Time-resolved targeted metabolomics shows an abrupt switch from Calvin- Benson-Bassham cycle to tricarboxylic acid cycle when the light is turned off

Time-resolved targeted metabolomics shows an abrupt switch from Calvin- Benson-Bassham cycle to tricarboxylic acid cycle when the light is turned off | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Time-resolved targeted metabolomics shows an abrupt switch from Calvin- Benson-Bassham cycle to tricarboxylic acid cycle when the light is turned off Yuan Xu, Stephanie C. Schmiege, Thomas D. Sharkey This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6839843/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Sep, 2025 Read the published version in Photosynthesis Research → Version 1 posted 9 You are reading this latest preprint version Abstract In leaves, major CO₂ fluxes alternate between fixation by the Calvin-Benson-Bassham (CBB) cycle during light and release by the tricarboxylic acid (TCA) cycle in darkness. The speed at which leaf metabolism transitions between these pathways likely influences plant tolerance to fluctuating light conditions. To investigate these rapid metabolic shifts, we exposed leaves to ¹³CO₂ for 20 minutes to establish a quasi-steady state before abruptly turning off the light while maintaining ¹³CO₂ feeding. Within 10 seconds of dark transition, 3-phosphoglycerate levels rose dramatically, while most other CBB cycle intermediates decreased by more than 90%. Simultaneously, carbon accumulated in alanine, likely via pyruvate. Over the subsequent 10 minutes, six- and five-carbon TCA cycle intermediates steadily increased. In contrast, four-carbon TCA intermediates peaked at one minute, declined by three minutes, and rose again at 10 minutes, a pattern mirrored by most measured amino acids. These results reveal an exceptionally rapid metabolic reconfiguration from CO₂ fixation by the CBB cycle in light to TCA cycle activation for energy production in darkness, accompanied by substantial changes in amino acid metabolism. Calvin Benson Bassham cycle Light transition Photosynthetic carbon metabolism Respiration Tricarboxylic Acid Cycle Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Plant leaves use energy in sunlight for metabolic needs and carbon fixation by the Calvin-Benson-Bassham (CBB) cycle in the light. In the dark they rely on energy from glycolysis and the tricarboxylic acid (TCA). A significant amount of carbon fixed using sunlight energy during the day is stored as starch, which is then broken down at night to supply sugars for glycolysis and the TCA cycle. The speed with which the CBB cycle stops and the TCA cycle begins is unknown but may be important for understanding how carbon metabolism responds to fluctuating light. Light fluctuations can occur over very short time periods, for example during leaf flutter of trembling aspen (Roden and Pearcy 1993) or over much longer periods, for example leaves at the bottom of the canopy (Shao et al. 2024; Durand et al. 2022) of trees and crop species. During the shortest light flecks, photosynthetic electron transport could be greatly affected (De Souza et al. 2022). The pools of the metabolites of the CBB cycle are small, most compounds have half-lives of 1 s or less (Szecowka et al. 2013; Arrivault et al. 2009) and so even short light flecks may have a big influence on carbon metabolism and the activity of the CBB and TCA cycles It has been assumed that ribulose 1,5-bisphosphate (RuBP) production ceases immediately when a leaf is exposed to darkness. In fact post illumination CO 2 uptake has been taken as a measure of the pool of RuBP present when the light is turned off (Laisk et al. 1984). The need for reducing power traps metabolites of the CBB cycle in 3-phosphoglycerate (PGA). This requires regulation of downstream reactions so that metabolism can be reoriented from CBB cycle to heterotrophy and the TCA cycle. These downstream reactions include lower glycolysis (from triose phosphates to pyruvate), which does not occur in chloroplasts (Stitt et al. 1978; Evans et al. 2024) preventing futile cycling. There is good evidence that the mitochondrial pyruvate dehydrogenase complex (mtPDC) is “inactivated by phosphorylation” in the light (Budde and Randall 1990). The chloroplast PDC is not inhibited allowing for fatty acid synthesis. While fatty acid synthesis is essential to the plant metabolic flux analysis indicates that it occurs at a low rate not likely to influence carbon metabolism rate in the light (Xu et al. 2022). Although a very small flow of recently fixed 1 3 CO 2 from citrate to glutamine and glutamate by way of a-ketoglutarate can be detected, for the four-carbon members of the TCA cycle, labeling is below the limits of detection (Xu et al. 2022; Szecowka et al. 2013). The near total lack of activity of the TCA cycle in the light has been known for many years Calvin and Massini (1952) saw a very rapid increase in label in citrate upon darkness. The small flux toward a-ketoglutarate in the light may allow stored citrate to contribute to nitrogen metabolism (Gauthier et al. 2010; Abadie et al. 2017). Recently it has been recognized that, during photorespiration, serine metabolism associated with photorespiration may play a large role in amino acid metabolism (Fu et al. 2023; Busch et al. 2018). In this study, we used time-resolved, targeted metabolomics to explore how carbon moves through metabolites during a transition from the CBB cycle to the TCA cycle, and how this transition influences energy metabolism and nitrogen assimilation. Unraveling these mechanisms is essential for understanding how plants maintain metabolic homeostasis and optimize energy utilization in dynamic light environments. The availability of stable isotopes (specifically 13 CO 2 ) provides a mechanism for following carbon when it is fed as 13 CO 2 to photosynthesizing leaves. This avoids potential complications when molecules are fed through the transpiration stream. Here we report on measurements in which 13 CO 2 was fed to a photosynthesizing leaf for 20 min to achieve a quasi-steady-state (Xu et al. 2025) then abruptly turned off the light. We predicted that the change from CBB To TCA cycle would occur quickly because: Citrate and other TCA metabolites exhibit almost no labeling in the light but a very rapid rise in citrate labeling within seconds of turning the light off (Calvin and Massini 1952; Szecowka et al. 2013; Ma et al. 2014; Xu et al. 2022). The metabolite pools of the CBB cycle are known to have very short half-lives (Arrivault et al. 2009; Badger et al. 1984; Hasunuma et al. 2010). We did not monitor CO 2 evolution, which results in a burst of CO 2 release when leaves are first darkened. We do not address respiration in the light, sometimes denoted R L but rely metabolic flux analysis that identified a time frame of 20 min as a semi steady state (Xu et al. 2022). Our intent was to resolve changes that occur in leaves over the first 10 min of darkness. Our results show a very rapid change from chloroplast CBB cycle carbon metabolism to mitochondria TCA metabolism. Methods Plant growth Tobacco ( Nicotiana tabacum , ‘Samsun’) plants transformed with Populus alba isoprene synthase (ISPS) were provided by Claudia Vickers from the University of Queensland. Details regarding plasmid design, vector construction, transformation, and line selection are described in Vickers et al. (2009). The IE lines were used as part of other experiments This made many very healthy plants available for this study. Plants were cultivated in greenhouses at the Michigan State University Plant Research Laboratory. The plants were maintained under a 16-hour photoperiod with a light intensity of 400–500 µmol m⁻² s⁻¹ provided by sunlight supplemented by sodium vapor lamps. day/night temperatures were set to 25–27°C/20–22°C, and additive relative humidity was set to 60–65%. Seeds were sown in Suremix growing medium (Michigan Grower Products, Galesburg, MI, USA) on separate trays. Fourteen days post-germination, seedlings were transplanted into small 3.5 L pots (five seedlings per pot) to ensure survival. Two weeks after transplantation, when seedlings were stable, they were transferred to larger 7 L pots (one plant per pot). Plants were watered with deionized water for the first two days, followed by one-half-strength Hoagland’s nutrient solution (Hoagland and Arnon 1938) for all subsequent days. Experiments were conducted on 6- to 8-week-old plants before flowering and seed development. Three biological replicates were used. Sampling method We employed an in-house-modified Fast Kill freeze clamp, as described by Sharkey et al. (2020), for this study. The setup integrated a LI-COR 6800 head with a 6 cm × 6 cm chamber. The chamber was sealed using cling film to create a closed environment and uniformly illuminated with two gooseneck fiber optic illuminators (Schott KL2500 LED lamps, obtained from Edmund Optics, https://www.edmundoptics.com) at a light intensity of 1,000 μmol m⁻² s⁻¹. Chamber temperature was controlled and monitored using a thermocouple connected to the LI-6800 portable gas exchange system. The LI-COR 6800 instrument was configured to scrub all CO₂ from the system before introducing CO₂ from one of two sources: 5% ¹²CO₂ in air with natural isotopic abundance from Airgas (www.airgas.com) or a lecture bottle containing 99+% ¹³CO₂ (Aldrich, Sigma-Aldrich.com) pressurized with N₂. Flow meters (Alicat Scientific) regulated the delivery of ¹²CO₂ and ¹³CO₂. To achieve the desired CO₂ concentration, the ¹²CO₂ flow rate was adjusted based on the ¹³CO₂ concentration in the pressurized tank, using flow meter settings. The two flow meters were connected to the air supply entering the LI-COR 6800 head via a four-way valve. Leaves were equilibrated in the chamber under illumination for 30–60 min until assimilation rates stabilized. At this point, the CO₂ source was switched to ¹³CO₂ and maintained for 20 min, corresponding to the second of three labeling phases (Xu et al. 2022). The ¹³CO₂ supply was regulated to maintain a consistent CO₂ concentration of 450 ppm, and the transition from ¹²CO₂ to ~90% ¹³CO₂ was achieved within one minute. After 20 min, the light was turned off, and the plants were kept in darkness under black light-blocking cloth for varying durations (0, 10, 30, 60, 180, or 600 sec). The Fast Kill mechanism was then triggered, using liquid-nitrogen-cooled copper blocks to rapidly freeze leaf samples. The interval between light interruption and the leaf sample reaching a temperature below 0 °C was measured at 35 ms (Sahu et al. 2023). Frozen leaf samples were collected in 2 mL microcentrifuge tubes and stored at −80 °C for subsequent mass spectrometry analysis. Mass spectrometry Metabolites were extracted from flash-frozen tissues according to established protocols (Xu et al., 2021). Subsequent analyses were conducted via mass spectrometry, as detailed in previous reports (Xu et al., 2021; Xu et al., 2022). Specific MS parameters for multiple reaction monitoring (MRM) with LC–MS/MS, selected ion monitoring (SIM), and GC–MS are provided in Supplemental Table S1. Phosphorylated metabolites in the CBB cycle were analyzed using ion-pair chromatography–tandem mass spectrometry (IPC–MS/MS). Separation was performed on a 2.1 × 50 mm ACQUITY UPLC BEH C18 column (Waters, Milford, MA, USA) connected to an ACQUITY UPLC pump system coupled with a Waters XEVO TQ-S UPLC/MS/MS (Waters, Milford, MA, USA). Nucleotide sugars and other phosphorylated intermediates were measured by anion exchange chromatography–tandem mass spectrometry (AEC–MS/MS). Samples were run on a 2 × 250 mm IonPac AS11 analytical column with a 2 × 50 mm IonPac AG11 guard column (Dionex), using an ACQUITY UPLC pump system (Waters, Milford, MA, USA) coupled to a Xevo ACQUITY TQ Triple Quadrupole Detector (Waters, Milford, MA, USA). A self-regenerating suppressor (Dionex ADRS 600, Thermo Scientific, Waltham, MA, USA) was employed post-column to neutralize the KOH eluent. Amino acids and organic acids were examined by GC–MS using an Agilent 7890 GC system interfaced with an Agilent 5975C inert XL Mass Selective Detector (Agilent, Santa Clara, CA, USA). Initial derivatization with methoxyamine hydrochloride in dry pyridine was followed by silylation of amino and organic acids to form TBDMS derivatives (using N-(tertbutyldimethylsilyl)-N-methyltrifluoroacetamide with 1% [w/v] tert-butyldimethylchlorosilane). Separation was achieved on an Agilent VF5ms GC column (Agilent, Santa Clara, CA, USA). Mass spectrometry data were analyzed to quantify pool size, mass isotopologue distributions (MIDs), and ¹³C enrichment, following previously described methods (Xu et al. 2022) LC–MS/MS data were acquired using MassLynx 4.0 (Agilent, Santa Clara, CA, USA), while GC–MS data were obtained with Agilent GC/MSD Chemstation (Agilent, Santa Clara, CA, USA). Peak detection and quantification were performed using the TargetLynx Application Manager within Waters MassLynx™ Software (Waters Corporation, MA). MIDs for GC–MS–measured metabolites were corrected for natural isotopic abundance using FluxFix (Trefely et al. 2016). Results Calvin-Benson-Bassham cycle decline s when the light is turned off We distinguish among three measures of each metabolite- (a) the total amount of the metabolite, nmol metabolite g -1 FW (b) the relative degree of label incorporation, 0 %, all carbons are 12 C; 100%, all carbons are 13 C (c) the amount of 13 C atoms in each metabolite pool, nmol 13 C g -1 FW (= a times b times number of carbons in the molecule). When the light was abruptly turned off the amount of ribulose 1,5-bisphosphate (RuBP) fell by almost 90% at the first time point of 10 s (Fig. 1). Other metabolites of the CBB cycle also fell except for PGA and sedoheptulose 7-phosphate (S7P). The increase in PGA was four-fold greater than the decrease in RuBP (two-fold greater would be expected because of RuBP carboxylation) indicating that pools other than RuBP contributed to the PGA accumulation. The S7P data differs from other CBB cycle intermediates, which may be related to a pool of sedoheptulose 1,7-bisphosphate (SBP) in the cytosol. This can cause the S7P labeling to be slow, as unlabeled SBP reenters the chloroplast (Xu et al. 2024) and so S7P is ignored in further analyses. The photorespiratory intermediate 2-phosphglycolate (2-PG) fell significantly in the first 10 s, mirroring the change in RuBP (Fig. 2). Glycine fell at 3 and 10 min but was stable up to 1 min. Serine and glycerate were present at much higher levels than 2PG and glycine (note the scales in Fig. 2). The amount of 13 C in RuBP fell 87% in the first 10 s of darkness (Fig. 3A). This decline was similar to the decline in RuBP content (Fig. 1), which reflected in a very small change in the degree of label in RuBP (Fig. 3B). The degree of label in RuBP and PGA (% 13 C) was similar at time zero (Fig. 3B). Over 10 min, the percentage of carbon atoms that were 13 C in RuBP fell from 95% to 77% but the degree of label was noticeably higher in PGA (Fig. 3B). Any carboxylation that may have occurred in the dark would be supplying 99% 13 CO 2 to the one-sixth of PGA resulting from carboxylation of RuBP. We expect slow fluxes of unlabeled carbon to RuBP possibly through the cytosolic G6P shunt (Xu et al. 2024) and other metabolism of unlabeled molecules. The initial increase in PGA (Fig. 1) coincided with an increase in PEP (Fig. 3C). Carbon eventually ended up in pyruvate The triose phosphate transporter transports PGA as efficiently as glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (Flügge and Heldt 1991). Conversion of PGA to pyruvate inside the chloroplasts is negligible (Evans et al. 2024). Presumably PEP acts as an intermediate, which after the first 10 sec, filling and emptying at rates that match, keeping the total pool size constant. The amount of PEP increased initially while the degree of label remained high (Fig. 3C), indicating that the carbon in PEP came from carbon in the highly labeled CBB cycle. On the other hand, label in pyruvate was low in the light (zero time) but increased for three min. This increase in 13 C was paralleled by an increase in total amount of pyruvate. (Fig. 3D). Transition from Calvin-Benson-Bassham cycle to the tricarboxylic acid cycle Labeling of pyruvate was limited during photosynthesis (Fig. 3D) given that PEP was heavily labeled (Fig. 3C). The degree of label in pyruvate never exceeded 62% (Supplemental Table S2). The large increase in PEP for the first few seconds did not result in any dilution of the label in PEP indicating that the increase in PEP resulted from conversion of the active pool of PGA to PEP. This reaction sequence is kept low in the light given that the amount of PEP was low at time zero. This suggests that pyruvate kinase in the cytosol is highly regulated and can become very active rapidly in the dark leading to an increase in heavily labeled PEP. There appeared to be a large unlabeled pool of pyruvate. When darkness was imposed, both the amount and degree of label in pyruvate increased for 3 min and then fell (Fig 1. and 3D). This indicates that much of the increase in pyruvate resulted from PEP conversion to pyruvate. Between 3 and 10 min, pyruvate declined. Citrate increased and the degree of increase in citrate exceeded the decline in 13 C in pyruvate. By 10 min the amount of pyruvate declined as did the degree of label indicating that there is a large metabolically inert pool of pyruvate. It is known that a large amount of pyruvate is in the vacuole (Szecowka et al. 2013). When the metabolites measured here were summed up, it appeared that the total 13 C content increased significantly in the first 10 s and climbed at a slower rate for the next min (Fig. 4) but changed very little between 1 and 10 min. Post-illumination CO 2 fixation attributable to carboxylation of RuBP (77.5 13 C nmol atoms g -1 FW, Fig. 2 and supplemental Table S2) was not sufficient to account for the extra 13 C atoms in citrate (12,398 13 C nmol atoms g -1 FW). We presume that a more comprehensive accounting, and accounting for other metabolites being converted to RuBP, might explain the higher-than-expected 13 C content of citrate. The tricarboxylic acid pathway response to darkness The amount of citrate jumped in the first 10 s but the additional 13 C accounted for just 2% of this increase in citrate amount (Fig. 5 and Supplemental Table S2). Citrate, glutamate (as a proxy for a-ketoglutarate), and glutamine increased over the whole 10 min although glutamine, and to a lesser degree glutamate, did not increase as much as would be expected at 3 min (Fig. 5). These compounds had a low degree of label (Fig. 6A) and so most of the change in total content was from unlabeled sources, in line with Gauthier et al. (2010) and Abadie et al. (2017).. The four-carbon metabolites succinate, fumarate and, to some degree malate, plus amino acids derived from oxaloacetate) exhibited a distinct peak at 1 min, then declined at 3 min but increased again at 10 min (Fig. 6 and Supplemental Table S2). The 4-carbon members of the TCA cycle, except malate, also had a low degree of label and low content of 13 C (Fig. 6C and 6D). Malate labeling was higher than succinate and fumarate but was still low (Fig. 7C) This could indicate an active PEP carboxylase. The low and relatively constant degree of label in succinate, fumarate, and malate resulted in little change to the amount 13 C in these metabolites (Fig. 6C). Amino acids The amount of glutamate increased over the 10 min of darkness, much like citrate (Fig. 5). On the other hand, changes in the amount of glutamine were more like other amino acids (Fig.5). Alanine and serine were heavily labeled and remained so for 10 min (Fig. 6E), but aspartate, asparagine and threonine were relatively unlabeled (Fig. 7E). Unlike the degree of label, the amount of label in serine peaked at 30 s while there was an even larger pool of alanine at 3 min (Fig. 6F). This indicated that these pools filled with carbon from the CBB cycle and then emptied. The increase happened later for alanine but to a much greater extent (Fig. 6F). The large amount of nitrogen in alanine at 3 min coincided with a dip in other amino acids (Fig. 5). To look for patterns we plotted the amount of 13 C atoms relative to the maximum for that metabolite (Fig. 7). Citrate and glutamate showed a steadily increasing relative amount of label while glutamine showed a temporary drop at 3 min (Fig. 7A), similar to most other amino acids. Succinate, fumarate, and malate all showed a dip at 3 min (Fig. 7B). Most of the amino acids had distinct minima at 3 min with the notable exception of alanine which had a very large peak in relative amount at 3 min (Fig.7C). This was also seen in the total amount of 13 C in alanine (Fig. 6F). The degree of label in alanine was high and did not change over time (Fig. 6E) indicating that the large increase in alanine came from other CBB cycle intermediates. Discussion The focus of this study was the change in carbon metabolism as the CBB cycle stops and TCA cycle begins. Carbon metabolism in leaves can be complex and involve many different pathways; how light or darkness will affect these pathways is difficult to discern (Tcherkez et al. 2024). There are many changes in plant cell metabolism that can be detected over a longer time frame. For example Abadie et al. (2021) found as many as 4,500 metabolic features when different CO 2 and O 2 levels were imposed on sunflower leaves for two hours followed by untargeted metabolomics. Dellero et al. (2024) found significant metabolic differences when leaves or leaf discs were fed 13 C-glucose for 30 min to 6 hr. The focus of the work reported here was on initial events in the switch from CBB cycle to TCA cycle using 13 CO 2 and a semi-targeted metabolic approach. Our study does not address longer term changes such as gene expression or translation effects. Postillumination burst and light enhanced dark respiration We did not measure gas exchange, but our results provided some insight into the metabolism changes during the post-illumination burst and light enhanced dark respiration. The post illumination burst (PIB) of CO 2 thought to originate in photorespiratory metabolism (Gregory et al. 2024; Decker 1955; Rawsthorne and Hylton 1991) as 2-PG is converted to glycine is decarboxylated. We measured both 2-PG and glycine. The amount of glycine did not decline but in fact increased over time (Table S2). The amount of 2-PG declined significantly (two tailed T-test p=0.003) from 27.1 ± 6.8 to 5.8 ± 0.7 nmol metabolites g -1 FW over the first 10 s. It then recovered slightly but remained below the value in the light. However, glycine increased after 10 s and remained above the level in the light until 3 min after darkening. Thus, we did not see behavior of either 2-PG nor glycine that would be expected if the post-illumination burst was fed by either of these compounds, but it is possible that other metabolites were converted to glycine supporting the burst. It is also possible that the oxidative pentose phosphate pathway in either the plastid or cytosol, providing CO 2 . The plastidial glucose-6-phosphate dehydrogenase is normally off in the light but could be activated quickly in the dark (Preiser et al. 2019). While we could not distinguish between glucose and fructose 6-phosphate nor between plastidial and cytosolic pools, we note that hexose phosphates, the substrate for the oxidative pentose phosphate pathways, declined monotonically by 30 nmol g -1 FW by 10 min while for 2-PG the decline was just 21 nmol g -1 FW at 10 s after which it recovered (Supplemental Table S2). Another behavior seen when the light is turned off is a stimulation of oxygen uptake called light enhanced dark respiration. This is more common in bacteria and algae than plants (Shimakawa et al. 2020). It is not clear whether the metabolite changes reported here provide insight into the oxygen exchange at a light to dark transition indicative of light enhanced dark respiration. ATP/NADPH dynamics In the light high ATP/ADP and NADPH/NADP + ratios enforce metabolism to proceed from PGA to triose phosphates. Our data suggests that reducing power has more effect than ATP in limiting the CBB cycle when light is first reduced. There are several lines of evidence supporting redox power being the most important limitation. The ATP turn over time is slower than NADPH turnover time (Szecowka et al. 2013; Arrivault et al. 2009) (turnover time will reflect pool size divided by rate of metabolism through that metabolite). Chlorophyll fluorescence studies indicate that some back reaction, from triose phosphates to PGA, can occur (light grey arrows in Fig. 1) providing some ATP. This can give rise to a transient in chlorophyll fluorescence that is affected by the presence or absence of fructose bisphosphate aldolase and the triose phosphate-phosphate transporter (Gotoh et al. 2010a). This behavior has been studied to learn about the pathways for cyclic electron flow (Gotoh et al. 2010b). Jagendorf and Uribe (1966) showed that there can be a short post-illumination ATP synthesis from the stored proton motive force gradient across the thylakoid membrane. In addition, conversion of PEP to pyruvate (plus alanine) could supply ATP by substrate phosphorylation. Mitochondrial electron transport activity could also supply ATP but at the expense of reducing power. The rapid changes in ATP/NADPH ratios highlight the critical role of energy balance in regulating metabolic transitions (Hoefnagel et al. 1998). These findings suggest that plants have evolved precise mechanisms that determine metabolite levels, and which maintain energy homeostasis during light-dark transitions, ensuring survival in environments with unpredictable light, such as forest canopies or dense crop fields. The largest initial increase of metabolites of the CBB cycle after entering darkness, among those we measured, was PGA. The accumulation of PGA is advantageous since it can refill the CBB cycle without delay if the leaf is reilluminated after one min or less (Sharkey et al. 1986; Stitt 1986) though after 10 min of darkness the PGA pool dissipates and so would not be available for refilling the CBB cycle (Fig. 1). Oxygen evolution can exhibit a greater-than-steady-state rate upon reillumination of a leaf while the excess PGA is reduced to triose phosphates (Stitt 1986; Kirschbaum and Pearcy 1988; Sharkey et al. 1986). From triose phosphates to pentose phosphates no energy is required. Because there are sources of ATP, carbon converted to pentose phosphates can be phosphorylated to RuBP. Rubisco and phosphoribulokinase can remain at least partially active for many minutes after switching to low light (Sassenrath-Cole and Pearcy 1994); pentoses produced after the light is off can be phosphorylated to RuBP and then carboxylated providing a highly labeled input into the PGA pool. We saw evidence for this as both RuBP and the pentose phosphate pool continued to decline for 10 min after the light was off. The significant decline in RuBP in the first 10 sec was followed by a further small but measurable decline out to 10 min (Fig. 3A). By 30 s the PGA pool began to fall; PEP increased at 10 s but then was constant while pyruvate, and then alanine, increased over 10 min. A large pool of PGA allows very rapid resumption of photosynthesis once reducing power (NADPH) becomes available but when the carbon moves on to pyruvate it becomes more difficult to repopulate the CBB cycle to restart the cycle. One mechanism that has been proposed for restarting the CBB cycle is a cytosolic shunt involving the oxidative pentose phosphate pathway (Xu et al. 2024). This would inject carbon as ribulose 5-phosphate that, because there is ATP available, would be easily converted to RuBP and so restart the cycle. This has been proposed for both plants (Xu et al. 2021) and cyanobacteria (Tanaka et al. 2022). C4 plants often have larger metabolite pools that could buffer the effects seen here and improve use of light flecks (Stitt and Zhu 2014). We can describe three fates for PGA (Fig. 1). The normal route (green curved arrow) is for PGA to be converted to triose phosphates and eventually back to RuBP plus end products, usually mostly starch and sucrose. However, when there is no NADPH, PGA cannot be converted to triose phosphates. In this case, another pathway for PGA metabolism comes into play, conversion to PEP and then to pyruvate plus ADP (Fig. 1 light gray). The small amount of PEP at zero time (in the light) might indicate that the flow of carbon from PGA to PEP is normally low though the high degree of label indicates that carbon in PEP is in isotopic equilibrium with the CBB cycle. Within 10 s the amount of PEP increased and remained constant throughout the following 10 min. Because PGA was declining and pyruvate was increasing, the elevated but constant PEP may indicate significant but balanced synthesis and catabolism of PEP. Pyruvate There was a large pool of unlabeled pyruvate in the light. In darkness total pyruvate increased further and the increase in pyruvate was labeled. Pyruvate began to fall by 10 min and the degree of label fell, consistent with a large, metabolically in inert, pool plus an active pool that occurred for three min following light off. Pyruvate kinase activity is catalyzed by any of a large number of pyruvate kinases with distinct kinetics and expression patterns (Wulfert et al. 2020). Some pyruvate kinases are particularly susceptible to inhibition by ATP. Very high levels of PGA and relatively oxidized NAD + could lead to phosphoglycerate dehydrogenase activity (Krämer et al. 2024). This would provide some reducing power. Serine, a product of the pathway that begins with phosphoglycerate dehydrogenase, tripled in amount at 1 min darkness (Supplemental Table S2). The lower part of glycolysis involves conversion of 3-PGA to 2-phosphoglycerate to PEP and then pyruvate. Because chloroplasts have little to no activity of enzymes in this pathway, the production of PEP and then pyruvate must occur in the cytosol of photosynthesizing leaves although some pyruvate can be made by rubisco (Evans et al. 2024). The amount of PEP in the leaf increased rapidly but the degree of label stayed the same and similar to CBB cycle intermediates (Fig. 3C). These findings suggest that PGA acts as a critical metabolic hub, enabling plants to efficiently manage carbon resources in dynamic light environments. When darkness was imposed there was a large increase in the amount of pyruvate. The increase coincided with an increase in the degree of label indicating that much of the increase results from highly labeled PEP conversion to pyruvate. Between 3 and 10 min the amount of pyruvate declined as did the degree of label indicating that there is a large metabolically inert pool of pyruvate. It is known that a large amount of pyruvate is in the vacuole (Szecowka et al. 2013). The mitochondrial pyruvate dehydrogenase complex is regulated; phosphorylation results in inactivation of the complex (Budde and Randall 1990). While some activity of the PDH complex may still occur in the light, the rate of labelling of citrate is exceedingly slow (Calvin and Massini 1952). The chloroplast pyruvate dehydrogenase complex is not regulated by phosphorylation and so can supply acetyl CoA from pyruvate for fatty acid synthesis etc. in the light (Camp and Randall 1985). The time course of 13 C content of sedoheptulose 7-phosphate did not resemble either the upstream triose phosphates or the downstream pentose phosphates. We concluded that this resulted from a metabolically inert pool of sedoheptulose 1,7-bisphosphate (SBP) in the cytosol that could slowly exchange with SBP in the plastid as described in Xu et al. (2024). The TCA cycle appeared to increase in activity almost immediately. The amount of citrate increased from almost nothing as soon as 10 s after turning off the light (Fig. 5). The citrate and a-ketoglutarate increased monotonically for 10 min but the degree of label remained low (Table S2). Succinate, fumarate, and malate all showed a decrease in amount at 3 min and recovery at 10 min. This increase in TCA cycle intermediates would require anaplerotic reactions such as PEP carboxylase and activation of cytosolic PEP carboxylase to provide acetyl CoA to make citrate. This could account for the declining pyruvate at min 10 and, if transaminases were active, the very large pool of alanine could feed the anaplerotic reactions. The rapid increase in pyruvate and TCA cycle activation demonstrates the metabolic flexibility of leaves in transitioning from CBB to TCA cycles, highlighting mitochondrial respiration as a critical energy source that is quickly activated when photosynthesis ceases. Nitrogen metabolism Glutamine, aspartate, asparagine, and threonine all showed increases at 1 min but then a decrease in amount at 3 min and recovery at 10 min. Alanine was the notable exception, being very abundant at 3 min when nearly all other metabolites were down. Serine reached a maximum even earlier, at 30 s. This may indicate some role for alanine aminotransferases, a large family of enzymes (McAllister et al. 2013). Breakdown of alanine when it is in excess may be the primary function of alanine aminotransferases (Miyashita et al. 2007). Serine production during photorespiration has been invoked as a mechanism for supplying amino groups during photosynthesis (Busch et al. 2018; Fu et al. 2023); in the absence of photorespiration serine can be made by two other pathways both of which begin with PGA (Zimmermann et al. 2021; Igamberdiev and Kleczkowski 2018). It may be that the very high concentration of PGA at 10 and 30 s stimulated serine synthesis by one of these alternate mechanisms since the rate of carboxylation (and oxygenation) would be very low after 10 s but serine levels were highest at 30 s (Fig. 8 C and supplemental Table S2). Significant changes over time, with amino groups on alanine increasing substantially at min 3 may reflect the adjustment of nitrogen metabolism from serine metabolism in the presence of photorespiration to GS-GOGAT directly during a light-dark transient. The shifts in nitrogen metabolism, particularly the accumulation of alanine and serine, highlight the tight coordination between carbon and nitrogen metabolism during light-dark transitions. These changes suggest that plants dynamically reallocate nitrogen resources to support metabolic reprogramming in response to light fluctuations, enhancing resilience and ensuring efficient resource use in dynamic light environments. Conclusion Our results provide new insights into the carbon metabolism changes that occur when leaves transition from light to darkness. We show that the rapid depletion of CBB cycle intermediates and the activation of the TCA cycle enable a swift switch from chloroplast-based photosynthesis to mitochondrial respiration. In addition to the changes in carbon metabolism, nitrogen metabolism appears to undergo a large shift, highlighting their interconnectedness. This work adds another dimension to the studies of metabolism in a stochastic light environment, highlighting the metabolic flexibility of leaves and their ability to maintain homeostasis under changing light environments. This work examined a single step change from light to darkness, characterized by rapid shifts in carbon flow, energy production, and nitrogen assimilation. The ability to swiftly transition between photosynthetic and respiratory metabolic states provides plants with a competitive advantage in dynamic ecosystems, with potential significant implications for crop resilience and agricultural productivity. Future work might involve looking at low light availability, for example, what is the metabolic state near the light compensation point. Finally, this study may provide useful information for understanding carbon metabolism changes in leaves experiencing fluctuating light of different durations and may point to a role for changing nitrogen metabolism during light flecks. Declarations Funding: YX was supported by a Department of Energy grant from the Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences of the United States Department of Energy (Grant DE-FG02-91ER20021), SCS was supported by the MSU Plant Resilience Institute. TDS received partial salary support from Michigan AgBioResearch. Author Contributions: YX, SCS, and TDS conceived the project, YX and SCS carried out the experiments. YX carried out the mass spectrometry. TDS wrote the paper and YX and SCS edited the paper. The authors declare no competing interests. Conflicts of Interests The authors have no conflicts of interest to declare that are relevant to the content of this article. Acknowledgements : This work was supported by a grant from the Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences of the United States Department of Energy (Grant DE-FG02-91ER20021) and the MSU Plant Resilience Institute. TDS received partial salary support from Michigan AgBioResearch. Ethics and Consent to Participate declarations : not applicable Data availability All the data are available in the main text and in the Supporting Information. 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Supplementary Files 20250517SupplementalTables.xlsx Cite Share Download PDF Status: Published Journal Publication published 29 Sep, 2025 Read the published version in Photosynthesis Research → Version 1 posted Editorial decision: Revision requested 25 Jul, 2025 Reviews received at journal 25 Jul, 2025 Reviews received at journal 22 Jul, 2025 Reviewers agreed at journal 02 Jul, 2025 Reviewers agreed at journal 01 Jul, 2025 Reviewers invited by journal 01 Jul, 2025 Editor assigned by journal 14 Jun, 2025 Submission checks completed at journal 10 Jun, 2025 First submitted to journal 06 Jun, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6839843","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":479650414,"identity":"910cd1bb-4e8d-4387-ae59-5e98cbc2f82e","order_by":0,"name":"Yuan Xu","email":"","orcid":"","institution":"Michigan State University","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Xu","suffix":""},{"id":479650415,"identity":"e94a110f-c5f6-4031-97aa-f03a7629654b","order_by":1,"name":"Stephanie C. 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Sharkey","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYFACHiA+wMDAD+ExE6MBqkWygWQtBgeI1WLPf/bg54ozNombrx1/JsFQYZ3YQNAWibxkyTM30hK33c4xk2A4k06MFh4DyYYPh0Fa2CQY2w4ToYX/jPHPhg//EzfPTn8mwfiPGC0MOWaSDTcOJG6QTjCTYGwgRsuNHDPLhjPJxjNu5xhbJBxLNyaohb3/jPHNhmN2sv2z0x/e+FBjLUtQCww4glUmEKscBOxJUTwKRsEoGAUjDAAAA9tBGrXvracAAAAASUVORK5CYII=","orcid":"","institution":"Michigan State University","correspondingAuthor":true,"prefix":"","firstName":"Thomas","middleName":"D.","lastName":"Sharkey","suffix":""}],"badges":[],"createdAt":"2025-06-07 00:08:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6839843/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6839843/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11120-025-01173-2","type":"published","date":"2025-09-29T15:56:51+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":85969265,"identity":"51c51c80-c0bb-42ea-bf09-55007b993929","added_by":"auto","created_at":"2025-07-03 17:57:33","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":110451,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e13\u003c/sup\u003eC content of selected Calvin-Benson-Bassham (CBB) cycle intermediates and phosphoenolpyruvate, pyruvate, and alanine. Alanine values were divided by 5. Leaves were fed 420 ppm of \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e for 20 min. At time 0 the light was turned off and pool sizes of various metabolites were measured for 10 min (600 s). Blue arrows indicate CBB cycle fluxes while grey arrows denote metabolism that is stimulated for the first 3 min of darkness. Bars show mean of three biological replicates ± standard deviation. RuBP = ribulose 1,5-bisphosphate, PGA = 3-phosphoglycerate, DHAP = dihydroxyacetone phosphate, GAP = glyceraldehyde 3-phosphate, PEP = phosphoenolpyruvate, S7P = sedoheptulose 7-phosphate, P5P = pentose phosphates\u003c/p\u003e","description":"","filename":"Fig1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6839843/v1/4faf3fdb9d4826ed0d9c8b61.jpeg"},{"id":85969267,"identity":"5dbb34d5-c401-470c-ae24-b48f710b6b80","added_by":"auto","created_at":"2025-07-03 17:57:33","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":98688,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e13\u003c/sup\u003eC content of selected photorespiratory cycle intermediates. Leaves were fed 420 ppm of \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e for 20 min. At time 0 the light was turned off \u003csup\u003e13\u003c/sup\u003eC contents of various metabolites were measured for 10 min (600 s). RuBP = ribulose 1,5-bisphosphate, PGA = 3-phosphoglycerate, DHAP = dihydroxyacetone phosphate, GAP = glyceraldehyde 3-phosphate, PEP = phospho\u003cem\u003eenol\u003c/em\u003epyruvate, 2-PG = 2-phosphoglycoltae, S7P = sedoheptulose 7-phosphate, P5P = pentose phosphates. Orange shows the oxidative pentose phosphate pathway that could contribute to the post-illumination burst of CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e","description":"","filename":"Fig2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6839843/v1/364e44eb9da115e6c5d9e87e.jpeg"},{"id":85969270,"identity":"e59bfbc7-b66d-4bb8-86b7-0bcf95dc44b9","added_by":"auto","created_at":"2025-07-03 17:57:33","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":81546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003csup\u003e13\u003c/sup\u003eC contents and total amounts of selected photosynthetic intermediates. Leaves were fed 420 ppm of \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e for 20 min. At time 0 the light was turned off. A, \u003csup\u003e13\u003c/sup\u003eC content of ribulose 1,5-bisphosphate (the same data as in figure 1 but plotted differently). B, Degree of label of RuBP (orange squares) and PGA (blue circles). C, Degree of label (blue circles) and pool size (orange squares, from Fig. 1) of PEP = (phosphoenolpyruvate). D, Degree of label of pyruvate (blue circles) and pool size (orange squares)\u003c/p\u003e","description":"","filename":"Fig3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6839843/v1/8dd26fefe4b5d775f7addc63.jpeg"},{"id":85969508,"identity":"5248b526-94b2-4c07-8047-99b3f87b4577","added_by":"auto","created_at":"2025-07-03 18:05:33","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":30625,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of \u003csup\u003e13\u003c/sup\u003eC-labeled atoms in all metabolites measured.\u003c/p\u003e","description":"","filename":"Fig4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6839843/v1/e5b720c8c3856cfc80e93bfe.jpeg"},{"id":85970101,"identity":"c16059b6-4461-430b-afd2-c3f1d78c4fd5","added_by":"auto","created_at":"2025-07-03 18:13:33","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":141581,"visible":true,"origin":"","legend":"\u003cp\u003ePool sizes of selected tricarboxylic acid (TCA) cycle intermediates plus pyruvate, and alanine (from Figure 1). Leaves were fed 420 ppm of \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e for 20 minutes. At time 0 the light was turned off and pool sizes of various metabolites were measured over the next 10 min. Red arrows indicate TCA cycle fluxes. 𝛼-ketoglutarate (𝛼-KG) could not be measured but amino acids derived from 𝛼-KG, glutamate and glutamine, are shown. Similarly, amino acids made from oxaloacetate are shown. Data is the mean of three biological replicates ± standard deviation\u003c/p\u003e","description":"","filename":"Fig5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6839843/v1/24384b79e741e42512423064.jpeg"},{"id":85969287,"identity":"ed8aba74-2610-459c-be29-135399323c68","added_by":"auto","created_at":"2025-07-03 17:57:34","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":102936,"visible":true,"origin":"","legend":"\u003cp\u003eDegree of label and total \u003csup\u003e13\u003c/sup\u003eC atoms in selected metabolites. \u003cstrong\u003eA, C, E\u003c/strong\u003e, Degree of label for citrate and 5-carbon amino acids, 4-carbon metabolites, and other amino acids respectively. \u003cstrong\u003eB, G, F, \u003c/strong\u003e\u003csup\u003e13\u003c/sup\u003eC atoms in the metabolites.\u003c/p\u003e","description":"","filename":"Fig6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6839843/v1/fda315d1db35a3ce18f2bc12.jpeg"},{"id":85970160,"identity":"4436ebf4-1f12-4c3f-8ea2-4acde3a07108","added_by":"auto","created_at":"2025-07-03 18:21:33","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":68563,"visible":true,"origin":"","legend":"\u003cp\u003eRelative number of ¹³C-labeled carbon atoms in the indicated metabolites, , normalized to their respective maximum values.\u003c/p\u003e","description":"","filename":"Fig7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6839843/v1/230e56fb030c37ec1eeb7787.jpeg"},{"id":92883571,"identity":"7176d2dd-1605-4b45-bb1f-6f140ac3ff31","added_by":"auto","created_at":"2025-10-06 16:00:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1159218,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6839843/v1/d282650f-90a1-40fe-a0c4-5295a763679e.pdf"},{"id":85969516,"identity":"eac88280-0891-415a-b7bb-18ccdaa4435c","added_by":"auto","created_at":"2025-07-03 18:05:34","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":290057,"visible":true,"origin":"","legend":"","description":"","filename":"20250517SupplementalTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6839843/v1/a529008ac50cfb05fe930339.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Time-resolved targeted metabolomics shows an abrupt switch from Calvin- Benson-Bassham cycle to tricarboxylic acid cycle when the light is turned off","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant leaves use energy in sunlight for metabolic needs and carbon fixation by the Calvin-Benson-Bassham (CBB) cycle in the light. In the dark they rely on energy from glycolysis and the tricarboxylic acid (TCA). A significant amount of carbon fixed using sunlight energy during the day is stored as starch, which is then broken down at night to supply sugars for glycolysis and the TCA cycle. The speed with which the CBB cycle stops and the TCA cycle begins is unknown but may be important for understanding how carbon metabolism responds to fluctuating light.\u003c/p\u003e\n\u003cp\u003eLight fluctuations can occur over very short time periods, for example during leaf flutter of trembling aspen (Roden and Pearcy 1993) or over much longer periods, for example leaves at the bottom of the canopy (Shao et al. 2024; Durand et al. 2022) of trees and crop species. During the shortest light flecks, photosynthetic electron transport could be greatly affected (De Souza et al. 2022). The pools of the metabolites of \u0026nbsp;the CBB cycle are small, most compounds have half-lives of 1 s or less (Szecowka et al. 2013; Arrivault et al. 2009) and so even short light flecks may have a big influence on carbon metabolism and the activity of the CBB and TCA cycles\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt has been assumed that ribulose 1,5-bisphosphate (RuBP) production ceases immediately when a leaf is exposed to darkness. In fact post illumination CO\u003csub\u003e2\u003c/sub\u003e uptake has been taken as a measure of the pool of RuBP present when the light is turned off (Laisk et al. 1984). The need for reducing power traps metabolites of the CBB cycle in 3-phosphoglycerate (PGA). This requires regulation of downstream reactions so that metabolism can be reoriented from CBB cycle to heterotrophy and the TCA cycle.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese downstream reactions include lower glycolysis (from triose phosphates to pyruvate), which does not occur in chloroplasts (Stitt et al. 1978; Evans et al. 2024) preventing futile cycling. There is good evidence that the mitochondrial pyruvate dehydrogenase complex (mtPDC) is “inactivated by phosphorylation” in the light (Budde and Randall 1990). The chloroplast PDC is not inhibited allowing for fatty acid synthesis. While fatty acid synthesis is essential to the plant metabolic flux analysis indicates that it occurs at a low rate not likely to influence carbon metabolism rate in the light (Xu et al. 2022). Although a very small flow of recently fixed\u0026nbsp;\u003csup\u003e1\u003c/sup\u003e\u003csup\u003e3\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e from citrate to glutamine and glutamate by way of\u0026nbsp;a-ketoglutarate can be detected, for the four-carbon members of the TCA cycle, labeling is below the limits of detection\u0026nbsp;(Xu et al. 2022; Szecowka et al. 2013). The near total lack of activity of the TCA cycle in the light has been known for many years\u0026nbsp;Calvin and Massini (1952)\u0026nbsp;saw a very rapid increase in label in citrate upon darkness. The small flux toward\u0026nbsp;a-ketoglutarate in the light may allow stored citrate to contribute to nitrogen metabolism\u0026nbsp;(Gauthier et al. 2010; Abadie et al. 2017). Recently it has been recognized that, during photorespiration, serine metabolism associated with photorespiration may play a large role in amino acid metabolism\u0026nbsp;(Fu et al. 2023; Busch et al. 2018).\u003c/p\u003e\n\u003cp\u003eIn this study, we used time-resolved, targeted metabolomics to explore how carbon moves through metabolites during a transition from the CBB cycle to the TCA cycle, and how this transition influences energy metabolism and nitrogen assimilation. Unraveling these mechanisms is essential for understanding how plants maintain metabolic homeostasis and optimize energy utilization in dynamic light environments. The availability of stable isotopes (specifically \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e) provides a mechanism for following carbon when it is fed as \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e to photosynthesizing leaves. This avoids potential complications when molecules are fed through the transpiration stream.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHere we report on measurements in which \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003ewas fed to a photosynthesizing leaf for 20 min to achieve a quasi-steady-state (Xu et al. 2025) then abruptly turned off the light. We predicted that the change from CBB To TCA cycle would occur quickly because:\u0026nbsp;\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eCitrate and other TCA metabolites exhibit almost no labeling in the light but a very rapid rise in citrate labeling within seconds of turning the light off (Calvin and Massini 1952; Szecowka et al. 2013; Ma et al. 2014; Xu et al. 2022).\u003c/li\u003e\n \u003cli\u003eThe metabolite pools of the CBB cycle are known to have very short half-lives (Arrivault et al. 2009; Badger et al. 1984; Hasunuma et al. 2010).\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eWe did not monitor CO\u003csub\u003e2\u003c/sub\u003e evolution, which results in a burst of CO\u003csub\u003e2\u003c/sub\u003e release when leaves are first darkened. We do not address respiration in the light, sometimes denoted \u003cem\u003eR\u003csub\u003eL\u003c/sub\u003e\u003c/em\u003e but rely metabolic flux analysis that identified a time frame of 20 min as a semi steady state (Xu et al. 2022). Our intent was to resolve changes that occur in leaves over the first 10 min of darkness. Our results show a very rapid change from chloroplast CBB cycle carbon metabolism to mitochondria TCA metabolism.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003ePlant growth\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTobacco (\u003cem\u003eNicotiana tabacum\u003c/em\u003e, \u0026lsquo;Samsun\u0026rsquo;) plants transformed with \u003cem\u003ePopulus alba\u003c/em\u003e isoprene synthase (ISPS) were provided by Claudia Vickers from the University of Queensland. Details regarding plasmid design, vector construction, transformation, and line selection are described in Vickers et al. (2009). The IE lines were used as part of other experiments This made many very healthy plants available for this study. Plants were cultivated in greenhouses at the Michigan State University Plant Research Laboratory. The plants were maintained under a 16-hour photoperiod with a light intensity of 400\u0026ndash;500 \u0026micro;mol m⁻\u0026sup2; s⁻\u0026sup1; provided by sunlight supplemented by sodium vapor lamps. day/night temperatures were set to 25\u0026ndash;27\u0026deg;C/20\u0026ndash;22\u0026deg;C, and additive relative humidity was set to 60\u0026ndash;65%. Seeds were sown in Suremix growing medium (Michigan Grower Products, Galesburg, MI, USA) on separate trays. Fourteen days post-germination, seedlings were transplanted into small 3.5 L pots (five seedlings per pot) to ensure survival. Two weeks after transplantation, when seedlings were stable, they were transferred to larger 7 L pots (one plant per pot). Plants were watered with deionized water for the first two days, followed by one-half-strength Hoagland\u0026rsquo;s nutrient solution (Hoagland and Arnon 1938) for all subsequent \u0026nbsp;days. Experiments were conducted on 6- to 8-week-old plants before flowering and seed development. Three biological replicates were used.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSampling method\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe employed an in-house-modified Fast Kill freeze clamp, as described by Sharkey et al. (2020), for this study. The setup integrated a LI-COR 6800 head with a 6 cm \u0026times; 6 cm chamber. The chamber was sealed using cling film to create a closed environment and uniformly illuminated with two gooseneck fiber optic illuminators (Schott KL2500 LED lamps, obtained from Edmund Optics, https://www.edmundoptics.com) at a light intensity of 1,000 \u0026mu;mol m⁻\u0026sup2; s⁻\u0026sup1;. Chamber temperature was controlled and monitored using a thermocouple connected to the LI-6800 portable gas exchange system. The LI-COR 6800 instrument was configured to scrub all CO₂ from the system before introducing CO₂ from one of two sources: 5% \u0026sup1;\u0026sup2;CO₂ in air with natural isotopic abundance from Airgas (www.airgas.com) or a lecture bottle containing 99+% \u0026sup1;\u0026sup3;CO₂ (Aldrich, Sigma-Aldrich.com) pressurized with N₂. Flow meters (Alicat Scientific) regulated the delivery of \u0026sup1;\u0026sup2;CO₂ and \u0026sup1;\u0026sup3;CO₂. To achieve the desired CO₂ concentration, the \u0026sup1;\u0026sup2;CO₂ flow rate was adjusted based on the \u0026sup1;\u0026sup3;CO₂ concentration in the pressurized tank, using flow meter settings. The two flow meters were connected to the air supply entering the LI-COR 6800 head via a four-way valve. Leaves were equilibrated in the chamber under illumination for 30\u0026ndash;60 min until assimilation rates stabilized. At this point, the CO₂ source was switched to \u0026sup1;\u0026sup3;CO₂ and maintained for 20 min, corresponding to the second of three labeling phases (Xu et al. 2022). The \u0026sup1;\u0026sup3;CO₂ supply was regulated to maintain a consistent CO₂ concentration of 450 ppm, and the transition from \u0026sup1;\u0026sup2;CO₂ to ~90% \u0026sup1;\u0026sup3;CO₂ was achieved within one minute. After 20 min, the light was turned off, and the plants were kept in darkness under black light-blocking cloth for varying durations (0, 10, 30, 60, 180, or 600 sec). The Fast Kill mechanism was then triggered, using liquid-nitrogen-cooled copper blocks to rapidly freeze leaf samples. The interval between light interruption and the leaf sample reaching a temperature below 0 \u0026deg;C was measured at 35 ms (Sahu et al. 2023). Frozen leaf samples were collected in 2 mL microcentrifuge tubes and stored at \u0026minus;80 \u0026deg;C for subsequent mass spectrometry analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMass spectrometry\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMetabolites were extracted from flash-frozen tissues according to established protocols (Xu et al., 2021). Subsequent analyses were conducted via mass spectrometry, as detailed in previous reports (Xu et al., 2021; Xu et al., 2022). Specific MS parameters for multiple reaction monitoring (MRM) with LC\u0026ndash;MS/MS, selected ion monitoring (SIM), and GC\u0026ndash;MS are provided in Supplemental Table S1. Phosphorylated metabolites in the CBB cycle were analyzed using ion-pair chromatography\u0026ndash;tandem mass spectrometry (IPC\u0026ndash;MS/MS). Separation was performed on a 2.1 \u0026times; 50 mm ACQUITY UPLC BEH C18 column (Waters, Milford, MA, USA) connected to an ACQUITY UPLC pump system coupled with a Waters XEVO TQ-S UPLC/MS/MS (Waters, Milford, MA, USA).\u003c/p\u003e\n\u003cp\u003eNucleotide sugars and other phosphorylated intermediates were measured by anion exchange chromatography\u0026ndash;tandem mass spectrometry (AEC\u0026ndash;MS/MS). Samples were run on a 2 \u0026times; 250 mm IonPac AS11 analytical column with a 2 \u0026times; 50 mm IonPac AG11 guard column (Dionex), using an ACQUITY UPLC pump system (Waters, Milford, MA, USA) coupled to a Xevo ACQUITY TQ Triple Quadrupole Detector (Waters, Milford, MA, USA). A self-regenerating suppressor (Dionex ADRS 600, Thermo Scientific, Waltham, MA, USA) was employed post-column to neutralize the KOH eluent.\u003c/p\u003e\n\u003cp\u003eAmino acids and organic acids were examined by GC\u0026ndash;MS using an Agilent 7890 GC system interfaced with an Agilent 5975C inert XL Mass Selective Detector (Agilent, Santa Clara, CA, USA). Initial derivatization with methoxyamine hydrochloride in dry pyridine was followed by silylation of amino and organic acids to form TBDMS derivatives (using N-(tertbutyldimethylsilyl)-N-methyltrifluoroacetamide with 1% [w/v] tert-butyldimethylchlorosilane). Separation was achieved on an Agilent VF5ms GC column (Agilent, Santa Clara, CA, USA).\u003c/p\u003e\n\u003cp\u003eMass spectrometry data were analyzed to quantify pool size, mass isotopologue distributions (MIDs), and \u0026sup1;\u0026sup3;C enrichment, following previously described methods (Xu et al. 2022) LC\u0026ndash;MS/MS data were acquired using MassLynx 4.0 (Agilent, Santa Clara, CA, USA), while GC\u0026ndash;MS data were obtained with Agilent GC/MSD Chemstation (Agilent, Santa Clara, CA, USA). Peak detection and quantification were performed using the TargetLynx Application Manager within Waters MassLynx\u0026trade; Software (Waters Corporation, MA). MIDs for GC\u0026ndash;MS\u0026ndash;measured metabolites were corrected for natural isotopic abundance using FluxFix (Trefely et al. 2016).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eCalvin-Benson-Bassham cycle decline\u003c/em\u003e\u003cem\u003es\u003c/em\u003e\u003cem\u003e\u0026nbsp;when the light is turned off\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe distinguish among three measures of each metabolite-\u003c/p\u003e\n\u003cp\u003e(a) the total amount of the metabolite, nmol metabolite g\u003csup\u003e-1\u003c/sup\u003e FW\u003c/p\u003e\n\u003cp\u003e(b) the relative degree of label incorporation, 0 %, all carbons are \u003csup\u003e12\u003c/sup\u003eC; 100%, all carbons are \u003csup\u003e13\u003c/sup\u003eC\u003c/p\u003e\n\u003cp\u003e(c) the amount of \u003csup\u003e13\u003c/sup\u003eC atoms in each metabolite pool, nmol \u003csup\u003e13\u003c/sup\u003eC g\u003csup\u003e-1\u003c/sup\u003e FW (= a times b times number of carbons in the molecule).\u003c/p\u003e\n\u003cp\u003eWhen the light was abruptly turned off the amount of ribulose 1,5-bisphosphate (RuBP) fell by almost 90% at the first time point of 10 s (Fig. 1). Other metabolites of the CBB cycle also fell except for PGA and sedoheptulose 7-phosphate (S7P). The increase in PGA was four-fold greater than the decrease in RuBP (two-fold greater would be expected because of RuBP carboxylation) indicating that pools other than RuBP contributed to the PGA accumulation. The S7P data differs from other CBB cycle intermediates, which may be related to a pool of sedoheptulose 1,7-bisphosphate (SBP) in the cytosol. This can cause the S7P labeling to be slow, as unlabeled SBP reenters the chloroplast (Xu et al. 2024) and so S7P is ignored in further analyses.\u003c/p\u003e\n\u003cp\u003eThe photorespiratory intermediate 2-phosphglycolate (2-PG) fell significantly in the first 10 s, mirroring the change in RuBP (Fig. 2). Glycine fell at 3 and 10 min but was stable up to 1 min. Serine and glycerate were present at much higher levels than 2PG and glycine (note the scales in Fig. 2). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe amount of \u003csup\u003e13\u003c/sup\u003eC in RuBP fell 87% in the first 10 s of darkness (Fig. 3A). This decline was similar to the decline in RuBP content (Fig. 1), which reflected in a very small change in the degree of label in RuBP (Fig. 3B). The degree of label in RuBP and PGA (%\u003csup\u003e13\u003c/sup\u003eC) was similar at time zero (Fig. 3B). Over 10 min, the percentage of carbon atoms that were \u003csup\u003e13\u003c/sup\u003eC in RuBP fell from 95% to 77% but the degree of label was noticeably higher in PGA (Fig. 3B). Any carboxylation that may have occurred in the dark would be supplying 99% \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e to the one-sixth of PGA resulting from carboxylation of RuBP. We expect slow fluxes of unlabeled carbon to RuBP possibly through the cytosolic G6P shunt (Xu et al. 2024) and other metabolism of unlabeled molecules.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe initial increase in PGA (Fig. 1) coincided with an increase in PEP (Fig. 3C). Carbon eventually ended up in pyruvate The triose phosphate transporter transports PGA as efficiently as glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (Flügge and Heldt 1991). Conversion of PGA to pyruvate inside the chloroplasts is negligible (Evans et al. 2024). Presumably PEP acts as an intermediate, which after the first 10 sec, filling and emptying at rates that match, keeping the total pool size constant. The amount of PEP increased initially while the degree of label remained high (Fig. 3C), indicating that the carbon in PEP came from carbon in the highly labeled CBB cycle. On the other hand, \u003cem\u003elabel\u003c/em\u003e in pyruvate was low in the light (zero time) but increased for three min. This increase in \u003csup\u003e13\u003c/sup\u003eC was paralleled by an increase in total \u003cem\u003eamount\u003c/em\u003e of pyruvate. (Fig. 3D).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eTransition from Calvin-Benson-Bassham cycle to the tricarboxylic acid cycle\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eLabeling of pyruvate was limited during photosynthesis (Fig. 3D) given that PEP was heavily labeled (Fig. 3C). The degree of label in pyruvate never exceeded 62% (Supplemental Table S2). The large increase in PEP for the first few seconds did not result in any dilution of the label in PEP indicating that the increase in PEP resulted from conversion of the active pool of PGA to PEP. This reaction sequence is kept low in the light given that the amount of PEP was low at time zero. This suggests that pyruvate kinase in the cytosol is highly regulated and can become very active rapidly in the dark leading to an increase in heavily labeled PEP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThere appeared to be a large unlabeled pool of pyruvate. When darkness was imposed, both the amount and degree of label in pyruvate increased for 3 min and then fell (Fig 1. and 3D). This indicates that much of the increase in pyruvate resulted from PEP conversion to pyruvate. Between 3 and 10 min, pyruvate declined. Citrate increased and the degree of increase in citrate exceeded the decline in \u003csup\u003e13\u003c/sup\u003eC in pyruvate. By 10 min the amount of pyruvate declined as did the degree of label indicating that there is a large metabolically inert pool of pyruvate. It is known that a large amount of pyruvate is in the vacuole (Szecowka et al. 2013).\u003c/p\u003e\n\u003cp\u003eWhen the metabolites measured here were summed up, it appeared that the total \u003csup\u003e13\u003c/sup\u003eC content increased significantly in the first 10 s and climbed at a slower rate for the next min (Fig. 4) but changed very little between 1 and 10 min. Post-illumination CO\u003csub\u003e2\u003c/sub\u003e fixation attributable to carboxylation of RuBP (77.5 \u003csup\u003e13\u003c/sup\u003eC nmol atoms g\u003csup\u003e-1\u003c/sup\u003e FW, Fig. 2 and supplemental Table S2) was not sufficient to account for the extra \u003csup\u003e13\u003c/sup\u003eC atoms in citrate (12,398 \u003csup\u003e13\u003c/sup\u003eC nmol atoms g\u003csup\u003e-1\u003c/sup\u003e FW). We presume that a more comprehensive accounting, and accounting for other metabolites being converted to RuBP, might explain the higher-than-expected \u003csup\u003e13\u003c/sup\u003eC content of citrate.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe tricarboxylic acid pathway response to darkness\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe amount of citrate jumped in the first 10 s but the additional \u003csup\u003e13\u003c/sup\u003eC accounted for just 2% of this increase in citrate amount (Fig. 5 and Supplemental Table S2). Citrate, glutamate (as a proxy for\u0026nbsp;a-ketoglutarate), and glutamine increased over the whole 10 min although glutamine, and to a lesser degree glutamate, did not increase as much as would be expected at 3 min (Fig. 5). These compounds had a low degree of label (Fig. 6A) and so most of the change in total content \u0026nbsp;was from unlabeled sources, in line with Gauthier et al. (2010) and Abadie et al. (2017).. The four-carbon metabolites succinate, fumarate and, to some degree malate, plus amino acids derived from oxaloacetate) exhibited a distinct peak at 1 min, then declined at 3 min but increased again at 10 min (Fig. 6 and Supplemental Table S2). The 4-carbon members of the TCA cycle, except malate, also had a low degree of label and low content of \u003csup\u003e13\u003c/sup\u003eC (Fig. 6C and 6D). Malate labeling was higher than succinate and fumarate but was still low (Fig. 7C) This could indicate an active PEP carboxylase. The low and relatively constant degree of label in succinate, fumarate, and malate resulted in little change to the amount \u003csup\u003e13\u003c/sup\u003eC in these metabolites (Fig. 6C).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAmino acids\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe amount of glutamate increased over the 10 min of darkness, much like citrate (Fig. 5). On the other hand, changes in the amount of glutamine were more like other amino acids (Fig.5). Alanine and serine were heavily labeled and remained so for 10 min (Fig. 6E), but aspartate, asparagine and threonine were relatively unlabeled (Fig. 7E). Unlike the \u003cem\u003edegree\u003c/em\u003e of label, the \u003cem\u003eamount\u003c/em\u003e of label in serine peaked at 30 s while there was an even larger pool of alanine at 3 min (Fig. 6F). This indicated that these pools filled with carbon from the CBB cycle and then emptied. The increase happened later for alanine but to a much greater extent (Fig. 6F). The large amount of nitrogen in alanine at 3 min coincided with a dip in other amino acids (Fig. 5).\u003c/p\u003e\n\u003cp\u003eTo look for patterns we plotted the amount of \u003csup\u003e13\u003c/sup\u003eC atoms relative to the maximum for that metabolite (Fig. 7). Citrate and glutamate showed a steadily increasing relative amount of label while glutamine showed a temporary drop at 3 min (Fig. 7A), similar to most other amino acids. Succinate, fumarate, and malate all showed a dip at 3 min (Fig. 7B). Most of the amino acids had distinct minima at 3 min with the notable exception of alanine which had a very large peak in relative amount at 3 min (Fig.7C). This was also seen in the total amount of \u003csup\u003e13\u003c/sup\u003eC in alanine (Fig. 6F). The degree of label in alanine was high and did not change over time (Fig. 6E) indicating that the large increase in alanine came from other CBB cycle intermediates.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe focus of this study was the change in carbon metabolism as the CBB cycle stops and TCA cycle begins. Carbon metabolism in leaves can be complex and involve many different pathways; how light or darkness will affect these pathways is difficult to discern (Tcherkez et al. 2024). There are many changes in plant cell metabolism that can be detected over a longer time frame. For example Abadie et al. (2021) found as many as 4,500 metabolic features when different CO\u003csub\u003e2\u003c/sub\u003e and O\u003csub\u003e2\u003c/sub\u003e levels were imposed on sunflower leaves for two hours followed by untargeted metabolomics. Dellero et al. (2024) found significant metabolic differences when leaves or leaf discs were fed \u003csup\u003e13\u003c/sup\u003eC-glucose for 30 min to 6 hr. The focus of the work reported here was on initial events in the switch from CBB cycle to TCA cycle using \u003csup\u003e13\u003c/sup\u003eCO\u003csub\u003e2\u003c/sub\u003e and a semi-targeted metabolic approach. Our study does not address longer term changes such as gene expression or translation effects.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePostillumination burst and light enhanced dark respiration\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe did not measure gas exchange, but our results provided some insight into the metabolism changes during the post-illumination burst and light enhanced dark respiration. The post\u0026nbsp;illumination burst (PIB)\u0026nbsp;of CO\u003csub\u003e2\u003c/sub\u003e thought to originate in photorespiratory metabolism (Gregory et al. 2024; Decker 1955; Rawsthorne and Hylton 1991) as 2-PG is converted to glycine is decarboxylated. We measured both 2-PG and glycine. The amount of glycine did not decline but in fact increased over time (Table S2). The amount of 2-PG declined significantly (two tailed T-test p=0.003) from 27.1\u0026nbsp;±\u0026nbsp;6.8 to 5.8\u0026nbsp;±\u0026nbsp;0.7 nmol metabolites g\u003csup\u003e-1\u003c/sup\u003e FW over the first 10 s. It then recovered slightly but remained below the value in the light. However, glycine increased after 10 s and remained above the level in the light until 3 min after darkening. Thus, we did not see behavior of either 2-PG nor glycine that would be expected if the post-illumination burst was fed by either of these compounds, but it is possible that other metabolites were converted to glycine supporting the burst. It is also possible that the oxidative pentose phosphate pathway in either the plastid or cytosol, providing CO\u003csub\u003e2\u003c/sub\u003e. The plastidial glucose-6-phosphate dehydrogenase is normally off in the light but could be activated quickly in the dark\u0026nbsp;(Preiser et al. 2019). While we could not distinguish between glucose and fructose 6-phosphate nor between plastidial and cytosolic pools, we note that hexose phosphates, the substrate for the oxidative pentose phosphate pathways, declined monotonically by 30 nmol g\u003csup\u003e-1\u003c/sup\u003e FW by 10 min while for 2-PG the decline was just 21 nmol g\u003csup\u003e-1\u003c/sup\u003e FW at 10 s after which it recovered (Supplemental Table S2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnother behavior seen when the light is turned off is a stimulation of oxygen uptake called light enhanced dark respiration. This is more common in bacteria and algae than plants (Shimakawa et al. 2020). It is not clear whether the metabolite changes reported here provide insight into the oxygen exchange at a light to dark transition indicative of light enhanced dark respiration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eATP/NADPH dynamics\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn the light high ATP/ADP and NADPH/NADP\u003csup\u003e+\u003c/sup\u003e ratios enforce metabolism to proceed from PGA to triose phosphates. Our data suggests that reducing power has more effect than ATP in limiting the CBB cycle when light is first reduced. There are several lines of evidence supporting redox power being the most important limitation.\u003c/p\u003e\n\u003col\u003e\n \u003cli\u003eThe ATP turn over time is slower than NADPH turnover time (Szecowka et al. 2013; Arrivault et al. 2009) (turnover time will reflect pool size divided by rate of metabolism through that metabolite).\u003c/li\u003e\n \u003cli\u003eChlorophyll fluorescence studies indicate that some back reaction, from triose phosphates to PGA, can occur (light grey arrows in Fig. 1) providing some ATP. This can give rise to a transient in chlorophyll fluorescence that is affected by the presence or absence of fructose bisphosphate aldolase and the triose phosphate-phosphate transporter (Gotoh et al. 2010a). This behavior has been studied to learn about the pathways for cyclic electron flow (Gotoh et al. 2010b).\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eJagendorf and Uribe (1966) showed that there can be a short post-illumination ATP synthesis from the stored proton motive force gradient across the thylakoid membrane. In addition, conversion of PEP to pyruvate (plus alanine) could supply ATP by substrate phosphorylation. Mitochondrial electron transport activity could also supply ATP but at the expense of reducing power.\u0026nbsp;\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003eThe rapid changes in ATP/NADPH ratios highlight the critical role of energy balance in regulating metabolic transitions (Hoefnagel et al. 1998). These findings suggest that plants have evolved precise mechanisms that determine metabolite levels, and which maintain energy homeostasis during light-dark transitions, ensuring survival in environments with unpredictable light, such as forest canopies or dense crop fields.\u003c/p\u003e\n\u003cp\u003eThe largest initial increase of metabolites of the CBB cycle after entering darkness, among those we measured, was PGA. The accumulation of PGA is advantageous since it can refill the CBB cycle without delay if the leaf is reilluminated after one min or less (Sharkey et al. 1986; Stitt 1986) though after 10 min of darkness the PGA pool dissipates and so would not be available for refilling the CBB cycle (Fig. 1). Oxygen evolution can exhibit a greater-than-steady-state rate upon reillumination of a leaf while the excess PGA is reduced to triose phosphates (Stitt 1986; Kirschbaum and Pearcy 1988; Sharkey et al. 1986).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFrom triose phosphates to pentose phosphates no energy is required. Because there are sources of ATP, carbon converted to pentose phosphates can be phosphorylated to RuBP. Rubisco and phosphoribulokinase can remain at least partially active for many minutes after switching to low light (Sassenrath-Cole and Pearcy 1994); pentoses produced after the light is off can be phosphorylated to RuBP and then carboxylated providing a highly labeled input into the PGA pool. We saw evidence for this as both RuBP and the pentose phosphate pool continued to decline for 10 min after the light was off. The significant decline in RuBP in the first 10 sec was followed by a further small but measurable decline out to 10 min (Fig. 3A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBy 30 s the PGA pool began to fall; PEP increased at 10 s but then was constant while pyruvate, and then alanine, increased over 10 min. A large pool of PGA allows very rapid resumption of photosynthesis once reducing power (NADPH) becomes available but when the carbon moves on to pyruvate it becomes more difficult to repopulate the CBB cycle to restart the cycle. One mechanism that has been proposed for restarting the CBB cycle is a cytosolic shunt involving the oxidative pentose phosphate pathway (Xu et al. 2024). This would inject carbon as ribulose 5-phosphate that, because there is ATP available, would be easily converted to RuBP and so restart the cycle. This has been proposed for both plants (Xu et al. 2021) and cyanobacteria (Tanaka et al. 2022). C4 plants often have larger metabolite pools that could buffer the effects seen here and improve use of light flecks (Stitt and Zhu 2014).\u003c/p\u003e\n\u003cp\u003eWe can describe three fates for PGA (Fig. 1). The normal route (green curved arrow) is for PGA to be converted to triose phosphates and eventually back to RuBP plus end products, usually mostly starch and sucrose. However, when there is no NADPH, PGA cannot be converted to triose phosphates. In this case, another pathway for PGA metabolism comes into play, conversion to PEP and then to pyruvate plus ADP (Fig. 1 light gray). The small amount of PEP at zero time (in the light) might indicate that the flow of carbon from PGA to PEP is normally low though the high degree of label indicates that carbon in PEP is in isotopic equilibrium with the CBB cycle. Within 10 s the amount of PEP increased and remained constant throughout the following 10 min. Because PGA was declining and pyruvate was increasing, the elevated but constant PEP may indicate significant but balanced synthesis and catabolism of PEP.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003ePyruvate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThere was a large pool of unlabeled pyruvate in the light. In darkness total pyruvate increased further and the increase in pyruvate was labeled. Pyruvate began to fall by 10 min and the degree of label fell, consistent with a large, metabolically in inert, pool plus an active pool that occurred for three min following light off.\u0026nbsp;Pyruvate kinase activity is catalyzed by any of a large number of pyruvate kinases with distinct kinetics and expression patterns (Wulfert et al. 2020). Some pyruvate kinases are particularly susceptible to inhibition by ATP.\u0026nbsp;Very high levels of PGA and relatively oxidized NAD\u003csup\u003e+\u003c/sup\u003e could lead to phosphoglycerate dehydrogenase activity\u0026nbsp;(Krämer et al. 2024). This would provide some reducing power. Serine, a product of the pathway that begins with phosphoglycerate dehydrogenase, tripled in amount at 1 min darkness (Supplemental Table S2).\u003c/p\u003e\n\u003cp\u003eThe lower part of glycolysis involves conversion of 3-PGA to 2-phosphoglycerate to PEP and then pyruvate. Because chloroplasts have little to no activity of enzymes in this pathway, the production of PEP and then pyruvate must occur in the cytosol of photosynthesizing leaves although some pyruvate can be made by rubisco (Evans et al. 2024). The amount of PEP in the leaf increased rapidly but the degree of label stayed the same and similar to CBB cycle intermediates (Fig. 3C). These findings suggest that PGA acts as a critical metabolic hub, enabling plants to efficiently manage carbon resources in dynamic light environments.\u003c/p\u003e\n\u003cp\u003eWhen darkness was imposed there was a large increase in the amount of pyruvate. The increase coincided with an increase in the degree of label indicating that much of the increase results from highly labeled PEP conversion to pyruvate. Between 3 and 10 min the amount of pyruvate declined as did the degree of label indicating that there is a large metabolically inert pool of pyruvate. It is known that a large amount of pyruvate is in the vacuole (Szecowka et al. 2013). The mitochondrial pyruvate dehydrogenase complex is regulated; phosphorylation results in inactivation of the complex (Budde and Randall 1990). While some activity of the PDH complex may still occur in the light, the rate of labelling of citrate is exceedingly slow (Calvin and Massini 1952). The chloroplast pyruvate dehydrogenase complex is not regulated by phosphorylation and so can supply acetyl CoA from pyruvate for fatty acid synthesis etc. in the light (Camp and Randall 1985).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe time course of \u003csup\u003e13\u003c/sup\u003eC content of sedoheptulose 7-phosphate did not resemble either the upstream triose phosphates or the downstream pentose phosphates. We concluded that this resulted from a metabolically inert pool of sedoheptulose 1,7-bisphosphate (SBP) in the cytosol that could slowly exchange with SBP in the plastid as described in Xu et al. (2024).\u003c/p\u003e\n\u003cp\u003eThe TCA cycle appeared to increase in activity almost immediately. The amount of citrate increased from almost nothing as soon as 10 s after turning off the light (Fig. 5). The citrate and\u0026nbsp;a-ketoglutarate increased monotonically for 10 min but the degree of label remained low (Table S2). Succinate, fumarate, and malate all showed a decrease in amount at 3 min and recovery at 10 min. This increase in TCA cycle intermediates would require anaplerotic reactions such as PEP carboxylase and activation of cytosolic PEP carboxylase to provide acetyl CoA to make citrate. This could account for the declining pyruvate at min 10 and, if transaminases were active, the very large pool of alanine could feed the anaplerotic reactions. The rapid increase in pyruvate and TCA cycle activation demonstrates the metabolic flexibility of leaves in transitioning from CBB to TCA cycles, highlighting mitochondrial respiration as a critical energy source that is quickly activated when photosynthesis ceases.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNitrogen metabolism\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eGlutamine, aspartate, asparagine, and threonine all showed increases at 1 min but then a decrease in amount at 3 min and recovery at 10 min. Alanine was the notable exception, being very abundant at 3 min when nearly all other metabolites were down. Serine reached a maximum even earlier, at 30 s. This may indicate some role for alanine aminotransferases, a large family of enzymes (McAllister et al. 2013). Breakdown of alanine when it is in excess may be the primary function of alanine aminotransferases (Miyashita et al. 2007). Serine production during photorespiration has been invoked as a mechanism for supplying amino groups during photosynthesis (Busch et al. 2018; Fu et al. 2023); in the absence of photorespiration serine can be made by two other pathways both of which begin with PGA \u0026nbsp;(Zimmermann et al. 2021; Igamberdiev and Kleczkowski 2018). It may be that the very high concentration of PGA at 10 and 30 s stimulated serine synthesis by one of these alternate mechanisms since the rate of carboxylation (and oxygenation) would be very low after 10 s but serine levels were highest at 30 s (Fig. 8 C and supplemental Table S2). Significant changes over time, with amino groups on alanine increasing substantially at min 3 may reflect the adjustment of nitrogen metabolism from serine metabolism in the presence of photorespiration to GS-GOGAT directly during a light-dark transient. The shifts in nitrogen metabolism, particularly the accumulation of alanine and serine, highlight the tight coordination between carbon and nitrogen metabolism during light-dark transitions. These changes suggest that plants dynamically reallocate nitrogen resources to support metabolic reprogramming in response to light fluctuations, enhancing resilience and ensuring efficient resource use in dynamic light environments.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur results provide new insights into the carbon metabolism changes that occur when leaves transition from light to darkness. We show that the rapid depletion of CBB cycle intermediates and the activation of the TCA cycle enable a swift switch from chloroplast-based photosynthesis to mitochondrial respiration. In addition to the changes in carbon metabolism, nitrogen metabolism appears to undergo a large shift, highlighting their interconnectedness. This work adds another dimension to the studies of metabolism in a stochastic light environment, highlighting the metabolic flexibility of leaves and their ability to maintain homeostasis under changing light environments. This work examined a single step change from light to darkness, characterized by rapid shifts in carbon flow, energy production, and nitrogen assimilation. The ability to swiftly transition between photosynthetic and respiratory metabolic states provides plants with a competitive advantage in dynamic ecosystems, with potential significant implications for crop resilience and agricultural productivity. Future work might involve looking at low light availability, for example, what is the metabolic state near the light compensation point. Finally, this study may provide useful information for understanding carbon metabolism changes in leaves experiencing fluctuating light of different durations and may point to a role for changing nitrogen metabolism during light flecks.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eFunding:\u0026nbsp;YX was supported by a Department of Energy grant from the Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences of the United States Department of Energy (Grant DE-FG02-91ER20021), SCS was supported by the MSU Plant Resilience Institute. TDS received partial salary support from Michigan AgBioResearch.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e YX, SCS, and TDS conceived the project, YX and SCS carried out the experiments. YX carried out the mass spectrometry. TDS wrote the paper and YX and SCS edited the paper.\u0026nbsp;The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare that are relevant to the content of this article.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: This work was supported by a grant from the Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences of the United States Department of Energy (Grant DE-FG02-91ER20021) and the MSU Plant Resilience Institute. TDS received partial salary support from Michigan AgBioResearch.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and Consent to Participate declarations\u003c/strong\u003e: not applicable\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAll the data are available in the main text and in the Supporting Information.\u003c/em\u003e\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAbadie C, Lalande J, Limami AM, Tcherkez G (2021) Non-targeted \u003csup\u003e13\u003c/sup\u003eC metabolite analysis demonstrates broad re‐orchestration of leaf metabolism when gas exchange conditions vary. 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Plant Physiol 186(3):1487\u0026ndash;1506. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1093/plphys/kiab167\u003c/span\u003e\u003cspan address=\"10.1093/plphys/kiab167\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"photosynthesis-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pres","sideBox":"Learn more about [Photosynthesis Research](http://link.springer.com/journal/11120)","snPcode":"11120","submissionUrl":"https://submission.nature.com/new-submission/11120/3","title":"Photosynthesis Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Calvin Benson Bassham cycle, Light transition, Photosynthetic carbon metabolism, Respiration, Tricarboxylic Acid Cycle","lastPublishedDoi":"10.21203/rs.3.rs-6839843/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6839843/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn leaves, major CO₂ fluxes alternate between fixation by the Calvin-Benson-Bassham (CBB) cycle during light and release by the tricarboxylic acid (TCA) cycle in darkness. The speed at which leaf metabolism transitions between these pathways likely influences plant tolerance to fluctuating light conditions. To investigate these rapid metabolic shifts, we exposed leaves to \u0026sup1;\u0026sup3;CO₂ for 20 minutes to establish a quasi-steady state before abruptly turning off the light while maintaining \u0026sup1;\u0026sup3;CO₂ feeding. Within 10 seconds of dark transition, 3-phosphoglycerate levels rose dramatically, while most other CBB cycle intermediates decreased by more than 90%. Simultaneously, carbon accumulated in alanine, likely via pyruvate. Over the subsequent 10 minutes, six- and five-carbon TCA cycle intermediates steadily increased. In contrast, four-carbon TCA intermediates peaked at one minute, declined by three minutes, and rose again at 10 minutes, a pattern mirrored by most measured amino acids. These results reveal an exceptionally rapid metabolic reconfiguration from CO₂ fixation by the CBB cycle in light to TCA cycle activation for energy production in darkness, accompanied by substantial changes in amino acid metabolism.\u003c/p\u003e","manuscriptTitle":"Time-resolved targeted metabolomics shows an abrupt switch from Calvin- Benson-Bassham cycle to tricarboxylic acid cycle when the light is turned off","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-03 17:57:29","doi":"10.21203/rs.3.rs-6839843/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-25T22:46:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-25T21:48:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-22T06:14:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"165931310745611411153809503969874411479","date":"2025-07-02T13:13:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"113922415412502531468663469640969501486","date":"2025-07-01T06:21:33+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-01T04:55:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-06-14T16:14:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-06-10T04:55:04+00:00","index":"","fulltext":""},{"type":"submitted","content":"Photosynthesis Research","date":"2025-06-07T00:01:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"photosynthesis-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pres","sideBox":"Learn more about [Photosynthesis Research](http://link.springer.com/journal/11120)","snPcode":"11120","submissionUrl":"https://submission.nature.com/new-submission/11120/3","title":"Photosynthesis Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c3424c05-e11b-4986-944c-b48b8bf25a36","owner":[],"postedDate":"July 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-10-06T15:58:34+00:00","versionOfRecord":{"articleIdentity":"rs-6839843","link":"https://doi.org/10.1007/s11120-025-01173-2","journal":{"identity":"photosynthesis-research","isVorOnly":false,"title":"Photosynthesis Research"},"publishedOn":"2025-09-29 15:56:51","publishedOnDateReadable":"September 29th, 2025"},"versionCreatedAt":"2025-07-03 17:57:29","video":"","vorDoi":"10.1007/s11120-025-01173-2","vorDoiUrl":"https://doi.org/10.1007/s11120-025-01173-2","workflowStages":[]},"version":"v1","identity":"rs-6839843","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6839843","identity":"rs-6839843","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}