Reaction-diffusion modeling provides insights into biophysical carbon concentrating mechanisms in land plants.

Carbon concentrating mechanisms (CCMs) have evolved numerous times in photosynthetic organisms. They elevate the concentration of CO2 around the carbon-fixing enzyme rubisco, thereby increasing CO2 assimilatory flux and reducing photorespiration. Biophysical CCMs, like the pyrenoid-based CCM of Chlamydomonas reinhardtii or carboxysome systems of cyanobacteria, are common in aquatic photosynthetic microbes, but in land plants appear only among the hornworts. To predict the likely efficiency of biophysical CCMs in C3 plants, we used spatially resolved reaction-diffusion models to predict rubisco saturation and light use efficiency. We found that the energy efficiency of adding individual CCM components to a C3 land plant is highly dependent on the permeability of lipid membranes to CO2, with values in the range reported in the literature that are higher than used in previous modeling studies resulting in low light use efficiency. Adding a complete pyrenoid-based CCM into the leaf cells of a C3 land plant was predicted to boost net CO2 fixation, but at higher energetic costs than those incurred by photorespiratory losses without a CCM. Two notable exceptions were when substomatal CO2 levels are as low as those found in land plants that already employ biochemical CCMs and when gas exchange is limited, such as with hornworts, making the use of a biophysical CCM necessary to achieve net positive CO2 fixation under atmospheric CO2 levels. This provides an explanation for the uniqueness of hornworts' CCM among land plants and evolution of pyrenoids multiple times.

Reimport of carbon from cytosolic and vacuolar sugar pools into the Calvin-Benson cycle explains photosynthesis labeling anomalies.

SignificancePhotosynthesis metabolites are quickly labeled when 13CO2 is fed to leaves, but the time course of labeling reveals additional contributing processes involved in the metabolic dynamics of photosynthesis. The existence of three such processes is demonstrated, and a metabolic flux model is developed to explore and characterize them. The model is consistent with a slow return of carbon from cytosolic and vacuolar sugars into the Calvin-Benson cycle through the oxidative pentose phosphate pathway. Our results provide insight into how carbon assimilation is integrated into the metabolic network of photosynthetic cells with implications for global carbon fluxes.

Accurate flux predictions using tissue-specific gene expression in plant metabolic modeling.

MOTIVATION: The accurate prediction of complex phenotypes such as metabolic fluxes in living systems is a grand challenge for systems biology and central to efficiently identifying biotechnological interventions that can address pressing industrial needs. The application of gene expression data to improve the accuracy of metabolic flux predictions using mechanistic modeling methods such as flux balance analysis (FBA) has not been previously demonstrated in multi-tissue systems, despite their biotechnological importance. We hypothesized that a method for generating metabolic flux predictions informed by relative expression levels between tissues would improve prediction accuracy. RESULTS: Relative gene expression levels derived from multiple transcriptomic and proteomic datasets were integrated into FBA predictions of a multi-tissue, diel model of Arabidopsis thaliana's central metabolism. This integration dramatically improved the agreement of flux predictions with experimentally based flux maps from 13C metabolic flux analysis compared with a standard parsimonious FBA approach. Disagreement between FBA predictions and MFA flux maps was measured using weighted averaged percent error values, and for parsimonious FBA this was169%-180% for high light conditions and 94%-103% for low light conditions, depending on the gene expression dataset used. This fell to 10%-13% and 9%-11% upon incorporating expression data into the modeling process, which also substantially altered the predicted carbon and energy economy of the plant. AVAILABILITY AND IMPLEMENTATION: Code and data generated as part of this study are available from https://github.com/Gibberella/ArabidopsisGeneExpressionWeights.

Daylength variation affects growth, photosynthesis, leaf metabolism, partitioning, and metabolic fluxes.

Daylength, a seasonal and latitudinal variable, exerts a substantial impact on plant growth. However, the relationship between daylength and growth is nonproportional, suggesting the existence of adaptive mechanisms. Thus, our study aimed to comprehensively investigate the adaptive strategies employed by plants in response to daylength variation. We grew false flax (Camelina sativa) plants, a model oilseed crop, under long-day (LD) and short-day (SD) conditions and used growth measurements, gas exchange measurements, and isotopic labeling techniques, including 13C, 14C, and 2H2O, to determine responses to different daylengths. Our findings revealed that daylength influences various growth parameters, photosynthetic physiology, carbon partitioning, metabolic fluxes, and metabolite levels. SD plants employed diverse mechanisms to compensate for reduced CO2 fixation in the shorter photoperiod. These mechanisms included enhanced photosynthetic rates and reduced respiration in the light (RL), leading to increased shoot investment. Additionally, SD plants exhibited reduced rates of the glucose 6-phosphate (G6P) shunt and greater partitioning of sugars into starch, thereby sustaining carbon availability during the longer night. Isotopic labeling results further demonstrated substantial alterations in the partitioning of amino acids and TCA cycle intermediates between rapidly and slowly turning over pools. Overall, the results point to multiple developmental, physiological, and metabolic ways in which plants adapt to different daylengths to maintain growth.

Kinetic complexities of triacylglycerol accumulation in developing embryos from Camelina sativa provide evidence for multiple biosynthetic systems.

Quantitative flux maps describing glycerolipid synthesis can be important tools for rational engineering of lipid content and composition in oilseeds. Lipid accumulation in cultured embryos of Camelina sativa is known to mimic that of seeds in terms of rate of lipid synthesis and composition. To assess the kinetic complexity of the glycerolipid flux network, cultured embryos were incubated with [14C/13C]glycerol, and initial and steady state rates of [14C/13Cglyceryl] lipid accumulation were measured. At steady state, the linear accumulations of labeled lipid classes matched those expected from mass compositions. The system showed an apparently simple kinetic precursor-product relationship between the intermediate pool, dominated by diacylglycerol (DAG) and phosphatidylcholine (PC), and the triacylglycerol (TAG) product. We also conducted isotopomer analyses on hydrogenated lipid class species. [13C3glyceryl] labeling of DAG and PC, together with estimates of endogenous [12C3glyceryl] dilution, showed that each biosynthetically active lipid pool is ∼30% of the total by moles. This validates the concept that lipid sub-pools can describe lipid biosynthetic networks. By tracking the kinetics of [13C3glyceryl] and [13C2acyl] labeling, we observed two distinct TAG synthesis components. The major TAG synthesis flux (∼75%) was associated with >95% of the DAG/PC intermediate pool, with little glycerol being metabolized to fatty acids, and with little dilution from endogenous glycerol; a smaller flux exhibited converse characteristics. This kinetic heterogeneity was further explored using postlabeling embryo dissection and differential lipid extractions. The minor flux was tentatively localized to surface cells across the whole embryo. Such heterogeneity must be recognized in order to construct accurate gene expression patterns and metabolic networks describing lipid biosynthesis in developing embryos.

13C-labeling reveals how membrane lipid components contribute to triacylglycerol accumulation in Chlamydomonas.

Lipid metabolism in microalgae has attracted much interest due to potential utilization of lipids as feedstocks for biofuels, nutraceuticals, and other high-value compounds. Chlamydomonas reinhardtii is a model organism for characterizing the synthesis of the neutral lipid triacylglycerol (TAG), from which biodiesel is made. While much of TAG accumulation under N-deprivation is the result of de novo fatty acid (FA) synthesis, recent work has revealed that approximately one-third of FAs, especially polyunsaturated FAs (PUFAs), come from preexisting membrane lipids. Here, we used 13C-isotopic labeling and mass spectrometry to analyze the turnover of glycerol backbones, headgroups, FAs, whole molecules, and molecular fragments of individual lipids. About one-third of the glyceryl backbones in TAG are derived from preexisting membrane lipids, as are approximately one-third of FAs. The different moieties of the major galactolipids turn over synchronously, while the FAs of diacylglyceryltrimethylhomoserine (DGTS), the most abundant extraplastidial lipid, turn over independently of the rest of the molecule. The major plastidic lipid monogalactosyldiacylglycerol (MGDG), whose predominant species is 18:3α/16:4, was previously shown to be a major source of PUFAs for TAG synthesis. This study reveals that MGDG turns over as whole molecules, the 18:3α/16:4 species is present in both DAG and TAG, and the positional distribution of these PUFAs is identical in MGDG, DAG, and TAG. We conclude that headgroup removal with subsequent acylation is the mechanism by which the major MGDG species is converted to TAG during N-deprivation. This has noteworthy implications for engineering the composition of microalgal TAG for food, fuel, and other applications.

The carbon-concentrating mechanism of the extremophilic red microalga Cyanidioschyzon merolae.

Cyanidioschyzon merolae is an extremophilic red microalga which grows in low-pH, high-temperature environments. The basis of C. merolae's environmental resilience is not fully characterized, including whether this alga uses a carbon-concentrating mechanism (CCM). To determine if C. merolae uses a CCM, we measured CO2 uptake parameters using an open-path infra-red gas analyzer and compared them to values expected in the absence of a CCM. These measurements and analysis indicated that C. merolae had the gas-exchange characteristics of a CCM-operating organism: low CO2 compensation point, high affinity for external CO2, and minimized rubisco oxygenation. The biomass δ13C of C. merolae was also consistent with a CCM. The apparent presence of a CCM in C. merolae suggests the use of an unusual mechanism for carbon concentration, as C. merolae is thought to lack a pyrenoid and gas-exchange measurements indicated that C. merolae primarily takes up inorganic carbon as carbon dioxide, rather than bicarbonate. We use homology to known CCM components to propose a model of a pH-gradient-based CCM, and we discuss how this CCM can be further investigated.

Large fluxes of fatty acids from membranes to triacylglycerol and back during N-deprivation and recovery in Chlamydomonas.

Microalgae accumulate triacylglycerol (TAG) during nutrient deprivation and break it down after nutrient resupply, and these processes involve dramatic shifts in cellular carbon allocation. Due to the importance of algae in the global carbon cycle, and the potential of algal lipids as feedstock for chemical and fuel production, these processes are of both ecophysiological and biotechnological importance. However, the metabolism of TAG is not well understood, particularly the contributions of fatty acids (FAs) from different membrane lipids to TAG accumulation and the fate of TAG FAs during degradation. Here, we used isotopic labeling time course experiments on Chlamydomonas reinhardtii to track FA synthesis and transfer between lipid pools during nitrogen (N)-deprivation and resupply. When cells were labeled before N-deprivation, total levels of label in cellular FAs were unchanged during subsequent N-deprivation and later resupply, despite large fluxes into and out of TAG and membrane lipid pools. Detailed analyses of FA levels and labeling revealed that about one-third of acyl chains accumulating in TAG during N-deprivation derive from preexisting membrane lipids, and in total, at least 45% of TAG FAs passed through membrane lipids at one point. Notably, most acyl chains in membrane lipids during recovery after N-resupply come from TAG. Fluxes of polyunsaturated FAs from plastidic membranes into TAG during N-deprivation were particularly noteworthy. These findings demonstrate a high degree of integration of TAG and membrane lipid metabolism and highlight a role for TAG in storage and supply of membrane lipid components.

The metabolic origins of non-photorespiratory CO2 release during photosynthesis: a metabolic flux analysis.

Respiration in the light (RL) releases CO2 in photosynthesizing leaves and is a phenomenon that occurs independently from photorespiration. Since RL lowers net carbon fixation, understanding RL could help improve plant carbon-use efficiency and models of crop photosynthesis. Although RL was identified more than 75 years ago, its biochemical mechanisms remain unclear. To identify reactions contributing to RL, we mapped metabolic fluxes in photosynthesizing source leaves of the oilseed crop and model plant camelina (Camelina sativa). We performed a flux analysis using isotopic labeling patterns of central metabolites during 13CO2 labeling time course, gas exchange, and carbohydrate production rate experiments. To quantify the contributions of multiple potential CO2 sources with statistical and biological confidence, we increased the number of metabolites measured and reduced biological and technical heterogeneity by using single mature source leaves and quickly quenching metabolism by directly injecting liquid N2; we then compared the goodness-of-fit between these data and data from models with alternative metabolic network structures and constraints. Our analysis predicted that RL releases 5.2 µmol CO2 g-1 FW h-1 of CO2, which is relatively consistent with a value of 9.3 µmol CO2 g-1 FW h-1 measured by CO2 gas exchange. The results indicated that ≤10% of RL results from TCA cycle reactions, which are widely considered to dominate RL. Further analysis of the results indicated that oxidation of glucose-6-phosphate to pentose phosphate via 6-phosphogluconate (the G6P/OPP shunt) can account for >93% of CO2 released by RL.

High Flux Through the Oxidative Pentose Phosphate Pathway Lowers Efficiency in Developing Camelina Seeds.

Many seeds are green during development, and light has been shown to play a role in the efficiency with which maternally supplied substrates are converted into storage compounds. However, the effects of light on the fluxes through central metabolism that determine this efficiency are poorly understood. Here, we used metabolic flux analysis to determine the effects of light on central metabolism in developing embryos of false flax (Camelina sativa). Metabolic efficiency in C. sativa is of interest because, despite its growing importance as a model oilseed and engineering target and its potential as a biofuel crop, its yields are lower than other major oilseed species. Culture conditions under which steady-state growth and composition of developing embryos match those in planta were used to quantify substrate uptake and respiration rates. The carbon conversion efficiency (CCE) was 21% ± 3% in the dark and 42% ± 4% under high light. Under physiological illumination, the CCE (32% ± 2%) was substantially lower than in green and nongreen oilseeds studied previously. 13C and 14C isotopic labeling experiments were used together with computer-aided modeling to map fluxes through central metabolism. Fluxes through the oxidative pentose phosphate pathway (OPPP) were the principal source of CO2 production and strongly negatively correlated with CCE across light levels. OPPP fluxes were greatly in excess of demand for NAD(P)H for biosynthesis and larger than those measured in other systems. Excess reductant appears to be dissipated via cyanide-insensitive respiration. OPPP enzymes therefore represent a potential target for increasing efficiency and yield in C. sativa.

Correction: Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga Nannochloropsis oceanica CCMP1779.

[This corrects the article DOI: 10.1371/journal.pgen.1003064.].

Unfair trade underground revealed by integrating data with Nash bargaining models.

Mutually beneficial resource exchange is fundamental to global biogeochemical cycles and plant and animal nutrition. However, there is inherent potential conflict in mutualisms, as each organism benefits more when the exchange ratio ('price') minimizes its own costs and maximizes its benefits. Understanding the bargaining power that each partner has in these interactions is key to our ability to predict the exchange ratio and therefore the functionality of the cell, organism, community and ecosystem. We tested whether partners have symmetrical ('fair') or asymmetrical ('unfair') bargaining power in a legume-rhizobia nitrogen-fixing symbiosis using measurements of carbon and nitrogen dynamics in a mathematical modeling framework derived from economic theory. A model of symmetric bargaining power was not consistent with our data. Instead, our data indicate that the growth benefit to the plant (Medicago truncatula) has greater weight in determining trade dynamics than the benefit to the bacteria. Quantitative estimates of the relative power of the plant revealed that the plant's influence rises as soil nitrogen availability decreases and trade benefits to both partners increase. Our finding that M. truncatula legumes have more bargaining power than their rhizobial partner at lower nitrogen availabilities highlights the importance of context-dependence for the evolution of mutualism with increasing nutrient deposition.

Modelling nutritional mutualisms: challenges and opportunities for data integration.

Nutritional mutualisms are ancient, widespread, and profoundly influential in biological communities and ecosystems. Although much is known about these interactions, comprehensive answers to fundamental questions, such as how resource availability and structured interactions influence mutualism persistence, are still lacking. Mathematical modelling of nutritional mutualisms has great potential to facilitate the search for comprehensive answers to these and other fundamental questions by connecting the physiological and genomic underpinnings of mutualisms with ecological and evolutionary processes. In particular, when integrated with empirical data, models enable understanding of underlying mechanisms and generalisation of principles beyond the particulars of a given system. Here, we demonstrate how mathematical models can be integrated with data to address questions of mutualism persistence at four biological scales: cell, individual, population, and community. We highlight select studies where data has been or could be integrated with models to either inform model structure or test model predictions. We also point out opportunities to increase model rigour through tighter integration with data, and describe areas in which data is urgently needed. We focus on plant-microbe systems, for which a wealth of empirical data is available, but the principles and approaches can be generally applied to any nutritional mutualism.

The Relationship of Triacylglycerol and Starch Accumulation to Carbon and Energy Flows during Nutrient Deprivation in Chlamydomonas reinhardtii.

Because of the potential importance of algae for green biotechnology, considerable effort has been invested in understanding their responses to nitrogen deprivation. The most frequently invoked reasons proposed for the accumulation of high cellular levels of triacylglycerol (TAG) and starch are variants of what may be termed the "overflow hypothesis." According to this, growth inhibition results in the rate of photosynthetic energy and/or carbon input exceeding cellular needs; the excess input is directed into the accumulation of TAG and/or starch to prevent damage. This study was aimed at providing a quantitative dataset and analysis of the main energy and carbon flows before and during nitrogen deprivation in a model system to assess alternative explanations. Cellular growth, biomass, starch, and lipid levels as well as several measures of photosynthetic function were recorded for cells of Chlamydomonas reinhardtii cultured under nine different autotrophic, mixotrophic, and heterotrophic conditions during nutrient-replete growth and for the first 4 d of nitrogen deprivation. The results of a (13)C labeling time course indicated that in mixotrophic culture, starch is predominantly made from CO2 and fatty acid synthesis is largely supplied by exogenous acetate, with considerable turnover of membrane lipids, so that total lipid rather than TAG is the appropriate measure of product accumulation. Heterotrophic cultures accumulated TAG and starch during N deprivation, showing that these are not dependent on photosynthesis. We conclude that the overflow hypothesis is insufficient and suggest that storage may be a more universally important reason for carbon compound accumulation during nutrient deprivation.

The response of Chlamydomonas reinhardtii to nitrogen deprivation: a systems biology analysis.

Drastic alteration in macronutrients causes large changes in gene expression in the photosynthetic unicellular alga Chlamydomonas reinhardtii. Preliminary data suggested that cells follow a biphasic response to this change hinging on the initiation of lipid accumulation, and we hypothesized that drastic repatterning of metabolism also followed this biphasic modality. To test this hypothesis, transcriptomic, proteomic, and metabolite changes that occur under nitrogen (N) deprivation were analyzed. Eight sampling times were selected covering the progressive slowing of growth and induction of oil synthesis between 4 and 6 h after N deprivation. Results of the combined, systems-level investigation indicated that C. reinhardtii cells sense and respond on a large scale within 30 min to a switch to N-deprived conditions turning on a largely gluconeogenic metabolic state, which then transitions to a glycolytic stage between 4 and 6 h after N depletion. This nitrogen-sensing system is transduced to carbon- and nitrogen-responsive pathways, leading to down-regulation of carbon assimilation and chlorophyll biosynthesis, and an increase in nitrogen metabolism and lipid biosynthesis. For example, the expression of nearly all the enzymes for assimilating nitrogen from ammonium, nitrate, nitrite, urea, formamide/acetamide, purines, pyrimidines, polyamines, amino acids and proteins increased significantly. Although arginine biosynthesis enzymes were also rapidly up-regulated, arginine pool size changes and isotopic labeling results indicated no increased flux through this pathway.

Metabolic flux analyses of Pseudomonas aeruginosa cystic fibrosis isolates.

Pseudomonas aeruginosa is a metabolically versatile wide-ranging opportunistic pathogen. In humans P. aeruginosa causes infections of the skin, urinary tract, blood, and the lungs of Cystic Fibrosis patients. In addition, P. aeruginosa's broad environmental distribution, relatedness to biotechnologically useful species, and ability to form biofilms have made it the focus of considerable interest. We used 13C metabolic flux analysis (MFA) and flux balance analysis to understand energy and redox production and consumption and to explore the metabolic phenotypes of one reference strain and five strains isolated from the lungs of cystic fibrosis patients. Our results highlight the importance of the oxidative pentose phosphate and Entner-Doudoroff pathways in P. aeruginosa growth. Among clinical strains we report two divergent metabolic strategies and identify changes between genetically related strains that have emerged during a chronic infection of the same patient. MFA revealed that the magnitude of fluxes through the glyoxylate cycle correlates with growth rates.

The regulation of photosynthetic structure and function during nitrogen deprivation in Chlamydomonas reinhardtii.

The accumulation of carbon storage compounds by many unicellular algae after nutrient deprivation occurs despite declines in their photosynthetic apparatus. To understand the regulation and roles of photosynthesis during this potentially bioenergetically valuable process, we analyzed photosynthetic structure and function after nitrogen deprivation in the model alga Chlamydomonas reinhardtii. Transcriptomic, proteomic, metabolite, and lipid profiling and microscopic time course data were combined with multiple measures of photosynthetic function. Levels of transcripts and proteins of photosystems I and II and most antenna genes fell with differing trajectories; thylakoid membrane lipid levels decreased, while their proportions remained similar and thylakoid membrane organization appeared to be preserved. Cellular chlorophyll (Chl) content decreased more than 2-fold within 24 h, and we conclude from transcript protein and (13)C labeling rates that Chl synthesis was down-regulated both pre- and posttranslationally and that Chl levels fell because of a rapid cessation in synthesis and dilution by cellular growth rather than because of degradation. Photosynthetically driven oxygen production and the efficiency of photosystem II as well as P700(+) reduction and electrochromic shift kinetics all decreased over the time course, without evidence of substantial energy overflow. The results also indicate that linear electron flow fell approximately 15% more than cyclic flow over the first 24 h. Comparing Calvin-Benson cycle transcript and enzyme levels with changes in photosynthetic (13)CO2 incorporation rates also pointed to a coordinated multilevel down-regulation of photosynthetic fluxes during starch synthesis before the induction of high triacylglycerol accumulation rates.

Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis.

The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularis genes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.

Identification of regulatory network hubs that control lipid metabolism in Chlamydomonas reinhardtii.

Microalgae-based biofuels are promising sources of alternative energy, but improvements throughout the production process are required to establish them as economically feasible. One of the most influential improvements would be a significant increase in lipid yields, which could be achieved by altering the regulation of lipid biosynthesis and accumulation. Chlamydomonas reinhardtii accumulates oil (triacylglycerols, TAG) in response to nitrogen (N) deprivation. Although a few important regulatory genes have been identified that are involved in controlling this process, a global understanding of the larger regulatory network has not been developed. In order to uncover this network in this species, a combined omics (transcriptomic, proteomic and metabolomic) analysis was applied to cells grown in a time course experiment after a shift from N-replete to N-depleted conditions. Changes in transcript and protein levels of 414 predicted transcription factors (TFs) and transcriptional regulators (TRs) were monitored relative to other genes. The TF and TR genes were thus classified by two separate measures: up-regulated versus down-regulated and early response versus late response relative to two phases of polar lipid synthesis (before and after TAG biosynthesis initiation). Lipidomic and primary metabolite profiling generated compound accumulation levels that were integrated with the transcript dataset and TF profiling to produce a transcriptional regulatory network. Evaluation of this proposed regulatory network led to the identification of several regulatory hubs that control many aspects of cellular metabolism, from N assimilation and metabolism, to central metabolism, photosynthesis and lipid metabolism.