Engineering topics

Walker, Berkley J.

Publications and source records attributed to Walker, Berkley J..

9 records

DOE OSTI2024

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. 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.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

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. 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 CO 2 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.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

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

C 3 photosynthesis can be complemented with a C 4 carbon concentrating mechanism (CCM) to minimize photorespiratory losses. C 4 photosynthesis is often more efficient than C 3 under steady-state conditions. However, the C 4 CCM depends on inter-cellular metabolite concentration gradients, which must increase following increases in light intensity and could decrease rates of C 4 photosynthesis under fluctuating light. Additionally, incomplete flux through photorespiration could prove beneficial to C 4 assimilation during light induction of the CCM. Here, we compare metabolic profiles in the closely related C 3 Flaveria robusta and C 4 Flaveria bidentis during a light transient from low to high light to determine if these non-steady state accumulation patterns provide insight to the induction of the metabolite gradients needed to drive C 4 intermediate transport and if there is incomplete cycling of photorespiratory intermediates. In these C 3 and C 4 species, metabolite steady-state pool sizes suggest that C 4 transport acids maintain concentration gradients across the bundle sheath and mesophyll cell types under these light fluctuations. However, there was incomplete flux through photorespiration in the C 4 F. bidentis, which could reduce photorespiratory CO 2 loss via glycine decarboxylation and help maintain higher rates of assimilation during following induction periods.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

The Dynamic Assimilation Technique measures photosynthetic CO 2 response curves with similar fidelity to steady-state approaches in half the time

The net CO 2 assimilation (A) response to intercellular CO 2 concentration (C i ) is a fundamental measurement in photosynthesis and plant physiology research. The conventional A/C i protocols rely on steady-state measurements and take 15–40 min per measurement, limiting data resolution or biological replication. Additionally, there are several CO 2 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 CO 2 protocols in steady-state measurements, and tested whether different CO 2 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 CO 2 protocols, with slight differences in Rubisco carboxylation limitation. The A/C i curves were not affected by the direction of the change of CO 2 concentration but rather the time spent under triose phosphate utilization (TPU)-limited conditions. Our results suggest that the maximum rate of Rubisco carboxylation (V cmax ), 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.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

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

A/Ci response↗

DOE OSTI2023

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

Abstract 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 (ϕ II ) at low photorespiratory flux was related to feedback regulation at the chloroplast ATP synthase. Additionally, decreased ϕ II 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.

Plant Sciences↗

DOE OSTI2023

Photosynthesis and rhizome carbohydrate concentrations of switchgrass grown from reserve-depleted rhizomes

A long-standing question in perennial grass breeding and physiology is whether yield improvement strategies could compromise winter survival. Perennial grasses rely on the pool of carbohydrates accumulated in storage organs from the previous growing season for winter maintenance and spring regrowth. Yield improvement strategies could reduce winter survival if they increase biomass and grain yields at the expense of carbon allocation to storage. Therefore, it is crucial to better understand the dependence of regrowth on storage reserves. We experimentally depleted switchgrass ( Panicum virgatum L.) rhizome reserves by storing rhizomes for two weeks at 5 °C (control treatment) and 25 °C (reserve-depleted treatment).

bioenergy↗

DOE OSTI2023

Increased activity of core photorespiratory enzymes and CO 2 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 3 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 CO 2 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 C 3 species to improve photosynthetic efficiency at high temperatures.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

A 30% reduction in switchgrass rhizome reserves did not decrease biomass yield

Abstract A long‐standing question in perennial grass breeding and physiology is whether yield improvement strategies could compromise winter survival. Since perennial grasses rely on stored carbohydrates for winter maintenance and spring regrowth, yield improvement strategies could reduce winter survival if they increase biomass and grain yields at the expense of carbon allocation to storage. Therefore, it is crucial to comprehend the dependence of regrowth on storage reserves. We experimentally depleted switchgrass ( Panicum virgatum L.) rhizome reserves by storing rhizomes for 2 weeks at 5°C (control treatment) and 25°C (reserve‐depleted treatment). During the storage period rhizome respiration was 5.3× higher at 25°C (0.010 μmol CO 2 g −1 min −1 at 5°C vs. 0.054 μmol CO 2 g −1 min −1 at 25°C; p < 0.0001) and the starch content was depleted by 30% by the end of storage. Surprisingly, reserve‐depleted switchgrass had 60% larger leaf area (LA; LA control = 149 cm 2 pot −1 vs. LA depleted = 239 cm 2 pot −1 ; p = 0.013) and produced ~40% more aboveground biomass than control plants (9.46 g pot −1 vs. 6.63 g pot −1 ; p = 0.112). In addition, reserve‐depleted switchgrass restored its rhizome starch reserves to pre‐storage levels. Switchgrass showed a large plasticity among its source‐sink components to buffer the imposed reserve depletion. It increased plant photosynthesis by increasing the photosynthetic leaf area while keeping photosynthesis constant on a leaf area basis and readjusted the timing and activity of sink organs. These results suggest that switchgrass, and potentially other perennial grasses, largely over‐invest in storage reserves. Therefore, current breeding strategies in perennial grasses aimed to extend the aboveground growing season should not compromise crop persistence. Our study also has implications on long‐term yield dynamics as it highlights sink limitations as potential driver of the yield decline commonly observed in perennial grasses 5+ years after cultivation.

Tejera‐Nieves, Mauricio↗