Luke M. Gregory’s research while affiliated with Michigan State University and other places

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. Alternatively, plants could maintain excess enzymatic capacity to manage unpredictable pathway influx. In this study, we use photorespiration as a case study to investigate these two hypotheses in Betula papyrifera. This involves altering photorespiratory influx by manipulating the growth environment, via changes in CO2 concentration and temperature, to determine how photorespiratory capacity acclimates to environmental treatments. Surprisingly, the results from these measurements indicate that there is no plasticity in photorespiratory capacity in B. papyrifera, and that a fixed capacity is maintained under each growth condition. The fixed capacity is likely due to the existence of reserve capacity in the pathway that manages unpredictable photorespiratory influx in dynamic environments. Additionally, we found that B. papyrifera had a constant net carbon assimilation under each growth condition due to an adjustment of functional rubisco activity driven by changes in activation state. These results provide insight into the acclimation ability and limitations of B. papyrifera to future climate scenarios currently predicted in the next century.

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.

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.

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.

The initial reaction of CO2 fixation has a shared specificity with oxygen, resulting in the formation of 2-phosphoglycolate, an inhibitory sugar. 2-phosphoglycolate is detoxified by photorespiration, but this recycling requires energy and releases carbon, substantially reducing rates of net CO2 fixation in many crop plants. Many strategies are in development to modify native photorespiration and more efficiently detoxify intermediates of photorespiration to boost subsequent net carbon assimilation and crop productivity. Current approaches include increasing the activities of enzymes present in native photorespiration to minimize the pool sizes of inhibitory intermediates or replacing native photorespiration with novel pathways that process 2-phosphoglycolate with improved carbon-conserving, or even fixing, series of reactions. There are also developments in improving photorespiration under elevated temperatures, which occur under typical growing conditions but are outside the scope of many lab-based studies. We finally discuss the potential for improving photorespiration under non-steady-state conditions, which are a hallmark of field crop production systems.

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.

The effect of including triose phosphate utilization (TPU) on parameterization of the Farquhar, von Caemmerer, Berry model of photosynthetic gas exchange measurements is explored. Better fits to data are found even though TPU rarely limits photosynthesis under physiological conditions.

Premise: Leaf morphology is dynamic, continuously deforming during leaf expansion and among leaves within a shoot. Here, we measured the leaf morphology of more than 200 grapevines (Vitis spp.) over four years and modeled changes in leaf shape along the shoot to determine whether a composite leaf shape comprising all the leaves from a single shoot can better capture the variation and predict species identity compared with individual leaves. Methods: Using homologous universal landmarks found in grapevine leaves, we modeled various morphological features as polynomial functions of leaf nodes. The resulting functions were used to reconstruct modeled leaf shapes across the shoots, generating composite leaves that comprehensively capture the spectrum of leaf morphologies present. Results: We found that composite leaves are better predictors of species identity than individual leaves from the same plant. We were able to use composite leaves to predict the species identity of previously unassigned grapevines, which were verified with genotyping. Discussion: Observations of individual leaf shape fail to capture the true diversity between species. Composite leaf shape-an assemblage of modeled leaf snapshots across the shoot-is a better representation of the dynamic and essential shapes of leaves, in addition to serving as a better predictor of species identity than individual leaves.