Robust Control of PEP Formation Rate in the Carbon Fixation Pathway of C4 Plants by a Bi-functional Enzyme

<p>Abstract</p> <p>Background</p> <p>C₄plants such as corn and sugarcane assimilate atmospheric CO₂ into biomass by means of the C₄carbon fixation pathway. We asked how PEP formation rate, a key step in the carbon fixation pathway, might work at a precise rate, regulated by light, despite fluctuations in substrate and enzyme levels constituting and regulating this process.</p> <p>Results</p> <p>We present a putative mechanism for robustness in C₄carbon fixation, involving a key enzyme in the pathway, pyruvate orthophosphate dikinase (PPDK), which is regulated by a bifunctional enzyme, Regulatory Protein (RP). The robust mechanism is based on avidity of the bifunctional enzyme RP to its multimeric substrate PPDK, and on a product-inhibition feedback loop that couples the system output to the activity of the bifunctional regulator. The model provides an explanation for several unusual biochemical characteristics of the system and predicts that the system's output, phosphoenolpyruvate (PEP) formation rate, is insensitive to fluctuations in enzyme levels (PPDK and RP), substrate levels (ATP and pyruvate) and the catalytic rate of PPDK, while remaining sensitive to the system's input (light levels).</p> <p>Conclusions</p> <p>The presented PPDK mechanism is a new way to achieve robustness using product inhibition as a feedback loop on a bifunctional regulatory enzyme. This mechanism exhibits robustness to protein and metabolite levels as well as to catalytic rate changes. At the same time, the output of the system remains tuned to input levels.</p

Multiscale metabolic modeling of C4 plants: connecting nonlinear genome-scale models to leaf-scale metabolism in developing maize leaves

C4 plants, such as maize, concentrate carbon dioxide in a specialized compartment surrounding the veins of their leaves to improve the efficiency of carbon dioxide assimilation. Nonlinear relationships between carbon dioxide and oxygen levels and reaction rates are key to their physiology but cannot be handled with standard techniques of constraint-based metabolic modeling. We demonstrate that incorporating these relationships as constraints on reaction rates and solving the resulting nonlinear optimization problem yields realistic predictions of the response of C4 systems to environmental and biochemical perturbations. Using a new genome-scale reconstruction of maize metabolism, we build an 18000-reaction, nonlinearly constrained model describing mesophyll and bundle sheath cells in 15 segments of the developing maize leaf, interacting via metabolite exchange, and use RNA-seq and enzyme activity measurements to predict spatial variation in metabolic state by a novel method that optimizes correlation between fluxes and expression data. Though such correlations are known to be weak in general, here the predicted fluxes achieve high correlation with the data, successfully capture the experimentally observed base-to-tip transition between carbon-importing tissue and carbon-exporting tissue, and include a nonzero growth rate, in contrast to prior results from similar methods in other systems. We suggest that developmental gradients may be particularly suited to the inference of metabolic fluxes from expression data.Comment: 57 pages, 14 figures; submitted to PLoS Computational Biology; source code available at http://github.com/ebogart/fluxtools and http://github.com/ebogart/multiscale_c4_sourc

Realization of a New-to-Nature Carboxylation Pathway

Most inorganic carbon enters the biosphere via the Calvin-Benson-Bassham (CBB) cycle by its key enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). An unproductive side reaction of RuBisCO with oxygen leads to the formation of 2-phosphoglycolate (2-PG), which is recycled via complex pathways into 3-phosphoglycerate (3-PGA), releasing carbon dioxide in the process. The tartronyl-CoA pathway represents a synthetic pathway that was designed to recycle 2-PG more efficiently, avoiding the release of carbon dioxide, and fixing carbon dioxide instead. It consists of four main reactions steps, which are not known to take part in any natural metabolic pathway. These steps are the activation of glycolate to glycolyl-CoA, the carboxylation of glycolyl-CoA to tartronyl-CoA as its key reaction, and the subsequent two reductions giving rise to glycerate. In this work, all required enzymes were identified or established by engineering and the tartronyl-CoA pathway was realized in vitro. Promiscuous enzyme candidates performing analogous reactions with similar substrates were screened and further improved to perform their desired functions. These include engineered glycolyl-CoA synthetase and glycolyl-CoA carboxylase (GCC), as well as a tartronyl-CoA reductase. For the engineering of GCC, rational design as well as high-throughput directed evolution was applied resulting in a new-to-nature carboxylase that matches the kinetic properties of natural carboxylases. Moreover, a 1.96 Å resolution cryogenic electron microscopy (cryo-EM) structure of GCC was obtained, highlighting and corroborating the effects of the introduced mutations. The concerted function of all tartronyl-CoA pathway enzymes was confirmed in the context of photorespiration in vitro. The in vitro reconstitution also included the optimization of reaction parameters as well as efficient cofactor recycling. Besides its function as photorespiratory bypass, the tartronyl-CoA pathway was shown to be functional as an additional carbon fixing module, able to connect a synthetic carbon dioxide fixation cycle to central carbon metabolism. Furthermore, the tartronyl-CoA pathway was successfully employed for the in vitro conversion of the plastic waste component ethylene glycol into the central carbon metabolite glycerate. In an initial attempt of an in vivo implementation of the tartronyl-CoA pathway for ethylene glycol assimilation, it was shown that GCC, the key enzyme of the tartronyl-CoA pathway, can be functionally produced in Pseudomonas putida

Climate change challenges, plant science solutions

Climate change is a defining challenge of the 21st century, and this decade is a critical time for action to mitigate the worst effects on human populations and ecosystems. Plant science can play an important role in developing crops with enhanced resilience to harsh conditions (e.g. heat, drought, salt stress, flooding, disease outbreaks) and engineering efficient carbon-capturing and carbon-sequestering plants. Here, we present examples of research being conducted in these areas and discuss challenges and open questions as a call to action for the plant science community

Dissecting heat and drought tolerance in wheat and maize using plant systems biology

Population growth and climate change pose serious threats to food security. Heat and drought are major abiotic constraints to crop production and their co-occurrence will increase during the cropping season in several regions. However, there is a lack of studies investigating their combined effect in crop physiological and biochemical processes. Aiming to close this gap, two of the main crops were investigated, wheat and maize, under these conditions. In the first results chapter, it is shown that these co-occurring stresses equally affect the photosynthetic efficiency of genotypes adapted to Mexico (Sokoll) and the UK (Paragon). However, Paragon recovered faster upon stress relief due to an increased PSII photoprotection and cytosolic Invertase activity, suggesting that optimal sucrose export/utilization and increased electron transport machinery photoprotection are essential to limit wheat yield fluctuations under these conditions. In the second results chapter, by studying maize genotypes with contrasting drought or heat tolerance, it was observed that limited transpiration under high temperature allowed water saving upon deficit without decreasing photosynthetic efficiency. This was sustained by higher phosphorylated PEPC and electron transport rate. Limited transpiration rate and synchronized regulation of the C4 carbon assimilation metabolism showed to be key traits for drought and heat tolerance in maize. In the third results chapter, by screening ten wheat genotypes with different tolerance to drought or heat, it was observed that leaf temperature and evapotranspiration expressed significant genotype-environment interactions. Low leaf number and transpiration efficiency were essential to balance water-saving strategies and biomass production. Changes in the carbohydrate (cytosolic Invertase, Hexokinase, Phosphofructokinase) and antioxidant metabolism (Peroxidases, phenolic compounds) were associated with tolerance mechanisms. Altogether, these results expand our knowledge about crops metabolic responses to high temperature and water deficit. These findings can be further explored in breeding programs to improve crop resilience to climate change and meet food security

An investigation into the chloroplast transformation of wheat, and the use of a cyanobacterial CCM gene for improving photosynthesis in a C3 plant

Wheat is a major component of the UK diet, and provides approximately 20% of global caloric intake. Wheat is grown on more land area than any other crop, and the continued supply of wheat is essential for global food security. Biotechnology is likely to play an important role in the sustainable increase of wheat yields, and the genetic manipulation of chloroplasts for photosynthetic improvement has many potential advantages over transformation of the nuclear genome. The genetic modification of the chloroplast genome via transformation was first demonstrated in the late 1980’s, and since then, chloroplast transformation of many Dicotyledonous (dicot) plant species such as Nicotiana tabaccum has been routinely performed. In comparison, the transformation of chloroplasts in Monocotyledons (monocot) plant species, which includes all cereal crops, has made far less progress. To date, there has been no reproducible homoplasmic plastid transformation event in the monocots. This study identifies a number of bottlenecks responsible for the prevention of chloroplast transformation in wheat. One such bottleneck is the lack of a suitable explant for plastid transformation, as traditional nuclear transformation targets are absent of metabolically active plastids. This study has developed a robust regeneration protocol for a previously undescribed tissue, termed the primary inflorescence leaf sheath (piLS), which is rich in active chloroplasts. Functional wheat specific chloroplast transformation vectors have been generated, and bombardment studies have been conducted with these on piLS and a second tissue, the immature embryosderived callus. Immature embryo callus (IEC) does not contain active plastids, however contains pro-plastids and is highly embryogenic. To uncover novel ways of increasing photosynthesis in C3 plants, a number of transplastomic tobacco lines expressing the Synechococcus elongatus PCC 7942 ictB gene were generated. Previous studies suggest that ictB may be an inorganic carbon transporter. In a number of transplastomic lines produced in this study, the intercellular carbon concentration (Ci) is significantly increased. This increased Ci did not result in an increased photosynthetic rate, however did cause a number of phenotypic differences, such as smaller plants, wider leaves, and earlier seed pod formation. The results, with regards to chloroplast transformation, and its implications in improving photosynthesis within C3 plants, are discussed in this thesis

Phosphorylation of C4 And Non-C4 Phosphoenolpyruvate Carboxylase from Panicum and the Kinetic Behaviour of Phosphorylated and Non-Phosphorylated PEPC

The C4 photosynthesis pathway is more efficient than C3 photosynthesis due to the capability of phosphoenolpyruvate carboxylase (PEPC) that provides a CO2 high concentration at Rubisco, thus reducing the photorespiration rate. PEPC is regulated by internal metabolite with malate or aspartate as its inhibitors, and glucose-6-phosphate (G-6-P) as its activator. Besides, PEPC also regulated by reversible phosphorylation by protein kinase known as phosphoenolpyruvate carboxylase kinase (PEPCK), that leads to an activation of the enzyme by G-6-P and decrease the sensitivity to malate or aspartate. PEPCK shows a high specificity towards PEPC and the reaction has been reported to be light controlled, but the details of mechanisms of PEPC phosphorylation are still unknown. In this study, a comparative analysis was performed between phosphorylated PEPCs from C3 Panicum pygmaeum and C4 Panicum queenslandicum produced either by PEPCK or Protein Kinase A (PKA). Over-expression of PEPCK with solubility tag NusA had produces soluble protein but in small amounts, and was insufficient for further analysis. Purifying PEPCK without the NusA tag, was unsuccessful because it exists as an insoluble protein. Thus, PKA was applied since it is known to phosphorylate PEPC. The phosphorylation of PEPC by PKA has been confirmed with fluorescence detection, by combining Pro-Q Diamond and SYPRO Ruby gel stain in SDS-PAGE gel. The phosphate affinity Phos-Tag™ was performed subsequently to ensure all PEPCs present were fully phosphorylated. Peptides resulting from the trypsin digestion of Phos-Tag™ SDS-PAGE gels were analysed by mass spectrometry to identify phosphorylation site on PEPC. Two phosphopeptides were detected in the PEPC from P. queenslandicum and six from the P. pygmaeum enzyme. Phosphorylation of PEPC changed the pattern of kinetic enzyme activity, as well as the malate and aspartate sensitivity when compared to the nonphosphorylated form. The enzyme activity (Vmax) of PEPC from the C4 species P. queenslandicum increased once phosphorylated, but this was not observed in the PEPC from the C3 species P. Pygmaeum. In terms of PEP Km, the phosphorylated P. queenslandicum PEPC, had a lower Km value when compared to the nonphosphorylated one. In the P. Pygmeaum PEPC, phosphorylation did not change the Km (PEP) value or the specific activity. Phosphorylation increased the specificity of PEPC to bicarbonate in the enzymes from both P. queenslandicum and P. pygmaeum at pH 8. Phosphorylated PEPC from P. queenslandicum becomes less sensitive to malate and aspartate inhibition at limiting PEP. In P. pygmaeum, phosphorylation of PEPC made it less sensitive to aspartate at limited PEP and to malate at both limited and saturated PEP. Together, these results lead to understanding how the phosphorylation influence the catalytic activity of PEPC in C3 and C4 plant species differently and protects the PEPCs against malate and aspartate inhibition

The Physiological Effects of Phycobilisome Antenna Modification on the Cyanobacterium Synechocystis sp. PCC 6803

Phycobilisomes are the large, membrane extrinsic light harvesting antenna of cyanobacteria. They function to absorb light energy and deliver it efficiently to the photosystems, thereby increasing photosynthetic light absorption. Wild type phycobilisomes in the model organism Synechocystis sp. PCC 6803: Synechocystis 6803) consist of a tricylindrical core from which six rods radiate. The colored phycobiliproteins are held together by colorless linker polypeptides. Several phycobilisome truncation mutants have been generated in Synechocystis 6803. The first, CB, has truncated phycobilisome rods; the second, CK, has only the phycobilisome core; and the third, PAL, has no phycobilisomes at all. Together, these mutants construct a series of increasingly truncated phycobilisomes which are useful for studying the physiology of antenna truncation in cyanobacteria. In this dissertation, the physiological effects of antenna truncation are examined from three perspectives. First, the effect of partial and complete phycobilisome removal on the expression and activity of photosystem II is examined using a variety of assays that center around fluorescence and oxygen evolution. Second, the overall effects of antenna truncation on thylakoid membrane spacing and structure is explored using electron microscopy and small angle neutron scattering. Finally, the effects of antenna truncation on culture-wide biomass productivity are examined in a variety of setting, including a bench-scale photobioreactor. Together, these studies represent a comprehensive examination of the physiological effects of antenna truncation on Synechocystis 6803

Understanding CO₂ diffusion in C₄ plants: An investigation of CO₂ permeable aquaporins and carbonic anhydrase in the C₄ grass Setaria viridis

The productive yield of key C₄ crops must increase in the future to meet the demands of an increasing global population. We are therefore endeavouring to improve the availability of CO₂ for photosynthesis, one of the fundamental limitations to photosynthetic carbon fixation. The initial steps of CO₂ assimilation in leaf mesophyll cells involve the diffusion of CO₂ from the intercellular airspace to the mesophyll cytosol (mesophyll conductance). This involves CO₂ passing through the liquid phase and the plasma membrane, a process believed to be both passive and possibly facilitated by protein pores, known as aquaporins. Within the cytosol of mesophyll cells, carbonic anhydrase (CA) catalyses the hydration of CO₂ to HCO₃- which PEP Carboxylase uses in the first CO₂ fixation step of C₄ photosynthesis. Here, I have examined the role of CO₂ permeable aquaporins and CA from a C₄ photosynthesis perspective using the model monocot species Setaria viridis (Foxtail millet). CO₂ permeable aquaporins have been demonstrated to increase CO₂ diffusion in C3 plants. However, to date very little is known about the role of CO₂ permeable aquaporins in the highly efficient and specialised C₄ photosynthetic pathway. After bioinformatic identification of all twelve Setaria PIPs (plasma membrane intrinsic proteins) I first used yeast as a heterologous expression system to confirm plasma membrane localisation and determine CO₂ permeability of the plasma membrane using CO₂ triggered intracellular acidification on a stopped flow spectrophotometry. This in vitro approach identified SiPIP2;7 as a putative CO₂ permeable aquaporin, adding a third CO₂ pore to the list of C₄ plant aquaporins characterised to date. I also examined the effect of PIP1 and PIP2 co-expression and found improved localisation to the plasma membrane but no improvement to CO₂ permeability compared to the single PIP1s. The effects of modifying CA activity in C₄ photosynthesis was examined in planta. I silenced the major leaf CA in Setaria viridis in three independent, stably transformed lines. At low CO₂ a strong correlation between photosynthetic assimilation rate and CA hydration rates was observed in the transformed lines, which have as little as 13% of wild type CA activity. Significantly, no visual phenotype or photosynthetic effect was observed in the transformed lines at ambient CO₂. C¹⁸O¹⁶O isotope discrimination was used to estimate the mesophyll conductance to CO₂ diffusion from the intercellular air space to the mesophyll cytosol in control plants, which allowed us to calculate CA activities in the mesophyll cytosol. These results indicated that CA is not rate limiting for C₄ photosynthesis in S. viridis under current atmospheric conditions. We conclude that CO₂ permeable aquaporins and CA activity are factors with variable importance to CO₂ diffusion in C₄ photosynthesis, with both factors becoming rate limiting under extreme environmental conditions that result in low intercellular CO₂ such as drought stress

Development of 13C Fingerprint Tool and Its Application for Exploring Carbon and Energy Metabolism in Cyanobacterium Synechocystis sp. PCC 6803

Cyanobacteria are important microbial cell factories that are widely used in the biotechnology filed nowadays. They can use light as the sole energy source to fix CO2, accumulate biomass, and produce various valuable bio-products. Engineered cyanobacterial species can uptake nutrients from wastes to further reduce the cost. Recently, it is reported that cyanobacteria will provide much higher carbon yield than heterotrophs by co-utilizing organic carbons and CO2. However, the quantitative information of such `photo-fermentation\u27 process is still limited. Decoding the carbon metabolism of cyanobacteria during the photo-fermentation process can reveal the functional pathways, carbon distribution, and the energy requirement, all of which will provide guidelines for rational design of metabolic engineering strategies. The emerging of multiple omics tools, e.g. genomics, transcriptomics, proteinomics, and metabolomics analysis, allowed the comprehensive determination of microbial metabolisms. This dissertation describes the development of 13C fingerprint-based method to characterize the carbon metabolic network in cyanobacteria model species Synechocystis sp. PCC 6803 and the integration of this method with metabolic flux analysis and transcriptomics analysis to quantify the diverse carbon and energy metabolism regulation under different internal or external stimuli. The project mainly consists of four aspects: (1) developing the GC-MS based low-cost 13C fingerprint method; (2) exploring the carbon metabolic network structure and quantifying the central carbon metabolism under different environmental conditions; (3) determining the energy requirement for cell maintenance in cyanobacteria; (4) investigating the effects of light conditions on cyanobacterial carbon metabolism. These new findings not only improve our understandings of the flexible carbon metabolism employed by cyanobacteria, but also offer evolutionary insight into photosynthesis and potential applications of photo-fermentation