Search

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. This study employs dynamic measurements of gas exchange and metabolic responses to examine the contribution of photorespiration in constraining net rates of carbon assimilation during light induction. This work indicates that photorespiratory glycine accumulation during the early light induction contributes 5-7% to the additional carbon fixed relative to the low light conditions. Mutants with large glycine pools under photorespiratory conditions (5-formyl THF cycloligase and hydroxypyruvate reductase 1) showed a transient spike in net CO 2 assimilation during light induction, with glycine buildup accounting for 22-36% of the extra carbon assimilated. Interestingly, levels of many C 3 cycle intermediates remained relatively constant in both mutants and wild-type throughout the light induction period where glycine accumulated, indicating that recycling of carbon into the C 3 cycle via photorespiration is not needed to maintain C3 cycle activity under transient conditions. Furthermore, our data show that oxygen transient experiments can be used as a proxy to identify the photorespiratory component of light-induced photosynthetic changes., (© 2024. The Author(s).)

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 K m and V max calculation app., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 2 assimilation to changes in photorespiratory fluxes manipulated by O 2 mole fractions. This technique allows for the screening of plant genotypes with variations in their efficiencies of photorespiration under nonsteady-state conditions., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 m and V max . 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., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Isotopically nonstationary metabolic flux analysis (INST-MFA) is a powerful technique for studying plant central metabolism, which involves introducing a 13 CO 2 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 3 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 13 CO 2 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 12 CO 2 to 13 CO 2 ., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Rapidly changing light conditions can reduce carbon gain and productivity in field crops because photosynthetic responses to light fluctuations are not instantaneous. Plant responses to fluctuating light occur across levels of organizational complexity from entire canopies to the biochemistry of a single reaction and across orders of magnitude of time. Although light availability and variation at the top of the canopy are largely dependent on the solar angle and degree of cloudiness, lower crop canopies rely more heavily on light in the form of sunflecks, the quantity of which depends mostly on canopy structure but also may be affected by wind. The ability of leaf photosynthesis to respond rapidly to these variations in light intensity is restricted by the relatively slow opening/closing of stomata, activation/deactivation of C 3 cycle enzymes, and up-regulation/down-regulation of photoprotective processes. The metabolic complexity of C 4 photosynthesis creates the apparently contradictory possibilities that C 4 photosynthesis may be both more and less resilient than C 3 to dynamic light regimes, depending on the frequency at which these light fluctuations occur. We review the current understanding of the underlying mechanisms of these limitations to photosynthesis in fluctuating light that have shown promise in improving the response times of photosynthesis-related processes to changes in light intensity., (© 2018 American Society of Plant Biologists. All Rights Reserved.)