Ludmila V Roze’s research while affiliated with Michigan State University and other places

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. The enzymes of photorespiration are targets for enhancing plant thermotolerance as this pathway limits carbon fixation at elevated temperatures. We explored the effects of temperature on the activity of the photorespiratory enzyme glycerate kinase (GLYK) from various organisms and the homologue from the thermophilic alga Cyanidioschyzon merolae was more thermotolerant than those from mesophilic plants, including Arabidopsis thaliana. To understand enzyme features underlying the thermotolerance of C. merolae GLYK (CmGLYK), we performed molecular dynamics simulations using AlphaFold‐predicted structures, which revealed greater movement of loop regions of mesophilic plant GLYKs at higher temperatures compared to CmGLYK. Based on these simulations, hybrid proteins were produced and analysed. These hybrid enzymes contained loop regions from CmGLYK replacing the most mobile corresponding loops of AtGLYK. Two of these hybrid enzymes had enhanced thermostability, with melting temperatures increased by 6 °C. One hybrid with three grafted loops maintained higher activity at elevated temperatures. Whilst this hybrid enzyme exhibited enhanced thermostability and a similar Km for ATP compared to AtGLYK, its Km for glycerate increased threefold. This study demonstrates that molecular dynamics simulation‐guided structure‐based recombination offers a promising strategy for enhancing the thermostability of other plant enzymes with possible application to increasing the thermotolerance of plants under warming climates.

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.

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.

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.

As global temperatures rise, maintaining and improving crop yields will require enhancing the thermotolerance of crops. One approach for improving thermotolerance is using bioengineering to increase the thermostability of enzymes catalyzing essential biological processes. Photorespiration is an essential recycling process in plants that is integral to photosynthesis and crop growth. The enzymes of photorespiration are targets for enhancing plant thermotolerance as this pathway limits carbon fixation at elevated temperatures. Exploring inter-specific variation of the key photorespiratory enzyme glycerate kinase (GLYK) from various photosynthetic organisms, we found that the homolog from the thermophilic alga Cyanidioschyzon merolae was more thermotolerant than those from mesophilic plants, including Arabidopsis thaliana. To understand factors influencing thermotolerance of C. merolae GLYK (CmGLYK), we performed molecular dynamics simulations using AlphaFold-predicted structures, which revealed greater movement of loop regions of mesophilic plant GLYKs at higher temperatures compared to CmGLYK. Based on these simulations, a series of hybrid proteins were produced and analyzed. These hybrid enzymes contained selected loop regions from CmGLYK replacing the most highly mobile corresponding loops of AtGLYK. Two of these hybrid enzymes had enhanced thermostability, with melting temperatures increased by 6 C. One hybrid with three grafted loops maintained higher activity at elevated temperatures. While this hybrid enzyme exhibited enhanced thermostability and a similar Km for ATP compared to AtGLYK, its Km for glycerate increased threefold. This study demonstrates that molecular dynamics simulation-guided structure-based recombination offers a promising strategy for enhancing thermostability of other plant enzymes.

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.