Modeling with uncertainty quantification reveals the essentials of a non-canonical algal carbon-concentrating mechanism.

The thermoacidophilic red alga Cyanidioschyzon merolae survives its challenging environment likely in part by operating a carbon-concentrating mechanism (CCM). Here, we demonstrated that C. merolae's cellular affinity for CO2 is stronger than the affinity of its rubisco for CO2.

Resilient plants, sustainable future.

The accelerated pace of climate change over the past several years should serve as a wake-up call for all scientists, farmers, and decision makers, as it severely threatens our food supply and could result in famine, migration, war, and an overall destabilization of our society. Rapid and significant changes are therefore needed in the way we conduct research on plant resilience, develop new crop varieties, and cultivate those crops in our agricultural systems. Here, we describe the main bottlenecks for these processes and outline a set of key recommendations on how to accelerate research in this critical area for our society.

Increasing thermostability of the key photorespiratory enzyme glycerate 3-kinase by structure-based recombination.

As global temperatures rise, improving crop yields will require enhancing the thermotolerance of crops. One approach for improving thermotolerance is using bioengineering to increase the thermostability of enzymes catalysing essential biological processes. Photorespiration is an essential recycling process in plants that is integral to photosynthesis and crop growth.

Rubisco activity and activation state dictate photorespiratory plasticity in Betula papyrifera acclimated to future climate conditions.

Plant metabolism faces a challenge of investing enough enzymatic capacity to a pathway without overinvestment. As it takes energy and resources to build, operate, and maintain enzymes, there are benefits and drawbacks to accurately matching capacity to the pathway influx. The relationship between functional capacity and physiological load could be explained through symmorphosis, which would quantitatively match enzymatic capacity to pathway influx.

Metabolomics of related C3 and C4 Flaveria species indicate differences in the operation of photorespiration under fluctuating light.

C photosynthesis can be complemented with a C carbon concentrating mechanism (CCM) to minimize photorespiratory losses. C photosynthesis is often more efficient than C under steady-state conditions. However, the C CCM depends on inter-cellular metabolite concentration gradients, which must increase following increases in light intensity and could decrease rates of C photosynthesis under fluctuating light.

Photorespiratory glycine contributes to photosynthetic induction during low to high light transition.

Leaves experience near-constant light fluctuations daily. Past studies have identified many limiting factors of slow photosynthetic induction when leaves transition from low light to high light. However, the contribution of photorespiration in influencing photosynthesis during transient light conditions is largely unknown.

Formate-tetrahydrofolate ligase: supplying the cytosolic one-carbon network in roots with one-carbon units originating from glycolate.

The metabolism of tetrahydrofolate (HPteGlu)-bound one-carbon (C) units (C metabolism) is multifaceted and required for plant growth, but it is unclear what of many possible synthesis pathways provide C units in specific organelles and tissues. One possible source of C units is via formate-tetrahydrofolate ligase, which catalyzes the reversible ATP-driven production of 10-formyltetrahydrofolate (10-formyl-HPteGlu) from formate and tetrahydrofolate (HPteGlu). Here, we report biochemical and functional characterization of the enzyme from Arabidopsis thaliana (AtFTHFL).

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.

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 CO 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 C 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.

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 CO assimilation to changes in photorespiratory fluxes manipulated by O mole fractions.

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

Measures of respiration in the light and C* are crucial to the modeling of photorespiration and photosynthesis. This chapter provides background on the equations used to model C 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 C photosynthesis models: respiration in the light (R) and C*.

Measuring and Quantifying Characteristics of the Post-illumination Burst.

Leaf-level gas exchange enables accurate measurements of net CO assimilation in the light, as well as CO respiration in the dark. Net positive CO assimilation in the light indicates that the gain of carbon by photosynthesis offsets the photorespiratory loss of CO and respiration of CO in the light (R), while the CO respired in the dark is mainly attributed to respiration in the dark (R). Measuring the CO release specifically from photorespiration in the light is challenging since net CO assimilation involves three concurrent processes (the velocity of rubisco carboxylation; v, velocity of rubisco oxygenation; v, and R).

High Throughput Glycerate Kinase Activity Assay Using Crude Leaf Extract and Recombinant Enzyme to Determine Kinetic Parameters K and V 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 K and V.

High Throughput Phosphoglycolate Phosphatase Activity Assay Using Crude Leaf Extract and Recombinant Enzyme to Determine Kinetic Parameters K and V 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.

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 (PCCM) of Chlamydomonas reinhardtii or carboxysome systems of cyanobacteria, are common in aquatic photosynthetic microbes, but in land plants appear only among the hornworts.

Re-evaluating the energy balance of the many routes of carbon flow through and from photorespiration.

Photorespiration is an essential process related to photosynthesis that is initiated following the oxygenation reaction catalyzed by rubisco, the initial enzyme of the Calvin-Benson-Bassham cycle. This reaction produces an inhibitory intermediate that is recycled back into the Calvin-Benson-Bassham cycle by photorespiration which requires the use of energy and the release of a portion of the carbon as CO. The energy use and CO release of canonical photorespiration form a foundation for biochemical models used to describe and predict leaf carbon exchange and energy use (ATP and NAPDH).

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.

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.

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.

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 CO around the carbon-fixing enzyme rubisco, thereby increasing CO assimilatory flux and reducing photorespiration. Biophysical CCMs, like the pyrenoid-based CCM of or carboxysome systems of cyanobacteria, are common in aquatic photosynthetic microbes, but in land plants appear only among the hornworts.

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.

Increased activity of core photorespiratory enzymes and CO 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 C extremophile, Rhazya stricta. These results demonstrate that R.

Laisk measurements in the nonsteady state: Tests in plants exposed to warming and variable CO2 concentrations.

Light respiration (RL) is an important component of plant carbon balance and a key parameter in photosynthesis models. RL is often measured using the Laisk method, a gas exchange technique that is traditionally employed under steady-state conditions. However, a nonsteady-state dynamic assimilation technique (DAT) may allow for more rapid Laisk measurements.