Thomas David Sharkey’s research while affiliated with Michigan State University and other places

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Publications (18)


Central carbon metabolic fluxes in photosynthetic Camelina sativa leaves under control (a) and high light high CO2 conditions (b). Fluxes, indicated by variable arrow width, are presented as numbers. Comparison between control and high light high CO2 fluxes is based on 95% confidence intervals by parameter continuation analysis, with significant differences highlighted by orange arrows. Flux units: μmol.metabolite.g⁻¹.FW.hr⁻¹. The model network is compartmentalized into cytosol (“.c”), chloroplast (“.p”), mitochondrion (“.m”), and vacuole (“.v”). See supplementary Table S5 for abbreviations.
(a) Heatmap of ¹³C enrichment of measured metabolites in plants grown in control and high light high CO2 conditions in a 0–30 min labeling experiment. The color bar from blue to red represents the value of ¹³C enrichment from low to high. (b) ¹³C enrichment of key metabolites in plants grown in control and high light high CO2 conditions in a 0–30 min labeling experiment (n = 3, ± standard deviation). See supplementary Table S5 for abbreviations. (b1) Metabolites that demonstrate faster labeling in HLHC at early time points, plateauing to a level similar to the control condition by 30 min. (b2) Metabolites that exhibit faster and higher ¹³C label incorporation under HLHC conditions. (b3) Metabolites that exhibit slower and lower ¹³C label incorporation under HLHC conditions. The x-axis represents the time of labeling (in minutes), while the y-axis represents the fraction of ¹³C enrichment. Monoisotopic mass of unlabeled fragment ions are shown in parentheses.
Averaged A/Cc data fitting points for four leaves and rate determining capacities determined by curve fitting. Data from four leaves were fit with the fitting routine available in Sharkey et al.⁷⁴ or from the authors and yielded seven fitting parameters. The averages of these fitted parameters were used to generate an averaged A/Cc curve (black circles). The blue line represents the classical RuBP regeneration limitation, while the reddish lines depict an alternative interpretation focusing on Rubisco activation limitation. The solid black arrow indicates the operating Cc under normal light and CO2 conditions while the dashed black arrow indicates the Cc at the high light and CO2 operational point. At normal light and CO2, the plants were operating very close to the crossover point where both Vcmax and J determine the rate. The middle Vcmax line, deactivated 1, assumed the rubisco activation state was 86% (chosen to allow the Vcmax line, to meet the operational point). The operating point at high light and high CO2 (dashed arrow) is very close to the junction of J and TPU. Rubisco activation state had to be reduced to 73% to match the photosynthetic rate at the operating point in high light and CO2. This is shown in the right-most Vcmax line labeled Deactivated 2.
The effects of photosynthetic rate on respiration in light, starch/sucrose partitioning, and other metabolic fluxes within photosynthesis
  • Article
  • Full-text available

March 2025

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47 Reads

Yuan Xu

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Sean E. Weise

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Thomas D. Sharkey

In the future, plants may encounter increased light and elevated CO2 levels. How consequent alterations in photosynthetic rates will impact fluxes in photosynthetic carbon metabolism remains uncertain. Respiration in light (RL) is pivotal in plant carbon balance and a key parameter in photosynthesis models. Understanding the dynamics of photosynthetic metabolism and RL under varying environmental conditions is essential for optimizing plant growth and agricultural productivity. However, measuring RL under high light and high CO2 (HLHC) conditions poses challenges using traditional gas exchange methods. In this study, we employed isotopically nonstationary metabolic flux analysis (INST-MFA) to estimate RL and investigate photosynthetic carbon flux, unveiling nuanced adjustments in Camelina sativa under HLHC. Despite numerous flux alterations in HLHC, RL remained stable. HLHC affects several factors influencing RL, such as starch and sucrose partitioning, vo/vc ratio, triose phosphate partitioning, and hexose kinase activity. Analysis of A/Ci curve operational points reveals that HLHC’s major changes primarily stem from CO2 suppressing photorespiration. Integration of these fluxes into a simplified model predicts changes in CBC labeling under HLHC. This study extends our prior discovery that incomplete CBC labeling is due to unlabeled carbon reimported during RL, offering insights into manipulating labeling through adjustments in photosynthetic rates.

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Fig. 4. Change in hormone levels in NE and IE leaves after worm feeding. Hormones were
Figures:
Isoprene deters insect herbivory by priming plant hormone responses

November 2024

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53 Reads

Isoprene, emitted by some plants, enhances plant abiotic resilience but its role in biotic stress resilience remains elusive. We used tobacco plants engineered to emit isoprene (IE) and the corresponding azygous non-emitting control (NE) to investigate isoprene emission and biotic stress resilience. IE plants were more resistant to insect herbivory than NE plants. Worms preferred to feed on NE rather than IE leaves. IE plants showed less decline in photosynthesis during worm feeding. Insect feeding increased jasmonate levels in IE leaves, suggesting isoprene-mediated priming of the jasmonic acid response. Wound-induced increase in isoprene emission corresponded with elevation of methyl-D-erythritol-4-phosphate pathway and Calvin-Benson cycle metabolites. The results highlight interactive functions of isoprene and jasmonic acid and advance our understanding of how isoprene emission enhances plant resilience.


Short-term salt stress reduces photosynthetic oscillations under triose phosphate utilization limitation in tomato

March 2024

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108 Reads

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1 Citation

Journal of Experimental Botany

Triose phosphate utilization (TPU) limitation is one of the three biochemical limitations of photosynthetic CO2 assimilation rate in C3 plants. Under TPU limitation, abrupt and large transitions in light intensity cause damped oscillations in photosynthesis. When plants are salt-stressed, photosynthesis is often down-regulated particularly under dynamic light intensity, but how salt stress affects TPU-related dynamic photosynthesis is still unknown. To elucidate this, tomato (Solanum lycopersicum) was grown with and without sodium chloride (NaCl, 100 mM) stress for 13 days. Under high CO2 partial pressure, rapid increases in light intensity caused profound photosynthetic oscillations. Salt stress reduced photosynthetic oscillations in leaves initially under both low- or high-light conditions and reduced the duration of oscillations by about two minutes. Besides, salt stress increased the threshold for CO2 partial pressure at which oscillations occurred. Salt stress increased TPU capacity without affecting Rubisco carboxylation and electron transport capacity, indicating the upregulation in end-product synthesis capacity in photosynthesis. Thus salt stress may reduce photosynthetic oscillations by either decreasing leaf internal CO2 partial pressure and/or increasing TPU capacity. Our results thus provide new insights in how salt stress modulates dynamic photosynthesis as controlled by CO2 availability and end-product synthesis.


Figure 2 Levels of MEP metabolites in the roots of non-emitting (EV) and emitting (B2 and C4)
Figure 4 Effects of root isoprene emission on the levels of stress-associated amino acids in the
Figure 5 The transcriptome profiles of isoprene non-emitter wild-type (WT) and empty vector
Figure 6 Differential expressed genes (DEGs) of isoprene non-emitter wild-type (WT) and
The effect of constitutive root isoprene emission on root phenotype and physiology under control and salt stress conditions

February 2024

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80 Reads

Isoprene, a volatile hydrocarbon, is typically emitted from the leaves and other aboveground plant organs; isoprene emission from roots is not well studied. Given its well-known function in plant growth and defense aboveground, isoprene may also be involved in shaping root physiology to resist belowground stress. We used isoprene-emitting transgenic lines (IE) and a non-emitting empty vector and/or wild type lines (NE) of Arabidopsis to elucidate the roles of isoprene in root physiology and salt stress resistance. We assessed root phenotype and metabolic changes, hormone biosynthesis and signaling, and stress-responses under normal and saline conditions of IE and NE lines. We also analyzed the root transcriptome in the presence or absence of salt stress. IE lines emitted isoprene from roots, which was associated with higher primary root growth, root biomass, and root/shoot biomass ratio under both control and salt stress conditions. Transcriptome data indicated that isoprene altered the expression of key genes involved in hormone metabolism and plant responses to stress factors. Our findings reveal that root constitutive isoprene emission sustains root growth also under salinity by regulating and/or priming hormone biosynthesis and signaling mechanisms, amino acids biosynthesis, and expression of key genes relevant to salt stress defense.


Rubisco supplies pyruvate for the 2-C-methyl-D-erythritol-4-phosphate pathway in Arabidopsis

January 2024

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25 Reads

Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) produces pyruvate in the chloroplast through beta-elimination of the aci-carbanion intermediate. Here we show that this side reaction supplies pyruvate for isoprenoid, fatty acid, and branched chain amino acid biosynthesis in photosynthetically active tissue. 13C labeling studies of whole Arabidopsis plants demonstrate that the total carbon commitment to pyruvate is too large for phosphoenolpyruvate (PEP) to serve as precursor. Low oxygen stimulates rubisco carboxylase activity and increased pyruvate production and flux through the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, which supplies the precursors for plastidic isoprenoid biosynthesis. Metabolome analysis of mutants defective in PEP or pyruvate import further supported rubisco as the main source of pyruvate in chloroplasts. Rubisco beta-elimination leading to pyruvate constituted 0.7% of the product profile in in vitro assays, which translates to 2% of the total carbon leaving the Calvin-Benson-Bassham cycle (CBC). These insights solve the so-called pyruvate paradox, improve the fit of metabolic models for central metabolism, and connect the MEP pathway directly to carbon assimilation.


The Discovery of Rubisco

June 2022

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85 Reads

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29 Citations

Journal of Experimental Botany

Rubisco is possibly the most important enzyme on Earth, certainly in terms of amount. This review describes the initial reports of ribulose 1,5-bisphosphate carboxylating activity. Discoveries of core concepts are described including its quaternary structure, the requirement for post-translational modification, and its role as an oxygenase as well as carboxylase. Finally, the requirement for numerous chaperonins for assembly of rubisco in plants is described.


Sites of cofactor consumption and production in central carbon metabolism. Previously, we estimated carbon fluxes in 4-week-old photosynthesising Camelina sativa leaves (Xu et al. 2022). According to this analysis, pathways in black carry significant flux. Pathways in grey can be expected to carry significant flux but were not considered in our previous analysis. Enzymes: 6PGD, 6-phosphogluconate dehydrogenase; ACC, acetyl-CoA carboxylase; ACPr, 2,3-trans-enoyl-ACP reductase; AGPase, ADP-glucose pyrophosphorylase; FK, fructokinase; G6PD, glucose-6-phosphate dehydrogenase; GAPC (cytosolic) and GAPDH (chloroplastic), phosphorylating glyceraldehyde-3-phosphate dehydrogenase; GAPN, non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase; GDC, glycine decarboxylase complex; GDH, glutamate dehydrogenase; GK, glycerate kinase; GOGAT, glutamine-α-ketoglutarate aminotransferase; GS, glutamine synthetase; HK, hexokinase; HPR, hydroxypyruvate reductase; IDH, isocitrate dehydrogenase; KAR, 3-ketoacyl-ACP reductase; PDC, pyruvate dehydrogenase complex; PGK, phosphoglycerate kinase; PK, pyruvate kinase; PRK, phosphoribulokinase; UGPase, UDP-glucose pyrophosphorylase. Metabolites: 1,3BPG, 1,3-bisphosphoglycerate; 2PGA, 2-phosphoglycerate; 3PGA, 3-phosphoglycerate; 6PG, 6-phosphogluconate; 6PGL, 6-phosphogluconolactone; ACA, acetyl coenzyme A; ADPG, ADP-glucose; AKG, α-ketoglutarate; F6P, fructose 6-phosphate; FRU, fructose; G1P, glucose 1-phosphate; G6P, glucose 6-phosphate; GA, glycerate; GLC, glucose; GLN, glutamine; GLO, glyoxylate; GLU, glutamate; GLY, glycine; HP, hydroxypyruvate; ICT, isocitrate; PEP, phosphoenolpyruvate; PYR, pyruvate; Ru5P, ribulose 5-phosphate; RuBP, ribulose 1,5-bisphosphate; SER, serine; SUC, sucrose; TP, triose phosphate (glyceraldehyde 3-phosphate and dihydroxyacetone phosphate); UDPG, UDP-glucose
Compartment-specific cofactor consumption (negative values) and production (positive values) by central carbon metabolism in 4-week-old photosynthesising Camelina sativa leaves. The figure shows the net effect of all the processes listed in Table 2. Error bars represent 95% confidence intervals. 1, malate valves exchange NADH through malate-oxaloacetate interconversion by malate dehydrogenase (Selinski and Scheibe 2019). A chloroplastic isoform accepts NADPH. 2 and 3, cofactor transfer through redox cycles proposed by Kelly and Gibbs (1973a) and Stocking and Larson (1969), respectively. 4, counter-exchange of cytosolic ADP for mitochondrial ATP by the ADP/ATP carrier (Klingenberg 2008). 5, conversion of ATP and NADH to NADPH by the cytosolic oxidation–reduction cycle (Wieloch 2021). 6, NADH to ATP conversion by oxidative phosphorylation. 7, conversion of cytosolic NAD(P)H to mitochondrial ATP by oxidative phosphorylation starting at type II NAD(P)H dehydrogenases and glycerol-3-phosphate dehydrogenase both located in the inner mitochondrial membrane facing the cytosol. Solid arrow, direct cofactor transport. Dotted arrow, indirect cofactor transport via redox reactions
Compartment-specific energy requirements of photosynthetic carbon metabolism in Camelina sativa leaves

May 2022

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44 Reads

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9 Citations

Planta

Main conclusion The oxidative pentose phosphate pathway provides cytosolic NADPH yet reduces carbon and energy use efficiency. Repressing this pathway and introducing cytosolic NADPH-dependent malate dehydrogenase may increase crop yields by ≈5%. Abstract Detailed knowledge about plant energy metabolism may aid crop improvements. Using published estimates of flux through central carbon metabolism, we phenotype energy metabolism in illuminated Camelina sativa leaves (grown at 22 °C, 500 µmol photons m ⁻² s ⁻¹ ) and report several findings. First, the oxidative pentose phosphate pathway (OPPP) transfers 3.3% of the NADPH consumed in the Calvin–Benson cycle to the cytosol. NADPH supply proceeds at about 10% of the rate of net carbon assimilation. However, concomitantly respired CO 2 accounts for 4.8% of total rubisco activity. Hence, 4.8% of the flux through the Calvin–Benson cycle and photorespiration is spent on supplying cytosolic NADPH, a significant investment. Associated energy requirements exceed the energy output of the OPPP. Thus, autotrophic carbon metabolism is not simply optimised for flux into carbon sinks but sacrifices carbon and energy use efficiency to support cytosolic energy metabolism. To reduce these costs, we suggest bioengineering plants with a repressed cytosolic OPPP, and an inserted cytosolic NADPH-dependent malate dehydrogenase tuned to compensate for the loss in OPPP activity (if required). Second, sucrose cycling is a minor investment in overall leaf energy metabolism but a significant investment in cytosolic energy metabolism. Third, leaf energy balancing strictly requires oxidative phosphorylation, cofactor export from chloroplasts, and peroxisomal NADH import. Fourth, mitochondria are energetically self-sufficient. Fifth, carbon metabolism has an ATP/NADPH demand ratio of 1.52 which is met if ≤ 21.7% of whole electron flux is cyclic. Sixth, electron transport has a photon use efficiency of ≥ 62%. Last, we discuss interactions between the OPPP and the cytosolic oxidation–reduction cycle in supplying leaf cytosolic NADPH.





Citations (7)


... Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the key enzyme responsible for catalysing the first step of the Calvin-Benson-Bassham (CBB) cycle in photosynthesis, which converts inorganic CO 2 into organic carbohydrates, and it is the major rate-limiting factor that determines carbon assimilation efficiency ( Bar-On and Milo, 2019;Sharkey, 2023). RuBisCO plays crucial roles in photosynthesis, biomass accumulation and global carbon cycling; however, it exhibits a low catalytic rate (3-20 CO 2 /s) and poor substrate specificity, due to the relatively ineffective discrimination between CO 2 and O 2 (Liu, 2022). ...

Reference:

Genetic engineering of RuBisCO by multiplex CRISPR editing small subunits in rice
The Discovery of Rubisco
  • Citing Article
  • June 2022

Journal of Experimental Botany

... This presents an opportunity to incorporate the new metabolism into existing models [e.g., (Noctor and Foyer, 1998)] and estimate the impact the synthetic pathways may have on cellular energetics. Several flux models exist quantifying metabolic flux under various environmental conditions (Ma et al., 2014;Wieloch and Sharkey, 2022;Fu et al., 2022a) and may help to gauge the impact introduction of new metabolic sinks may have on energetics relative to native metabolism. This could also serve as a starting point for the design of accessory metabolism to offset any energetic imbalances imposed by the introduced metabolism. ...

Compartment-specific energy requirements of photosynthetic carbon metabolism in Camelina sativa leaves

Planta

... Chloroplast genomes have numerous applications, including in plant systematics to elucidate evolutionary relationships among species [75], [76] and in barcoding for species identification and biodiversity conservation [77]. Chloroplast genetic engineering has also been used to enhance crop traits, such as resistance to pests and diseases, and to produce biopharmaceuticals and industrial enzymes [73], [78], For instance, inserting commercially valuable traits such as herbicide and insect resistance into soybeans plastid genome led to high-level expression and excellent transgene containment [79], [80]. The complete chloroplast genomes of 43 Saudi plant species, primarily of medicinal value, could provide a foundation for developing improved medicinal plants with enhanced traits, contributing to the sustainable use and preservation of Saudi Arabia's plant biodiversity. ...

Editorial: Chloroplast Biotechnology for Crop Improvement

... extracted from tree rings to preclude error due to variation in wood composition (arguments given below apply to cellulose but not necessarily to wood) (6). Tree-ring cellulose 13 C/ 12 C data are commonly expressed in terms of 13 C discrimination, Δtrc, denoting carbon isotope changes caused by physiological processes (9). Current data interpretations invoke a simple mechanistic model accounting for 13 C discrimination accompanying two processes: CO2 diffusion from ambient air into leaf intercellular air spaces (or chloroplasts) and carbon assimilation by rubisco (6,10,11), collectively termed diffusion-rubisco (DR) discrimination (12). ...

Intramolecular carbon isotope signals reflect metabolite allocation in plants

Journal of Experimental Botany

... The Farquhar-von-Caemmerer-Berry (FvCB) model for C 3 net CO 2 assimilation is a cornerstone 30 of modern plant biology, ecology, and climate science, having been highly successful in 31 explaining experimental measurements and making predictions at scales ranging from single 32 cells to the entire globe (Farquhar et al., 2001;Von Caemmerer, 2013). Although commonly 33 referred to by the names of the authors of a key 1980 publication (Farquhar et al., 1980), the 34 FvCB model is nonetheless built on decades of research that predates 1980, and it has been 35 improved by other researchers since its initial description (Yin et al., 2021). It is a mechanistic 36 model based on a simplified version of the light-dependent reactions, the Calvin-Benson-37 Bassham (CBB) cycle, and the photorespiratory cycle, and it consists of a small set of equations 38 for predicting net CO 2 assimilation rates (Farquhar et al., 1980;Farquhar and von Caemmerer, 39 1982; Kirschbaum and Farquhar, 1984). ...

Evolution of a biochemical model of steady‐state photosynthesis

... Climate change has led to HS becoming a significant challenge, with negative impacts on crop growth at different developmental stages. Tomato plants are sensitive to high temperatures, and even mild HS can reduce yield and quality, while more severe stress can lead to plant death (Jagadish et al. 2021). Therefore, identifying heat-tolerant genes, breeding superior varieties, and enhancing tomato resilience to high temperatures are effective strategies for adapting to climate change and improving yields. ...

Plant heat stress: Concepts directing future research
  • Citing Article
  • March 2021

... This approach has the effect of constraining the potential for data to be shared between sites or projects. The aggregation of extensive trait databases from disparate studies offers promising avenues for macro-ecological inquiry (e.g., Ely et al. 2021) and the evaluation of hypotheses pertaining to ecological restoration (e.g., Crowther et al. 2022). Nevertheless, it remains uncertain to what extent these data can be employed in fine-grained inquiries, such as plant selection at either interspecific or intraspecific levels in complex stress environments, including urban forests. ...

A reporting format for leaf-level gas exchange data and metadata

Ecological Informatics