway genes encoding NAD+ biosynthesis enzymes, demonstrating the transcriptional control mediated by NDT2 activity over these genes. Taken together, our results suggest that NDT2 expression is fundamental for maintaining NAD+ balance amongst organelles that modulate metabolism, physiology and developmental processes of heterotrophic tissues.

The sugar transporter SWEET10 acts downstream of FLOWERING LOCUS T during floral transition of Arabidopsis thaliana.

BACKGROUND: Floral transition initiates reproductive development of plants and occurs in response to environmental and endogenous signals. In Arabidopsis thaliana, this process is accelerated by several environmental cues, including exposure to long days. The photoperiod-dependent promotion of flowering involves the transcriptional induction of FLOWERING LOCUS T (FT) in the phloem of the leaf. FT encodes a mobile protein that is transported from the leaves to the shoot apical meristem, where it forms part of a regulatory complex that induces flowering. Whether FT also has biological functions in leaves of wild-type plants remains unclear. RESULTS: In order to address this issue, we first studied the leaf transcriptomic changes associated with FT overexpression in the companion cells of the phloem. We found that FT induces the transcription of SWEET10, which encodes a bidirectional sucrose transporter, specifically in the leaf veins. Moreover, SWEET10 is transcriptionally activated by long photoperiods, and this activation depends on FT and one of its earliest target genes SUPPRESSOR OF CONSTANS OVEREXPRESSION 1 (SOC1). The ectopic expression of SWEET10 causes early flowering and leads to higher levels of transcription of flowering-time related genes in the shoot apex. CONCLUSIONS: Collectively, our results suggest that the FT-signaling pathway activates the transcription of a sucrose uptake/efflux carrier during floral transition, indicating that it alters the metabolism of flowering plants as well as reprogramming the transcription of floral regulators in the shoot meristem.

Cold Acclimation of the Thermoacidophilic Red Alga Galdieria sulphuraria: Changes in Gene Expression and Involvement of Horizontally Acquired Genes.

Galdieria sulphuraria is a unicellular red alga that lives in hot, acidic, toxic metal-rich, volcanic environments, where few other organisms survive. Its genome harbors up to 5% of genes that were most likely acquired through horizontal gene transfer. These genes probably contributed to G.sulphuraria's adaptation to its extreme habitats, resulting in today's polyextremophilic traits. Here, we applied RNA-sequencing to obtain insights into the acclimation of a thermophilic organism towards temperatures below its growth optimum and to study how horizontally acquired genes contribute to cold acclimation. A decrease in growth temperature from 42�C/46�C to 28�C resulted in an upregulation of ribosome biosynthesis, while excreted proteins, probably components of the cell wall, were downregulated. Photosynthesis was suppressed at cold temperatures, and transcript abundances indicated that C-metabolism switched from gluconeogenesis to glycogen degradation. Folate cycle and S-adenosylmethionine cycle (one-carbon metabolism) were transcriptionally upregulated, probably to drive the biosynthesis of betaine. All these cold-induced changes in gene expression were reversible upon return to optimal growth temperature. Numerous genes acquired by horizontal gene transfer displayed temperature-dependent expression changes, indicating that these genes contributed to adaptive evolution in G.sulphuraria.

NADP-MALIC ENZYME 1 Affects Germination after Seed Storage in Arabidopsis thaliana.

Aging decreases the quality of seeds and results in agricultural and economic losses. The damage that occurs at the biochemical level can alter the seed physiological status. Although loss of viability has been investigated frequently, little information exists on the molecular and biochemical factors involved in seed deterioration and loss of viability. Oxidative stress has been implicated as a major contributor to seed deterioration, and several pathways are involved in protection against this. In this study, we show that seeds of Arabidopsis thaliana lacking a functional NADP-MALIC ENZYME 1 (NADP-ME1) have reduced seed viability relative to the wild type. Seeds of the NADP-ME1 loss-of-function mutant display higher levels of protein carbonylation than those of the wild type. NADP-ME1 catalyzes the oxidative decarboxylation of malate to pyruvate with the simultaneous production of CO2 and NADPH. Upon seed imbibition, malate and amino acids accumulate in embryos of aged seeds of the NADP-ME1 loss-of-function mutant compared with those of the wild type. NADP-ME1 expression is increased in imbibed aged as compared with non-aged seeds. NADP-ME1 activity at testa rupture promotes normal germination of aged seeds. In seedlings of aged seeds, NADP-ME1 is specifically active in the root meristematic zone. We propose that NADP-ME1 activity is required for protecting seeds against oxidation during seed dry storage.

Efficient 2-phosphoglycolate degradation is required to maintain carbon assimilation and allocation in the C4 plant Flaveria bidentis.

Photorespiration is indispensable for oxygenic photosynthesis since it detoxifies and recycles 2-phosphoglycolate (2PG), which is the primary oxygenation product of Rubisco. However, C4 plant species typically display very low rates of photorespiration due to their efficient biochemical carbon-concentrating mechanism. Thus, the broader relevance of photorespiration in these organisms remains unclear. In this study, we assessed the importance of a functional photorespiratory pathway in the C4 plant Flaveria bidentis using knockdown of the first enzymatic step, namely 2PG phosphatase (PGLP). The isolated RNAi lines showed strongly reduced amounts of PGLP protein, but distinct signs of the photorespiratory phenotype only emerged below 5% residual PGLP protein. Lines with this characteristic were stunted in growth, had strongly increased 2PG content, exhibited accelerated leaf senescence, and accumulated high amounts of branched-chain and aromatic amino acids, which are both characteristics of incipient carbon starvation. Oxygen-dependent gas-exchange measurements consistently suggested the cumulative impairment of ribulose-1,5-bisphosphate regeneration with increased photorespiratory pressure. Our results indicate that photorespiration is essential for maintaining high rates of C4 photosynthesis by preventing the 2PG-mediated inhibition of carbon utilization efficiency. However, considerably higher 2PG accumulation can be tolerated compared to equivalent lines of C3 plants due to the differential distribution of specific enzymatic steps between the mesophyll and bundle sheath cells.

The Starch Granule-Associated Protein EARLY STARVATION1 Is Required for the Control of Starch Degradation in Arabidopsis thaliana Leaves.

To uncover components of the mechanism that adjusts the rate of leaf starch degradation to the length of the night, we devised a screen for mutant Arabidopsis thaliana plants in which starch reserves are prematurely exhausted. The mutation in one such mutant, named early starvation1 (esv1), eliminates a previously uncharacterized protein. Starch in mutant leaves is degraded rapidly and in a nonlinear fashion, so that reserves are exhausted 2 h prior to dawn. The ESV1 protein and a similar uncharacterized Arabidopsis protein (named Like ESV1 [LESV]) are located in the chloroplast stroma and are also bound into starch granules. The region of highest similarity between the two proteins contains a series of near-repeated motifs rich in tryptophan. Both proteins are conserved throughout starch-synthesizing organisms, from angiosperms and monocots to green algae. Analysis of transgenic plants lacking or overexpressing ESV1 or LESV, and of double mutants lacking ESV1 and another protein necessary for starch degradation, leads us to propose that these proteins function in the organization of the starch granule matrix. We argue that their misexpression affects starch degradation indirectly, by altering matrix organization and, thus, accessibility of starch polymers to starch-degrading enzymes.

Exogenous vasopressin dose-dependently modulates gastric microcirculatory oxygenation in dogs via V1A receptor.

BACKGROUND: Hypercapnia improves gastric microcirculatory oxygenation (µHbO2) and increases vasopressin plasma levels, whereas V1A receptor blockade abolishes the increase of µHbO2. The aim of this study was to evaluate the effect of exogenous vasopressin (AVP) in increasing doses on microcirculatory perfusion and oxygenation and systemic hemodynamic variables. Furthermore, we evaluated the role of the vasopressin V1A receptor in mediating the effects. METHODS: In repetitive experiments, six anesthetized dogs received a selective vasopressin V1A receptor inhibitor ([Pmp1, Tyr (Me)2]-Arg8-Vasopressin) or sodium chloride (control groups). Thereafter, a continuous infusion of AVP was started with dose escalation every 30 min (0.001 ng/kg/min-1 ng/kg/min). Microcirculatory variables of the oral and gastric mucosa were measured with reflectance spectrometry, laser Doppler flowmetry, and incident dark field imaging. Transpulmonary thermodilution was used to measure systemic hemodynamic variables. AVP plasma concentrations were measured during baseline conditions and 30 min after each dose escalation. RESULTS: During control conditions, gastric µHbO2 did not change during the course of experiments. Infusion of 0.001 ng/kg/min and 0.01 ng/kg/min AVP increased gastric µHbO2 to 87 ± 4% and 87 ± 6%, respectively, compared to baseline values (80 ± 7%), whereas application of 1 ng/kg/min AVP strongly reduced gastric µHbO2 (59 ± 16%). V1A receptor blockade prior to AVP treatment abolished these effects on µHbO2. AVP dose-dependently enhanced systemic vascular resistance (SVR) and decreased cardiac output (CO). After prior V1A receptor blockade, SVR was reduced and CO increased (0.1 ng/kg/min + 1 ng/kg/min AVP). CONCLUSIONS: Exogenous AVP dose-dependently modulates gastric µHbO2, with an increased µHbO2 with ultra-low dose AVP. The effects of AVP on µHbO2 are abolished by V1A receptor inhibition. These effects are independent of a modulation of systemic hemodynamic variables.

A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle.

Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2 Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2 We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1's four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency.

Natural variation in stomata size contributes to the local adaptation of water-use efficiency in Arabidopsis thaliana.

Stomata control gas exchanges between the plant and the atmosphere. How natural variation in stomata size and density contributes to resolve trade-offs between carbon uptake and water loss in response to local climatic variation is not yet understood. We developed an automated confocal microscopy approach to characterize natural genetic variation in stomatal patterning in 330 fully sequenced Arabidopsis thaliana accessions collected throughout the European range of the species. We compared this to variation in water-use efficiency, measured as carbon isotope discrimination (δ13 C). We detect substantial genetic variation for stomata size and density segregating within Arabidopsis thaliana. A positive correlation between stomata size and δ13 C further suggests that this variation has consequences on water-use efficiency. Genome wide association analyses indicate a complex genetic architecture underlying not only variation in stomatal patterning but also to its covariation with carbon uptake parameters. Yet, we report two novel QTL affecting δ13 C independently of stomatal patterning. This suggests that, in A. thaliana, both morphological and physiological variants contribute to genetic variance in water-use efficiency. Patterns of regional differentiation and covariation with climatic parameters indicate that natural selection has contributed to shape some of this variation, especially in Southern Sweden, where water availability is more limited in spring relative to summer. These conditions are expected to favour the evolution of drought avoidance mechanisms over drought escape strategies.

Quantification of Polyphosphate in Microalgae by Raman Microscopy and by a Reference Enzymatic Assay.

Polyphosphates have occurred in living cells early in evolution and microalgae contain these important polymers in their cells. Progress in research of polyphosphate metabolism of these ecologically as well as biotechnologically important microorganisms is hampered by the lack of rapid quantification methods. Experiments with the green alga Chlorella vulgaris presented here compared polyphosphate extraction in water, methanol-chloroform, and phenol-chloroform followed by polyphosphate purification by binding to silica columns or ethanol precipitation. The phenol-chloroform extraction of C. vulgaris followed by ethanol precipitation of polyphosphate was shown to be superior to the other tested method variants. Recovery test of added polyphosphate standard to algal biomass showed that the method is accurate. Using this biochemical assay as a validated reference, we show that 2-dimensional, confocal Raman microscopy can serve as a linear proxy for polyphosphate in C. vulgaris with R2 up to 0.956. With this, polyphosphate quantification can be shortened by use of Raman microscopy from days to hours and, additionally, information about intracellular distribution of polyphosphate and heterogeneity among individual cells in algal culture can be obtained. This offers new insights into the dynamics and role of these polymers crucial for phosphorus uptake and storage. This analytical capability is of particular practical importance because algae aid phosphorus sequestration from wastewater and the thus enriched biomass may serve as organic fertilizer. Both these applications have a strong potential in a future sustainable, circular bioeconomy.

Melatonin pretreatment improves gastric mucosal blood flow and maintains intestinal barrier function during hemorrhagic shock in dogs.

OBJECTIVE: Melatonin improves hepatic perfusion after hemorrhagic shock and may reduce stress-induced gastric lesions. This study was designed to investigate whether pretreatment with melatonin may influence gastric mucosal microcirculatory perfusion (µflow), oxygenation (µHbO2 ), or intestinal barrier function during physiological and hemorrhagic conditions in dogs. METHODS: In a randomized crossover study, five anesthetized foxhounds received melatonin 100 µg kg-1 or vehicle (ethanol 5%) intravenously in the absence or presence of hemorrhagic shock (60 minutes, -20% blood volume). Systemic hemodynamic variables, gastric mucosal perfusion, and oxygenation were recorded continuously; intestinal barrier function was assessed intermittently via xylose absorption. RESULTS: During hemorrhagic shock, melatonin significantly attenuated the decrease in µflow, compared with vehicle (-19±9 vs -43±10 aU, P<.05), without influence on µHbO2 . A significant increase in xylose absorption was detected during hemorrhage in vehicle-treated dogs, compared with sham-operated animals (13±2 vs 8±1 relative amounts, P<.05); this was absent in melatonin-treated animals (6±1 relative amounts). Melatonin did not influence macrocirculation. CONCLUSIONS: Melatonin improves regional blood flow suggesting improved oxygen delivery in gastric mucosa during hemorrhagic shock. This could provide a mechanism for the observed protection of intestinal barrier function in dogs.

Reversible Burst of Transcriptional Changes during Induction of Crassulacean Acid Metabolism in Talinum triangulare.

Drought tolerance is a key factor for agriculture in the 21st century as it is a major determinant of plant survival in natural ecosystems as well as crop productivity. Plants have evolved a range of mechanisms to cope with drought, including a specialized type of photosynthesis termed Crassulacean acid metabolism (CAM). CAM is associated with stomatal closure during the day as atmospheric CO2 is assimilated primarily during the night, thus reducing transpirational water loss. The tropical herbaceous perennial species Talinum triangulare is capable of transitioning, in a facultative, reversible manner, from C3 photosynthesis to weakly expressed CAM in response to drought stress. The transcriptional regulation of this transition has been studied. Combining mRNA-Seq with targeted metabolite measurements, we found highly elevated levels of CAM-cycle enzyme transcripts and their metabolic products in T. triangulare leaves upon water deprivation. The carbohydrate metabolism is rewired to reduce the use of reserves for growth to support the CAM-cycle and the synthesis of compatible solutes. This large-scale expression dataset of drought-induced CAM demonstrates transcriptional regulation of the C3-CAM transition. We identified candidate transcription factors to mediate this photosynthetic plasticity, which may contribute in the future to the design of more drought-tolerant crops via engineered CAM.

Effect of Topical Iloprost and Nitroglycerin on Gastric Microcirculation and Barrier Function during Hemorrhagic Shock in Dogs.

BACKGROUND: Topical drug application is used to avoid systemic side effects. The aim of this study was to analyze whether locally applied iloprost or nitroglycerin influence gastric mucosal perfusion, oxygenation, and barrier function during physiological and hemorrhagic conditions. METHODS: In repeated experiments, 5 anesthetized dogs received iloprost, nitroglycerin, or normal saline during physiological and hemorrhagic (-20% blood volume) conditions. Macro- and microcirculatory variables were recorded continuously. Gastric barrier function was assessed via translocation of sucrose into the blood. RESULTS: During hemorrhage, gastric mucosal oxygenation decreased from 77 ± 4 to 37 ± 7%. This effect was attenuated by nitroglycerin (78 ± 6 to 47 ± 13%) and iloprost (82 ± 4 to 54 ± 9%). Sucrose plasma levels increased during hemorrhage from 7 ± 4 to 55 ± 15 relative amounts. This was alleviated by nitroglycerin (5 ± 8 to 29 ± 38 relative amounts). These effects were independent of systemic hemodynamic variables. CONCLUSIONS: During hemorrhage, topical nitroglycerin and iloprost improve regional gastric oxygenation without affecting perfusion. Nitroglycerin attenuated the shock-induced impairment of the mucosal barrier integrity. Thus, local drug application improves gastric microcirculation without compromising systemic hemodynamic variables, and it may also protect mucosal barrier function.

Photosynthesis in C3-C4 intermediate Moricandia species.

Evolution of C4 photosynthesis is not distributed evenly in the plant kingdom. Particularly interesting is the situation in the Brassicaceae, because the family contains no C4 species, but several C3-C4 intermediates, mainly in the genus Moricandia Investigation of leaf anatomy, gas exchange parameters, the metabolome, and the transcriptome of two C3-C4 intermediate Moricandia species, M. arvensis and M. suffruticosa, and their close C3 relative M. moricandioides enabled us to unravel the specific C3-C4 characteristics in these Moricandia lines. Reduced CO2 compensation points in these lines were accompanied by anatomical adjustments, such as centripetal concentration of organelles in the bundle sheath, and metabolic adjustments, such as the balancing of C and N metabolism between mesophyll and bundle sheath cells by multiple pathways. Evolution from C3 to C3-C4 intermediacy was probably facilitated first by loss of one copy of the glycine decarboxylase P-protein, followed by dominant activity of a bundle sheath-specific element in its promoter. In contrast to recent models, installation of the C3-C4 pathway was not accompanied by enhanced activity of the C4 cycle. Our results indicate that metabolic limitations connected to N metabolism or anatomical limitations connected to vein density could have constrained evolution of C4 in Moricandia.

Photorespiratory glycolate oxidase is essential for the survival of the red alga Cyanidioschyzon merolae under ambient CO2 conditions.

Photorespiration is essential for all organisms performing oxygenic photosynthesis. The evolution of photorespiratory metabolism began among cyanobacteria and led to a highly compartmented pathway in plants. A molecular understanding of photorespiration in eukaryotic algae, such as glaucophytes, rhodophytes, and chlorophytes, is essential to unravel the evolution of this pathway. However, mechanistic detail of the photorespiratory pathway in red algae is scarce. The unicellular red alga Cyanidioschyzon merolae represents a model for the red lineage. Its genome is fully sequenced, and tools for targeted gene engineering are available. To study the function and importance of photorespiration in red algae, we chose glycolate oxidase (GOX) as the target. GOX catalyses the conversion of glycolate into glyoxylate, while hydrogen peroxide is generated as a side-product. The function of the candidate GOX from C. merolae was verified by the fact that recombinant GOX preferred glycolate over L-lactate as a substrate. Yellow fluorescent protein-GOX fusion proteins showed that GOX is targeted to peroxisomes in C. merolae The GOX knockout mutant lines showed a high-carbon-requiring phenotype with decreased growth and reduced photosynthetic activity compared to the wild type under ambient air conditions. Metabolite analyses revealed glycolate and glycine accumulation in the mutant cells after a shift from high CO2 conditions to ambient air. In summary, or results demonstrate that photorespiratory metabolism is essential for red algae. The use of a peroxisomal GOX points to a high photorespiratory flux as an ancestral feature of all photosynthetic eukaryotes.

Plants Possess a Cyclic Mitochondrial Metabolic Pathway similar to the Mammalian Metabolic Repair Mechanism Involving Malate Dehydrogenase and l-2-Hydroxyglutarate Dehydrogenase.

Enzymatic side reactions can give rise to the formation of wasteful and toxic products that are removed by metabolite repair pathways. In this work, we identify and characterize a mitochondrial metabolic repair mechanism in Arabidopsis thaliana involving malate dehydrogenase (mMDH) and l-2-hydroxyglutarate dehydrogenase (l-2HGDH). We analyze the kinetic properties of both A. thaliana mMDH isoforms, and show that they produce l-2-hydroxyglutarate (l-2HG) from 2-ketoglutarate (2-KG) at low rates in side reactions. We identify A. thaliana l-2HGDH as a mitochondrial FAD-containing oxidase that converts l-2HG back to 2-KG. Using loss-of-function mutants, we show that the electrons produced in the l-2HGDH reaction are transferred to the mitochondrial electron transport chain through the electron transfer protein (ETF). Thus, plants possess the biochemical components of an l-2HG metabolic repair system identical to that found in mammals. While deficiencies in the metabolism of l-2HG result in fatal disorders in mammals, accumulation of l-2HG in plants does not adversely affect their development under a range of tested conditions. However, orthologs of l-2HGDH are found in all examined genomes of viridiplantae, indicating that the repair reaction we identified makes an essential contribution to plant fitness in as yet unidentified conditions in the wild.