Evaluating the contribution of plant metabolic pathways in the light to the ATP:NADPH demand using a meta-analysis of isotopically non-stationary metabolic flux analyses.

Balancing the ATP: NADPH demand from plant metabolism with supply from photosynthesis is essential for preventing photodamage and operating efficiently, so understanding its drivers is important for integrating metabolism with the light reactions of photosynthesis and for bioengineering efforts that may radically change this demand. It is often assumed that the C3 cycle and photorespiration consume the largest amount of ATP and reductant in illuminated leaves and as a result mostly determine the ATP: NADPH demand. However, the quantitative extent to which other energy consuming metabolic processes contribute in large ways to overall ATP: NADPH demand remains unknown. Here, we used the metabolic flux networks of numerous recently published isotopically non-stationary metabolic flux analyses (INST-MFA) to evaluate flux through the C3 cycle, photorespiration, the oxidative pentose phosphate pathway, the tricarboxylic acid cycle, and starch/sucrose synthesis and characterize broad trends in the demand of energy across different pathways and compartments as well as in the overall ATP:NADPH demand. These data sets include a variety of species including Arabidopsis thaliana, Nicotiana tabacum, and Camelina sativa as well as varying environmental factors including high/low light, day length, and photorespiratory levels. Examining these datasets in aggregate reveals that ultimately the bulk of the energy flux occurred in the C3 cycle and photorespiration, however, the energy demand from these pathways did not determine the ATP: NADPH demand alone. Instead, a notable contribution was revealed from starch and sucrose synthesis which might counterbalance photorespiratory demand and result in fewer adjustments in mechanisms which balance the ATP deficit.

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.

High Throughput Phosphoglycolate Phosphatase Activity Assay Using Crude Leaf Extract and Recombinant Enzyme to Determine Kinetic Parameters Km and Vmax Using a Microplate Reader.

Determining enzyme activities involved in photorespiration, either in a crude plant tissue extract or in a preparation of a recombinant enzyme, is time-consuming, especially when large number of samples need to be processed. This chapter presents a phosphoglycolate phosphatase (PGLP) activity assay that is adapted for use in a 96-well microplate format. The microplate format for the assay requires fewer enzymes and reagents and allows rapid and less expensive measurement of PGLP enzyme activity. The small volume of reaction mix in a 96-well microplate format enables the determination of PGLP enzyme activity for screening many plant samples, multiple enzyme activities using the same protein extract, and/or identifying kinetic parameters for a recombinant enzyme. To assist in preparing assay reagents, we also present an R Shiny buffer preparation app for PGLP and other photorespiratory enzyme activities and a Km and Vmax calculation app.

High Throughput Glycerate Kinase Activity Assay Using Crude Leaf Extract and Recombinant Enzyme to Determine Kinetic Parameters Km and Vmax Using a Microplate Reader.

We describe an assay for measuring the activity of D-glycerate 3-kinase (GLYK) in a 96-well microplate format with the use of a set of coupling enzymes. The assay is appropriate for use with a crude protein extract prepared from leaf tissue and with the recombinant purified enzyme. The 96-well microplate format reduces the needed amounts of reagents and coupling enzymes, making the assay less expensive, high throughput, and suitable for the determination of kinetic parameters Km and Vmax. In addition, we provide a two-step discontinuous assay modified from past work, making it possible to measure the activity of GLYK at temperatures higher than 45 °C.

Measuring and Quantifying Characteristics of the Post-illumination Burst.

Leaf-level gas exchange enables accurate measurements of net CO2 assimilation in the light, as well as CO2 respiration in the dark. Net positive CO2 assimilation in the light indicates that the gain of carbon by photosynthesis offsets the photorespiratory loss of CO2 and respiration of CO2 in the light (RL), while the CO2 respired in the dark is mainly attributed to respiration in the dark (RD). Measuring the CO2 release specifically from photorespiration in the light is challenging since net CO2 assimilation involves three concurrent processes (the velocity of rubisco carboxylation; vc, velocity of rubisco oxygenation; vo, and RL). However, by employing a rapid light-dark transient, it is possible to transiently measure some of the CO2 release from photorespiration without the background of vc-based assimilation in the dark. This method is commonly known as the post-illumination CO2 burst (PIB) and results in a "burst" of CO2 immediately after the transition to the dark. This burst can be quantitatively characterized using several approaches. Here, we describe how to set up a PIB measurement and provide some guidelines on how to analyze and interpret the data obtained using a PIB analysis application developed in R.

Using Dynamic Gas Exchange Measurements During Oxygen Transients to Study Nonsteady-State Photorespiration.

Leaf-level gas exchange is widely used to investigate the largest carbon fluxes in illuminated leaves, offering a nondestructive way to investigate the impact of photorespiration on plant carbon balance. Modern commercial gas exchange systems allow high temporal resolution measurements under changing environments, aiding the development of nonsteady-state approaches for measuring dynamic photosynthetic responses. Here, we describe a nonsteady-state technique for acquiring the dynamic response of net CO2 assimilation to changes in photorespiratory fluxes manipulated by O2 mole fractions. This technique allows for the screening of plant genotypes with variations in their efficiencies of photorespiration under nonsteady-state conditions.

Combining Gas Exchange and Rapid Quenching of Leaf Tissue for Mass Spectrometry Analysis Directly in Gas Exchange Cuvette.

Isotopically nonstationary metabolic flux analysis (INST-MFA) is a powerful technique for studying plant central metabolism, which involves introducing a 13CO2 tracer to plant leaves and sampling the labeled metabolic intermediates during the transient period before reaching an isotopic steady state. The metabolic intermediates involved in the C3 cycle have exceptionally fast turnover rates, with some intermediates turning over many times a second. As a result, it is necessary to rapidly introduce the label and then rapidly quench the plant tissue to determine concentrations in the light or capture the labeling kinetics of these intermediates at early labeling time points. Here, we describe a rapid quenching (0.1-0.5 s) system for 13CO2 labeling experiments in plant leaves to minimize metabolic changes during labeling and quenching experiments. This system is integrated into a commercially available gas exchange analyzer to measure initial rates of gas exchange, precisely control ambient conditions, and monitor the conversion from 12CO2 to 13CO2.

Using Gas Exchange to Study CO2 Release During Photosynthesis with Steady- and Nonsteady-State Approaches.

Measures of respiration in the light and Ci* are crucial to the modeling of photorespiration and photosynthesis. This chapter provides background on the equations used to model C3 photosynthesis and the history of the incorporation of the effects of rubisco oxygenation into these models. It then describes three methods used to determine two key parameters necessary to incorporate photorespiratory effects into C3 photosynthesis models: respiration in the light (RL) and Ci*. These methods include the Laisk, Yin, and isotopic methods. For the Laisk method, we also introduce a new rapid measurement technique.

Tools for Measuring Photosynthesis at Different Scales.

Measurements of in vivo photosynthesis are powerful tools that probe the largest fluxes of carbon and energy in an illuminated leaf, but often the specific techniques used are so varied and specialized that it is difficult for researchers outside the field to select and perform the most useful assays for their research questions. The goal of this chapter is to provide a broad overview of the current tools available for the study of photosynthesis, both in vivo and in vitro, so as to provide a foundation for selecting appropriate techniques, many of which are presented in detail in subsequent chapters. This chapter will also organize current methods into a comparative framework and provide examples of how they have been applied to research questions of broad agronomical, ecological, or biological importance. This chapter closes with an argument that the future of in vivo measurements of photosynthesis lies in the ability to use multiple methods simultaneously and discusses the benefits of this approach to currently open physiological questions. This chapter, combined with the relevant methods chapters, could serve as a laboratory course in methods in photosynthesis research or as part of a more comprehensive laboratory course in general plant physiology methods.

The Dynamic Assimilation Technique measures photosynthetic CO2 response curves with similar fidelity to steady-state approaches in half the time.

The net CO2 assimilation (A) response to intercellular CO2 concentration (Ci) is a fundamental measurement in photosynthesis and plant physiology research. The conventional A/Ci protocols rely on steady-state measurements and take 15-40 min per measurement, limiting data resolution or biological replication. Additionally, there are several CO2 protocols employed across the literature, without clear consensus as to the optimal protocol or systematic biases in their estimations. We compared the non-steady-state Dynamic Assimilation Technique (DAT) protocol and the three most used CO2 protocols in steady-state measurements, and tested whether different CO2 protocols lead to systematic differences in estimations of the biochemical limitations to photosynthesis. The DAT protocol reduced the measurement time by almost half without compromising estimation accuracy or precision. The monotonic protocol was the fastest steady-state method. Estimations of biochemical limitations to photosynthesis were very consistent across all CO2 protocols, with slight differences in Rubisco carboxylation limitation. The A/Ci curves were not affected by the direction of the change of CO2 concentration but rather the time spent under triose phosphate utilization (TPU)-limited conditions. Our results suggest that the maximum rate of Rubisco carboxylation (Vcmax), linear electron flow for NADPH supply (J), and TPU measured using different protocols within the literature are comparable, or at least not systematically different based on the measurement protocol used.

A guide to photosynthetic gas exchange measurements: Fundamental principles, best practice and potential pitfalls.

Gas exchange measurements enable mechanistic insights into the processes that underpin carbon and water fluxes in plant leaves which in turn inform understanding of related processes at a range of scales from individual cells to entire ecosytems. Given the importance of photosynthesis for the global climate discussion it is important to (a) foster a basic understanding of the fundamental principles underpinning the experimental methods used by the broad community, and (b) ensure best practice and correct data interpretation within the research community. In this review, we outline the biochemical and biophysical parameters of photosynthesis that can be investigated with gas exchange measurements and we provide step-by-step guidance on how to reliably measure them. We advise on best practices for using gas exchange equipment and highlight potential pitfalls in experimental design and data interpretation. The Supporting Information contains exemplary data sets, experimental protocols and data-modelling routines. This review is a community effort to equip both the experimental researcher and the data modeller with a solid understanding of the theoretical basis of gas-exchange measurements, the rationale behind different experimental protocols and the approaches to data interpretation.

Biophysical carbon concentrating mechanisms in land plants: insights from reaction-diffusion modeling.

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 find 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 is predicted to boost net CO2 fixation, but at higher energetic costs than those incurred by photorespiratory losses without a CCM. Two notable exceptions are 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.

The role of photorespiration in preventing feedback regulation via ATP synthase in Nicotiana tabacum.

Photorespiration consumes substantial amounts of energy in the forms of adenosine triphosphate (ATP) and reductant making the pathway an important component in leaf energetics. Because of this high reductant demand, photorespiration is proposed to act as a photoprotective electron sink. However, photorespiration consumes more ATP relative to reductant than the C3 cycle meaning increased flux disproportionally increases ATP demand relative to reductant. Here we explore how energetic consumption from photorespiration impacts the flexibility of the light reactions in nicotiana tabacum. Specifically, we demonstrate that decreased photosynthetic efficiency (ϕII ) at low photorespiratory flux was related to feedback regulation at the chloroplast ATP synthase. Additionally, decreased ϕII at high photorespiratory flux resulted in the accumulation of photoinhibition at photosystem II centers. These results are contrary to the proposed role of photorespiration as a photoprotective electron sink. Instead, our results suggest a novel role of ATP consumption from photorespiration in maintaining ATP synthase activity, with implications for maintaining energy balance and preventing photodamage that will be critical for plant engineering strategies.

Growth in fluctuating light buffers plants against photorespiratory perturbations.

Photorespiration (PR) is the pathway that detoxifies the product of the oxygenation reaction of Rubisco. It has been hypothesized that in dynamic light environments, PR provides a photoprotective function. To test this hypothesis, we characterized plants with varying PR enzyme activities under fluctuating and non-fluctuating light conditions. Contrasting our expectations, growth of mutants with decreased PR enzyme levels was least affected in fluctuating light compared with wild type. Results for growth, photosynthesis and metabolites combined with thermodynamics-based flux analysis revealed two main causal factors for this unanticipated finding: reduced rates of photosynthesis in fluctuating light and complex re-routing of metabolic fluxes. Only in non-fluctuating light, mutants lacking the glutamate:glyoxylate aminotransferase 1 re-routed glycolate processing to the chloroplast, resulting in photooxidative damage through H2O2 production. Our results reveal that dynamic light environments buffer plant growth and metabolism against photorespiratory perturbations.

Increased activity of core photorespiratory enzymes and CO2 transfer conductances are associated with higher and more optimal photosynthetic rates under elevated temperatures in the extremophile Rhazya stricta.

Increase photorespiration and optimising intrinsic water use efficiency are unique challenges to photosynthetic carbon fixation at elevated temperatures. To determine how plants can adapt to facilitate high rates of photorespiration at elevated temperatures while also maintaining water-use efficiency, we performed in-depth gas exchange and biochemical assays of the C3 extremophile, Rhazya stricta. These results demonstrate that R. stricta supports higher rates of photorespiration under elevated temperatures and that these higher rates of photorespiration correlate with increased activity of key photorespiratory enzymes; phosphoglycolate phosphatase and catalase. The increased photorespiratory enzyme activities may increase the overall capacity of photorespiration by reducing enzymatic bottlenecks and allowing minimal inhibitor accumulation under high photorespiratory rates. Additionally, we found the CO2 transfer conductances (stomatal and mesophyll) are re-allocated to increase the water-use efficiency in R. stricta but not necessarily the photosynthetic response to temperature. These results suggest important adaptive strategies in R. stricta that maintain photosynthetic rates under elevated temperatures with optimal water loss. The strategies found in R. stricta may inform breeding and engineering efforts in other C3 species to improve photosynthetic efficiency at high temperatures.

Energetic considerations for engineering novel biochemistries in photosynthetic organisms.

Humans have been harnessing biology to make valuable compounds for generations. From beer and biofuels to pharmaceuticals, biology provides an efficient alternative to industrial processes. With the continuing advancement of molecular tools to genetically modify organisms, biotechnology is poised to solve urgent global problems related to environment, increasing population, and public health. However, the light dependent reactions of photosynthesis are constrained to produce a fixed stoichiometry of ATP and reducing equivalents that may not match the newly introduced synthetic metabolism, leading to inefficiency or damage. While photosynthetic organisms have evolved several ways to modify the ATP/NADPH output from their thylakoid electron transport chain, it is unknown if the native energy balancing mechanisms grant enough flexibility to match the demands of the synthetic metabolism. In this review we discuss the role of photosynthesis in the biotech industry, and the energetic considerations of using photosynthesis to power synthetic biology.

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.

The role of photorespiration in plant immunity.

To defend themselves in the face of biotic stresses, plants employ a sophisticated immune system that requires the coordination of other biological and metabolic pathways. Photorespiration, a byproduct pathway of oxygenic photosynthesis that spans multiple cellular compartments and links primary metabolisms, plays important roles in defense responses. Hydrogen peroxide, whose homeostasis is strongly impacted by photorespiration, is a crucial signaling molecule in plant immunity. Photorespiratory metabolites, interaction between photorespiration and defense hormone biosynthesis, and other mechanisms, are also implicated. An improved understanding of the relationship between plant immunity and photorespiration may provide a much-needed knowledge basis for crop engineering to maximize photosynthesis without negative tradeoffs in plant immunity, especially because the photorespiratory pathway has become a major target for genetic engineering with the goal to increase photosynthetic efficiency.

Integrated flux and pool size analysis in plant central metabolism reveals unique roles of glycine and serine during photorespiration.

Photorespiration is an essential process juxtaposed between plant carbon and nitrogen metabolism that responds to dynamic environments. Photorespiration recycles inhibitory intermediates arising from oxygenation reactions catalysed by Rubisco back into the C3 cycle, but it is unclear what proportions of its nitrogen-containing intermediates (glycine and serine) are exported into other metabolisms in vivo and how these pool sizes affect net CO2 gas exchange during photorespiratory transients. Here, to address this uncertainty, we measured rates of amino acid export from photorespiration using isotopically non-stationary metabolic flux analysis. This analysis revealed that ~23-41% of the photorespiratory carbon was exported from the pathway as serine under various photorespiratory conditions. Furthermore, we determined that the build-up and relaxation of glycine pools constrained a large portion of photosynthetic acclimation during photorespiratory transients. These results reveal the unique and important roles of glycine and serine in successfully maintaining various photorespiratory fluxes that occur under environmental fluctuations in nature and providing carbon and nitrogen for metabolism.