The oxidative pentose phosphate pathway in photosynthesis: a tale of two shunts.

CO2 release in the light (RL) and its presumed source, oxidative pentose phosphate pathways, were found to be insensitive to CO2 concentration. The oxidative pentose phosphate pathways form glucose 6-phosphate (G6P) shunts that bypass the nonoxidative pentose phosphate reactions of the Calvin-Benson cycle. Using adenosine diphosphate glucose and uridine diphosphate glucose as proxies for labeling of G6P in the stroma and cytosol respectively, it was found that only the cytosolic shunt was active. Uridine diphosphate glucose, a proxy for cytosolic G6P, and 6-phosphogluconate (6PG) were significantly less labeled than Calvin-Benson cycle intermediates in the light. But ADP glucose, a proxy for stromal G6P, is labeled to the same degree as Calvin-Benson cycle intermediates and much greater than 6PG. A metabolically inert pool of sedoheptulose bisphosphate can slowly equilibrate keeping the label in sedoheptulose lower than in other stromal metabolites. Finally, phosphorylation of fructose 6-phosphate (F6P) in the cytosol can allow some unlabeled carbon in cytosolic F6P to dilute label in phosphenolpyruvate. The results clearly show that there is oxidative pentose phosphate pathway activity in the cytosol that provides a shunt around the nonoxidative pentose phosphate pathway reactions of the Calvin-Benson cycle and is not strongly CO2-sensitive.

Engineered poplar for bioproduction of the triterpene squalene.

Building sustainable platforms to produce biofuels and specialty chemicals has become an increasingly important strategy to supplement and replace fossil fuels and petrochemical-derived products. Terpenoids are the most diverse class of natural products that have many commercial roles as specialty chemicals. Poplar is a fast growing, biomassdense bioenergy crop with many species known to produce large amounts of the hemiterpene isoprene, suggesting an inherent capacity to produce significant quantities of other terpenes. Here we aimed to engineer poplar with optimized pathways to produce squalene, a triterpene commonly used in cosmetic oils, a potential biofuel candidate, and the precursor to the further diversified classes of triterpenoids and sterols. The squalene production pathways were either re-targeted from the cytosol to plastids or co-produced with lipid droplets in the cytosol. Squalene and lipid droplet co-production appeared to be toxic, which we hypothesize to be due to disruption of adventitious root formation, suggesting a need for tissue specific production. Plastidial squalene production enabled up to 0.63 mg/g fresh weight in leaf tissue, which also resulted in reductions in isoprene emission and photosynthesis. These results were also studied through a technoeconomic analysis, providing further insight into developing poplar as a production host.

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

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.

The end game(s) of photosynthetic carbon metabolism.

The year 2024 marks 70 years since the general outline of the carbon pathway in photosynthesis was published. Although several alternative pathways are now known, it is remarkable how many organisms use the reaction sequence described 70 yrs ago, which is now known as the Calvin-Benson cycle or variants such as the Calvin-Benson-Bassham cycle or Benson-Calvin cycle. However, once the carbon has entered the Calvin-Benson cycle and is converted to a 3-carbon sugar, it has many potential fates. This review will examine the last stages of photosynthetic metabolism in leaves. In land plants, this process mostly involves the production of sucrose provided by an endosymbiont (the chloroplast) to its host for use and transport to the rest of the plant. Photosynthetic metabolism also usually involves the synthesis of starch, which helps maintain respiration in the dark and enables the symbiont to supply sugars during both the day and night. Other end products made in the chloroplast are closely tied to photosynthetic CO2 assimilation. These include serine from photorespiration and various amino acids, fatty acids, isoprenoids, and shikimate pathway products. I also describe 2 pathways that can short circuit parts of the Calvin-Benson cycle. These final processes of photosynthetic metabolism play many important roles in plants.

The isoprene-responsive phosphoproteome provides new insights into the putative signalling pathways and novel roles of isoprene.

Many plants, especially trees, emit isoprene in a highly light- and temperature-dependent manner. The advantages for plants that emit, if any, have been difficult to determine. Direct effects on membranes have been disproven. New insights have been obtained by RNA sequencing, proteomic and metabolomic studies. We determined the responses of the phosphoproteome to exposure of Arabidopsis leaves to isoprene in the gas phase for either 1 or 5 h. Isoprene effects that were not apparent from RNA sequencing and other methods but were apparent in the phosphoproteome include effects on chloroplast movement proteins and membrane remodelling proteins. Several receptor kinases were found to have altered phosphorylation levels. To test whether potential isoprene receptors could be identified, we used molecular dynamics simulations to test for proteins that might have strong binding to isoprene and, therefore might act as receptors. Although many Arabidopsis proteins were found to have slightly higher binding affinities than a reference set of Homo sapiens proteins, no specific receptor kinase was found to have a very high binding affinity. The changes in chloroplast movement, photosynthesis capacity and so forth, found in this work, are consistent with isoprene responses being especially useful in the upper canopy of trees.

Hydroxymethylbutenyl diphosphate accumulation reveals MEP pathway regulation for high CO2-induced suppression of isoprene emission.

Isoprene is emitted by some plants and is the most abundant biogenic hydrocarbon entering the atmosphere. Multiple studies have elucidated protective roles of isoprene against several environmental stresses, including high temperature, excessive ozone, and herbivory attack. However, isoprene emission adversely affects atmospheric chemistry by contributing to ozone production and aerosol formation. Thus, understanding the regulation of isoprene emission in response to varying environmental conditions, for example, elevated CO2, is critical to comprehend how plants will respond to climate change. Isoprene emission decreases with increasing CO2 concentration; however, the underlying mechanism of this response is currently unknown. We demonstrated that high-CO2-mediated suppression of isoprene emission is independent of photosynthesis and light intensity, but it is reduced with increasing temperature. Furthermore, we measured methylerythritol 4-phosphate (MEP) pathway metabolites in poplar leaves harvested at ambient and high CO2 to identify why isoprene emission is reduced under high CO2. We found that hydroxymethylbutenyl diphosphate (HMBDP) was increased and dimethylallyl diphosphate (DMADP) decreased at high CO2. This implies that high CO2 impeded the conversion of HMBDP to DMADP, possibly through the inhibition of HMBDP reductase activity, resulting in reduced isoprene emission. We further demonstrated that although this phenomenon appears similar to abscisic acid (ABA)-dependent stomatal regulation, it is unrelated as ABA treatment did not alter the effect of elevated CO2 on the suppression of isoprene emission. Thus, this study provides a comprehensive understanding of the regulation of the MEP pathway and isoprene emission in the face of increasing CO2.

Daylength variation affects growth, photosynthesis, leaf metabolism, partitioning, and metabolic fluxes.

Daylength, a seasonal and latitudinal variable, exerts a substantial impact on plant growth. However, the relationship between daylength and growth is nonproportional, suggesting the existence of adaptive mechanisms. Thus, our study aimed to comprehensively investigate the adaptive strategies employed by plants in response to daylength variation. We grew false flax (Camelina sativa) plants, a model oilseed crop, under long-day (LD) and short-day (SD) conditions and used growth measurements, gas exchange measurements, and isotopic labeling techniques, including 13C, 14C, and 2H2O, to determine responses to different daylengths. Our findings revealed that daylength influences various growth parameters, photosynthetic physiology, carbon partitioning, metabolic fluxes, and metabolite levels. SD plants employed diverse mechanisms to compensate for reduced CO2 fixation in the shorter photoperiod. These mechanisms included enhanced photosynthetic rates and reduced respiration in the light (RL), leading to increased shoot investment. Additionally, SD plants exhibited reduced rates of the glucose 6-phosphate (G6P) shunt and greater partitioning of sugars into starch, thereby sustaining carbon availability during the longer night. Isotopic labeling results further demonstrated substantial alterations in the partitioning of amino acids and TCA cycle intermediates between rapidly and slowly turning over pools. Overall, the results point to multiple developmental, physiological, and metabolic ways in which plants adapt to different daylengths to maintain growth.

Laisk measurements in the nonsteady state: Tests in plants exposed to warming and variable CO2 concentrations.

Light respiration (RL) is an important component of plant carbon balance and a key parameter in photosynthesis models. RL is often measured using the Laisk method, a gas exchange technique that is traditionally employed under steady-state conditions. However, a nonsteady-state dynamic assimilation technique (DAT) may allow for more rapid Laisk measurements. In 2 studies, we examined the efficacy of DAT for estimating RL and the parameter Ci* (the intercellular CO2 concentration where Rubisco's oxygenation velocity is twice its carboxylation velocity), which is also derived from the Laisk technique. In the first study, we compared DAT and steady-state RL and Ci* estimates in paper birch (Betula papyrifera) growing under control and elevated temperature and CO2 concentrations. In the second, we compared DAT-estimated RL and Ci* in hybrid poplar (Populus nigra L. × P. maximowiczii A. Henry "NM6") exposed to high or low CO2 concentration pre-treatments. The DAT and steady-state methods provided similar RL estimates in B. papyrifera, and we found little acclimation of RL to temperature or CO2; however, Ci* was higher when measured with DAT compared to steady-state methods. These Ci* differences were amplified by the high or low CO2 pre-treatments. We propose that changes in the export of glycine from photorespiration may explain these apparent differences in Ci*.

Nanoscale simulation of the thylakoid membrane response to extreme temperatures.

The thylakoid membrane is in a temperature-sensitive equilibrium that shifts repeatedly during the life cycle in response to ambient temperature or solar irradiance. Plants respond to seasonal temperature variation by changing their thylakoid lipid composition, while a more rapid mechanism for short-term heat exposure is required. The emission of the small organic molecule isoprene has been postulated as one such possible rapid mechanism. The protective mechanism of isoprene is unknown, but some plants emit isoprene at high temperature. We investigate the dynamics and structure for lipids within a thylakoid membrane across temperatures and varied isoprene content using classical molecular dynamics simulations. The results are compared with experimental findings for temperature-dependent changes in the lipid composition and shape of thylakoids. The surface area, volume, and flexibility of the membrane, as well as the lipid diffusion, increase with temperature, while the membrane thickness decreases. Saturated thylakoid 34:3 glycolipids derived from eukaryotic synthesis pathways exhibit altered dynamics relative to lipids from prokaryotic synthesis paths, which could explain the upregulation of specific lipid synthesis pathways at different temperatures. Increasing isoprene concentration was not observed to have a significant thermoprotective effect on the thylakoid membranes, and that isoprene readily permeated the membrane models tested.

Rapid CO2 changes cause oscillations in photosynthesis that implicate PSI acceptor-side limitations.

Oscillations in CO2 assimilation rate and associated fluorescence parameters have been observed alongside the triose phosphate utilization (TPU) limitation of photosynthesis for nearly 50 years. However, the mechanics of these oscillations are poorly understood. Here we utilize the recently developed dynamic assimilation techniques (DATs) for measuring the rate of CO2 assimilation to increase our understanding of what physiological condition is required to cause oscillations. We found that TPU-limiting conditions alone were insufficient, and that plants must enter TPU limitation quickly to cause oscillations. We found that ramps of CO2 caused oscillations proportional in strength to the speed of the ramp, and that ramps induce oscillations with worse outcomes than oscillations induced by step change of CO2 concentration. An initial overshoot is caused by a temporary excess of available phosphate. During the overshoot, the plant outperforms steady-state TPU and ribulose 1,5-bisphosphate regeneration limitations of photosynthesis, but cannot exceed the rubisco limitation. We performed additional optical measurements which support the role of PSI reduction and oscillations in availability of NADP+ and ATP in supporting oscillations.

Designing plant-transparent agrivoltaics.

Covering greenhouses and agricultural fields with photovoltaics has the potential to create multipurpose agricultural systems that generate revenue through conventional crop production as well as sustainable electrical energy. In this work, we evaluate the effects of wavelength-selective cutoffs of visible and near-infrared (biologically active) radiation using transparent photovoltaic (TPV) absorbers on the growth of three diverse, representative, and economically important crops: petunia, basil, and tomato. Despite the differences in TPV harvester absorption spectra, photon transmission of photosynthetically active radiation (PAR; 400-700 nm) is the most dominant predictor of crop yield and quality. This indicates that different wavebands of blue, red, and green are essentially equally important to these plants. When the average photosynthetic daily light integral is > 12 mol m-2 d-1, basil and petunia yield and quality is acceptable for commercial production. However, even modest decreases in TPV transmission of PAR reduces tomato growth and fruit yield. These results identify crop-specific design requirements that exist for TPV harvester transmission and the necessity to maximize transmission of PAR to create the most broadly applicable TPV greenhouse harvesters for diverse crops and geographic locations. We determine that the deployment of 10% power conversion efficiency (PCE) plant-optimized TPVs over approximately 10% of total agricultural and pasture land in the U.S. would generate 7 TW, nearly double the entire energy demand of the U.S.

Characterization of promoter elements of isoprene-responsive genes and the ability of isoprene to bind START domain transcription factors.

Isoprene has recently been proposed to be a signaling molecule that can enhance tolerance of both biotic and abiotic stress. Not all plants make isoprene, but all plants tested to date respond to isoprene. We hypothesized that isoprene interacts with existing signaling pathways rather than requiring novel mechanisms for its effect on plants. We analyzed the cis-regulatory elements (CREs) in promoters of isoprene-responsive genes and the corresponding transcription factors binding these promoter elements to obtain clues about the transcription factors and other proteins involved in isoprene signaling. Promoter regions of isoprene-responsive genes were characterized using the Arabidopsis cis-regulatory element database. CREs bind ARR1, Dof, DPBF, bHLH112, GATA factors, GT-1, MYB, and WRKY transcription factors, and light-responsive elements were overrepresented in promoters of isoprene-responsive genes; CBF-, HSF-, WUS-binding motifs were underrepresented. Transcription factors corresponding to CREs overrepresented in promoters of isoprene-responsive genes were mainly those important for stress responses: drought-, salt/osmotic-, oxidative-, herbivory/wounding and pathogen-stress. More than half of the isoprene-responsive genes contained at least one binding site for TFs of the class IV (homeodomain leucine zipper) HD-ZIP family, such as GL2, ATML1, PDF2, HDG11, ATHB17. While the HD-zipper-loop-zipper (ZLZ) domain binds to the L1 box of the promoter region, a special domain called the steroidogenic acute regulatory protein-related lipid transfer, or START domain, can bind ligands such as fatty acids (e.g., linolenic and linoleic acid). We tested whether isoprene might bind in such a START domain. Molecular simulations and modeling to test interactions between isoprene and a class IV HD-ZIP family START-domain-containing protein were carried out. Without membrane penetration by the HDG11 START domain, isoprene within the lipid bilayer was inaccessible to this domain, preventing protein interactions with membrane bound isoprene. The cross-talk between isoprene-mediated signaling and other growth regulator and stress signaling pathways, in terms of common CREs and transcription factors could enhance the stability of the isoprene emission trait when it evolves in a plant but so far it has not been possible to say what how isoprene is sensed to initiate signaling responses.

The discovery of rubisco.

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 a carboxylase. Finally, the requirement for numerous chaperonins for assembly of rubisco in plants is described.

The time course of acclimation to the stress of triose phosphate use limitation.

Triose phosphate utilisation (TPU) limits the maximum rate at which plants can photosynthesise. However, TPU is almost never found to be limiting photosynthesis under ambient conditions for plants. This, along with previous results showing adaptability of TPU at low temperature, suggest that TPU capacity is regulated to be just above the photosynthetic rate achievable under the prevailing conditions. A set of experiments were performed to study the adaptability of TPU capacity when plants are acclimated to elevated CO2 concentrations. Plants held at 1500 ppm CO2 were initially TPU limited. After 30 h they no longer exhibited TPU limitations but they did not elevate their TPU capacity. Instead, the maximum rates of carboxylation and electron transport declined. A timecourse of regulatory responses was established. A step increase of CO2 first caused PSI to be oxidised but after 40 s both PSI and PSII had excess electrons as a result of acceptor-side limitations. Electron flow to PSI slowed and the proton motive force increased. Eventually, non-photochemical quenching reduced electron flow sufficiently to balance the TPU limitation. Over several minutes rubisco deactivated contributing to regulation of metabolism to overcome the TPU limitation.

Isoprene measurements to assess plant hydrocarbon emissions and the methylerythritol pathway.

Isoprene is the most abundant non-methane hydrocarbon emitted to the atmosphere and a target of biotechnology as a source of biofuels or chemical feedstock. Measurements of the amount of isoprene or the rate of production of isoprene are important for atmospheric chemistry, evaluating biotechnology processes, and can provide information on the capacity and regulation of the methyl erythritol 4-phosphate pathway found in plants and bacteria. In this chapter we discuss techniques, and their strengths and weaknesses, of methods in common use for measuring isoprene. There are many sources of isoprene for measurements including emissions from leaves and head space analysis of reactions involving recombinant enzymes or bacterial/fungal cultures. Similarly, there are a variety of detection methods including several mass spectrometer methods that are useful for examining rates of labeling of isoprene when carbon isotopes are used.

Role of guard cell- or mesophyll cell-localized phytochromes in stomatal responses to blue, red, and far-red light.

MAIN CONCLUSION: Guard cell- or mesophyll cell-localized phytochromes do not have a predominant direct light sensory role in red- or blue-light-mediated stomatal opening or far-red-light-mediated stomatal closure of Arabidopsis. The role of phytochromes in blue- and red-light-mediated stomatal opening, and far-red-light- mediated decrease in opening, is still under debate. It is not clear whether reduced stomatal opening in a phytochrome B (phyB) mutant line, is due to phytochrome acting as a direct photosensor or an indirect growth effect. The exact tissue localization of the phytochrome photoreceptor important for stomatal opening is also not known. We studied differences in stomatal opening in an Arabidopsis phyB mutant, and lines showing mesophyll cell-specific or guard cell-specific inactivation of phytochromes. Stomatal conductance (gs) of intact leaves was measured under red, blue, and blue + far-red light. Lines exhibiting guard cell-specific inactivation of phytochrome did not show a change in gs under blue or red light compared to Col-0. phyB consistently exhibited a reduction in gs under both blue and red light. Addition of far-red light did not have a significant impact on the blue- or red-light-mediated stomatal response. Treatment of leaves with DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea), a photosynthetic electron transport (PET) inhibitor, eliminated the response to red light in all lines, indicating that stomatal opening under red light is controlled by PET, and not directly by phytochrome. Similar to previous studies, leaves of the phyB mutant line had fewer stomata. Overall, phytochrome does not appear have a predominant direct sensory role in stomatal opening under red or blue light. However, phytochromes likely have an indirect effect on the degree of stomatal opening under light through effects on leaf growth and stomatal development.

Pathway Engineering, Re-targeting, and Synthetic Scaffolding Improve the Production of Squalene in Plants.

Plants are increasingly becoming an option for sustainable bioproduction of chemicals and complex molecules like terpenoids. The triterpene squalene has a variety of biotechnological uses and is the precursor to a diverse array of triterpenoids, but we currently lack a sustainable strategy to produce large quantities for industrial applications. Here, we further establish engineered plants as a platform for production of squalene through pathway re-targeting and membrane scaffolding. The squalene biosynthetic pathway, which natively resides in the cytosol and endoplasmic reticulum, was re-targeted to plastids, where screening of diverse variants of enzymes at key steps improved squalene yields. The highest yielding enzymes were used to create biosynthetic scaffolds on co-engineered, cytosolic lipid droplets, resulting in squalene yields up to 0.58 mg/gFW or 318% higher than a cytosolic pathway without scaffolding during transient expression. These scaffolds were also re-targeted to plastids where they associated with membranes throughout, including the formation of plastoglobules or plastidial lipid droplets. Plastid scaffolding ameliorated the negative effects of squalene biosynthesis and showed up to 345% higher rates of photosynthesis than without scaffolding. This study establishes a platform for engineering the production of squalene in plants, providing the opportunity to expand future work into production of higher-value triterpenoids.

Reimport of carbon from cytosolic and vacuolar sugar pools into the Calvin-Benson cycle explains photosynthesis labeling anomalies.

SignificancePhotosynthesis metabolites are quickly labeled when 13CO2 is fed to leaves, but the time course of labeling reveals additional contributing processes involved in the metabolic dynamics of photosynthesis. The existence of three such processes is demonstrated, and a metabolic flux model is developed to explore and characterize them. The model is consistent with a slow return of carbon from cytosolic and vacuolar sugars into the Calvin-Benson cycle through the oxidative pentose phosphate pathway. Our results provide insight into how carbon assimilation is integrated into the metabolic network of photosynthetic cells with implications for global carbon fluxes.

Isoprene enhances leaf cytokinin metabolism and induces early senescence.

Isoprene, a major biogenic volatile hydrocarbon of climate-relevance, indisputably mitigates abiotic stresses in emitting plants. However functional relevance of constitutive isoprene emission in unstressed plants remains contested. Isoprene and cytokinins (CKs) are synthesized from a common substrate and pathway in chloroplasts. It was postulated that isoprene emission may affect CK-metabolism. Using transgenic isoprene-emitting (IE) Arabidopsis and isoprene nonemitting (NE) RNA-interference grey poplars (paired with respective NE and IE genotypes), the life of individual IE and NE leaves from emergence to abscission was followed under stress-free conditions. We monitored plant growth rate, aboveground developmental phenotype, modelled leaf photosynthetic energy status, quantified the abundance of leaf CKs, analysed Arabidopsis and poplar leaf transcriptomes by RNA-sequencing in presence and absence of isoprene during leaf senescence. Isoprene emission by unstressed leaves enhanced the abundance of CKs (isopentenyl adenine and its precursor) by > 200%, significantly upregulated genes coding for CK-synthesis, CK-signalling and CK-degradation, hastened plant development, increased chloroplast metabolic rate, altered photosynthetic energy status, induced early leaf senescence in both Arabidopsis and poplar. IE leaves senesced sooner even in decapitated poplars where source-sink relationships and hormone homeostasis were perturbed. Constitutive isoprene emission significantly accelerates CK-led leaf and organismal development and induces early senescence independent of growth constraints. Isoprene emission provides an early-riser evolutionary advantage and shortens lifecycle duration to assist rapid diversification in unstressed emitters.