From Photochemistry to Flux: Toward Quantitative Photosynthetic Bioenergetics

1. Introduction

Reduction of one molecule of CO₂ through the Calvin–Benson–Bassham (CBB) cycle requires approximately three ATP and two NADPH, corresponding to an ATP/NADPH demand ratio of about 1.5 (Kramer and Evans, 2010; Walker et al., 2020). Linear electron flow (LEF) is estimated to generate an ATP/NADPH ratio of approximately 1.28-1.5, depending on the assumed proton-to-ATP stoichiometry (Kramer and Evans, 2010). However, cellular energetic demand is highly dynamic because photorespiration, nutrient assimilation, protein repair, ion transport, carbon-concentrating mechanisms and stress metabolism continuously alter both ATP and NADPH requirements (Kramer and Evans, 2010; Walker et al., 2020).

Photosynthetic organisms compensate through a network of alternative electron pathways. Cyclic electron flow (CEF) around PSI increases proton-motive force without net NADPH production (Yamori and Shikanai, 2016; Yamori et al., 2016), flavodiiron-dependent electron transport can divert electrons toward O₂ (Chaux et al., 2017), plastid terminal oxidase (PTOX) can contribute to plastoquinol oxidation (Krieger-Liszkay and Feilke, 2016), and Mehler-type reactions provide additional oxygen-dependent electron sinks (Badger et al., 2000; Makino, Miyake and Yokota, 2002). Reducing equivalents can also be exchanged between chloroplasts and mitochondria through metabolite shuttles (Raghavendra and Padmasree, 2003; Fakhimi and Grossman, 2024). These pathways therefore operate as an interconnected network in which perturbation of one component can be compensated by others (Levin, 2024; Peltier et al., 2024).

Although genetic disruption of individual pathways has enabled their characterisation, compensation makes their quantitative contribution under physiological or stress conditions difficult to determine (Walker et al., 2020; Peltier et al., 2024). Chlorophyll fluorescence, P700 absorbance and electrochromic-shift measurements effectively describe PSII, PSI and proton-motive-force states (Klughammer and Schreiber, 1994; Baker, 2008; Mathiot and Alric, 2021), but do not independently quantify ATP production, NADPH consumption, carbon allocation or reductant export. Conversely, metabolomics reveals energetic and carbon-metabolic states, but metabolite abundance does not directly report turnover (Ratcliffe and Shachar-Hill, 2006; Sweetlove and Ratcliffe, 2011; Kruger and Ratcliffe, 2021).

The central challenge is therefore quantitative: how much electron flux enters each pathway, how much ATP is generated, where reducing power is consumed or exported, and how these processes influence carbon allocation and growth. Recent studies have quantified contributions of CEF, pseudocyclic electron flow (PCEF) and chloroplast-to-mitochondrial electron flow (CMEF) under carbon-concentrating conditions and demonstrated substantial functional compensation among them (Burlacot et al., 2022; Peltier et al., 2024).

Addressing this challenge requires integrating photochemical measurements with metabolomics and flux-based approaches. Stable-isotope tracing and metabolic-flux analysis can resolve pathway turnover and carbon partitioning (Ratcliffe and Shachar-Hill, 2006; Ma et al., 2014; Kruger and Ratcliffe, 2021), while membrane-inlet mass spectrometry can quantify simultaneous O₂ evolution and consumption (Burlacot et al., 2022; Peltier et al., 2024). Here, I examine how combining these approaches could enable quantitative chloroplast energy budgets and reveal the physiological functions of alternative electron pathways.

2. What Do Conventional Photosynthetic Measurements Actually Tell Us?

Conventional approaches include chlorophyll fluorescence, P700 absorption spectroscopy, electrochromic-shift (ECS) measurements and gas exchange. Pulse-amplitude-modulated (PAM) fluorescence is widely used to quantify PSII efficiency, excitation pressure and non-photochemical quenching (NPQ), thereby describing fate of the absorbed energy (Genty, Briantais and Baker, 1989; Baker, 2008; Murchie and Lawson, 2013). Time-resolved methods, such as, TCSPC and streak-camera spectroscopy can additionally resolve excitation-transfer and trapping kinetics of PSI and PSII (Chukhutsina, Holzwarth and Croce, 2019). Interestingly, similar fluorescence phenotypes can arise from very different metabolic states. Reduced PSII yield, for example, may reflect photoprotection, carbon-sink limitation, altered ATP/NADPH demand or changes in downstream electron allocation (Baker, 2008; Murchie and Lawson, 2013; Bag et al., 2020). Fluorescence therefore reveals the state of the photosynthetic apparatus but cannot directly determine ATP production, NADPH consumption or the identity of downstream electron sinks. P700 measurements complement fluorescence by reporting PSI redox state and distinguishing donor-side from acceptor-side limitation (Klughammer and Schreiber, 1994; Schreiber and Klughammer, 2016). Acceptor-side limitation can arise when electrons reach PSI faster than NADP+ and other stromal acceptors can be regenerated. Nevertheless, P700 oxidation does not identify where those electrons ultimately go. Increased P700 oxidation could result from enhanced carbon metabolism, alternative electron sinks, altered cyclic electron flow (CEF) or export of reducing equivalents. P700 therefore reports PSI redox poise rather than the metabolic fate of photosynthetic reducing power.

Electrochromic-shift (ECS) measurements provide information about proton-motive force (pmf), proton flux and thylakoid proton conductivity (Sacksteder and Kramer, 2000; Avenson et al., 2005; Mathiot and Alric, 2021). Because ATP synthesis is driven by proton flow through the chloroplast ATP synthase, ECS brings photosynthetic analysis closer to the energetic output of the light reactions than electron transport measurements alone. This becomes particularly important when alternative electron pathways modify proton translocation relative to NADPH formation. CEF, for example, can enhance pmf formation without net NADPH production and therefore adjust the ATP/NADPH output (Avenson et al., 2005; Kramer and Evans, 2010). However, increased pmf or proton flux cannot be interpreted as a quantitative measurement of metabolic ATP availability. ATP production is ultimately coupled to ATP synthase activity and downstream ATP consumption, while the resulting ATP pool is simultaneously influenced by numerous competing metabolic processes. ECS therefore provides an essential component of chloroplast energetic accounting, but not the complete energy budget.

CO₂ exchange provides a measure of net carbon assimilation and its biochemical or diffusional limitations (Long and Bernacchi, 2003). O₂ exchange, in contrast, contains contributions from photosynthetic water oxidation (Shevela et al., 2016) together with oxygen-consuming processes including mitochondrial respiration (Smith, Strand and Walker, 2024), photorespiration (Bauwe, Hagemann and Fernie, 2010) and alternative electron-transfer pathways (Burlacot et al., 2022). Membrane-inlet mass spectrometry (MIMS), particularly when combined with isotopic O₂ or CO₂ exchange, can resolve simultaneous gas production and consumption and quantify oxygen-dependent electron sinks that are difficult to distinguish (Beckmann et al., 2009; Burlacot, Burlacot, et al., 2020). Together, fluorescence, P700, ECS and gas exchange provide complementary information on photochemistry, redox state, proton energetics and gas flux. Their major limitation is that none independently determines how ATP, NADPH and fixed carbon are subsequently consumed or redistributed through metabolism.

3. From Photochemistry to Metabolic Flux

A complete understanding of photosynthetic acclimation requires connecting regulation of the light reactions with chloroplast metabolic homeostasis (Figure 1). Metabolite profiling provides an important bridge by revealing whether changes in electron transport are accompanied by altered pools of Calvin-Benson-Bassham (CBB) cycle intermediates, organic acids, sugars and other metabolites (Szecowka et al., 2013; Westhoff and Weber, 2024). Targeted metabolite measurements and genetically encoded biosensors can further resolve energetic and redox components such as ATP, ADP and pyridine nucleotides (De Col et al., 2017; Voon et al., 2018). Such measurements therefore reveal metabolic consequences of altered photochemistry that cannot be inferred from fluorescence, P700 or ECS alone (Bailleul et al., 2015).

ATP provides a particularly clear example. Photophosphorylation generates ATP, whereas carbon assimilation, ion transport and numerous other metabolic processes simultaneously consume it. Measurements of stromal ATP or ATP/ADP status therefore provide information that cannot be obtained directly from measurements of electron or proton transport (De Col et al., 2017; Voon et al., 2018). Recently, Degen et al. (2026) showed using a chloroplast-targeted MgATP^2- biosensor that Arabidopsis mutants impaired in cyclic electron flow (CEF) maintained stromal MgATP^2- levels comparable with wild type despite substantially reduced proton-motive force. This disequilibrium challenges the assumption that CEF-dependent increases in proton-motive force necessarily translate proportionally into increased steady-state stromal ATP and highlights the importance of distinguishing energetic state from energetic flux (Degen, Schwarzländer and Johnson, 2026).

A similar problem applies to NADP⁺/NADPH. NADPH supplies reducing power to the CBB cycle and other stromal reactions, while NADP⁺ regeneration maintains electron withdrawal from PSI. Chloroplast NADP(H) status is consequently closely linked to redox homeostasis (Hashida et al., 2018). However, increased NADPH cannot distinguish enhanced production from reduced consumption or impaired reductant redistribution, particularly under stress when carbon assimilation is restricted and alternative electron pathways are activated.

More generally, metabolite abundance should not be equated with metabolic flux. ATP, NADPH, CBB-cycle intermediates, malate and carbohydrate pools may remain relatively stable despite substantial changes in turnover, whereas metabolite accumulation may result from reduced downstream consumption rather than increased production. Time-resolved ¹³C labelling has demonstrated that substantial flux can occur through pools whose steady-state abundance changes only modestly (Szecowka et al., 2013; Ma et al., 2014). Consequently, metabolomics provides essential information about metabolic state but cannot by itself determine whether changes in photosynthesis alter ATP/NADPH production or consumption, redirect reducing power towards alternative sinks, or modify carbon export.

Subcellular compartmentation adds a further layer of complexity. Whole-cell metabolite measurements may not accurately reflect chloroplast energy or redox state because photosynthetic metabolism is distributed across chloroplasts, mitochondria, peroxisomes and the cytosol (Kueger et al., 2012; Dietz, 2017). The malate/oxaloacetate system can redistribute reducing equivalents between compartments, triose-phosphate export connects chloroplast carbon fixation with cytosolic metabolism, and photorespiration couples chloroplast, peroxisomal and mitochondrial metabolism through rapid exchange of intermediates (Selinski and Scheibe, 2019) (Figure 1). Thus, understanding photosynthetic energy balance ultimately requires information not only on metabolite abundance, but also on the rate and location of metabolic turnover.

The key distinction is therefore between metabolic state and metabolic flux. Metabolomics can reveal whether photosynthetic perturbations are accompanied by changes in ATP, NADPH, carbon and redox pools, but determining where photosynthetically derived energy and carbon ultimately go requires flux-based measurements. Stable-isotope tracing, metabolic-flux analysis and complementary approaches can provide this missing quantitative layer by resolving rates of production, consumption, export and recycling.

4. Flux-Based Approaches: Resolving Rate and Fate

The next step is to move from describing photosynthetic and metabolic states towards determining the rates at which energy and carbon move through the system. No single technique can provide a complete chloroplast energy budget. Instead, complementary approaches are required to determine where newly fixed carbon is allocated, how rapidly metabolic pathways operate, and how changes in flux influence chloroplast ATP and redox state.

4.1. Stable-Isotope Tracing: Following Newly Fixed Carbon

Stable-isotope labelling provides a direct means of distinguishing metabolite abundance from metabolic turnover. Pulse labelling with ¹³CO₂ in plants or ¹³C -bicarbonate in photosynthetic microorganisms can follow newly fixed carbon through the CBB cycle, photorespiration, starch and sucrose synthesis, organic-acid metabolism and amino-acid biosynthesis (Rühl et al., 2012; Szecowka et al., 2013; Ma et al., 2014; Niedenführ, Wiechert and Nöh, 2015). When combined with time-resolved LC-MS metabolomics, the appearance and redistribution of labelled isotopolouges can reveal pathway dynamics that remain invisible in steady-state metabolite pools (Rühl et al., 2012; Szecowka et al., 2013; Emwas et al., 2022).

This distinction is particularly important in photosynthetic systems, where central metabolites may turn over rapidly while maintaining relatively stable concentrations. Stable-isotope tracing can therefore establish whether an apparently unchanged metabolite pool is metabolically static or instead sustained by rapid and balanced production and consumption. It also provides information on carbon partitioning between competing metabolic destinations, allowing photochemical changes to be connected with downstream carbon allocation.

4.2. From Isotope Enrichment to Metabolic Flux

Isotope tracing becomes most powerful when incorporated into metabolic-flux analysis. Rather than simply determining whether a metabolite becomes labelled, mathematical modelling of the temporal appearance and redistribution of labelled isotopologues can be used to estimate reaction and pathway fluxes (Ratcliffe and Shachar-Hill, 2006; Szecowka et al., 2013).

For example, isotopically non-stationary ¹³C metabolic-flux analysis revealed substantial reorganisation of central carbon metabolism during high-light acclimation in Arabidopsis that could not be inferred from metabolite concentrations alone (Ma et al., 2014). Such approaches begin to provide the quantitative information required for photosynthetic energy accounting: how rapidly fixed carbon enters competing pathways, whether metabolic demand changes following perturbation of electron transport, and whether apparently similar photosynthetic phenotypes are associated with fundamentally different downstream fluxes.

An important future objective will therefore be to integrate estimates of electron and proton transport with carbon-flux measurements obtained under the same physiological conditions. Such integration could provide a quantitative connection between changes in photosynthetic energy production and the metabolic processes ultimately consuming that energy.

4.3. Linking Flux to Energetic and Redox State

Flux measurements alone do not reveal whether changing pathway activity alters the energetic state of the chloroplast. Genetically encoded sensors provide a complementary layer by enabling ATP and redox metabolites to be followed in specific cellular compartments. Fluorescent sensors have been used to monitor MgATP^2- dynamics in plant cells and have revealed substantial differences in ATP status between cellular compartments (De Col et al., 2017; Voon et al., 2018). Similarly, iNAP and SoNar have enabled monitoring of NADPH abundance and NADH/NAD⁺ status during photosynthesis and photorespiration, while NERNST permits monitoring of NADP(H) redox poise within living cells, including chloroplasts (Lim et al., 2020; Molinari et al., 2023).

These measurements are particularly valuable because changes in electron or carbon flux need not produce proportional changes in steady-state ATP or NADPH pools. Conversely, apparently stable energetic pools may conceal substantial changes in their rates of production and consumption. Sensors therefore provide the energetic and redox context needed to interpret flux measurements, especially when combined with fluorescence, P700 and ECS measurements on comparable material (Degen, Schwarzländer and Johnson, 2026). Their greatest value may consequently arise from integration with isotope tracing and metabolic-flux analysis. Photochemical measurements can define the operating state of the light reactions, sensors can reveal the corresponding energetic and redox state, and isotope-based approaches can determine how rapidly carbon is redistributed through metabolism. Together, these measurements begin to connect photosynthetic state, rate and fate.

Oxygen-resolved measurements can provide an additional, pathway-specific layer. Membrane-inlet mass spectrometry (MIMS), particularly with isotopic O₂, can distinguish simultaneous O₂ evolution and consumption and has been valuable for quantifying oxygen-dependent electron sinks such as FLV-mediated photoreduction (Chaux et al., 2017; Burlacot, Richaud, et al., 2020; Bag et al., 2023). However, because O₂ consumption can arise from several processes, MIMS is most informative when combined with genetic or physiological perturbations and with fluorescence, P700 or ECS measurements (Burlacot, Richaud, et al., 2020; Fitzpatrick, Aro and Tiwari, 2022; Bag et al., 2023). It therefore complements, rather than replaces, the broader metabolic and flux-based framework described above.

5. Test Cases: Pathway Activity Does Not Equal Metabolic Function

Alternative electron pathways illustrate why pathway activation cannot be interpreted independently of metabolic context. The same pathway may contribute to ATP supply, redox balancing or photoprotection depending on cellular demand.

CEF around PSI provides an important example. Because electrons are recycled into the plastoquinone/cytochrome b₆f pathway rather than used for net NADPH production, CEF can increase pmf formation relative to NADPH output (Shikanai, 2014; Alric and Johnson, 2017). However, CEF also contributes to photosynthetic control, PSI protection and redox regulation (Dang et al., 2014; Johnson et al., 2014; Kono et al., 2017; Buchert et al., 2020; Bag et al., 2025; Degen, Schwarzländer and Johnson, 2026). Recent measurements showing near-wild-type stromal MgATP²⁻ levels in CEF-impaired Arabidopsis despite reduced proton-motive force further demonstrate that CEF activity cannot be equated directly with ATP availability (Buchert et al., 2020; Degen, Schwarzländer and Johnson, 2026). Determining its metabolic function therefore requires linking CEF and proton flux with ATP status, carbon assimilation and metabolic turnover.

Oxygen-dependent electron sinks provide a similar example. Flavodiiron proteins can divert electrons from PSI towards O₂, relieving acceptor-side pressure while maintaining upstream electron and proton transfer (Chaux et al., 2017; Ilík et al., 2017; Bag et al., 2023). Because this pathway draws electrons away from PSI acceptors while allowing continued upstream electron and proton transfer, it can simultaneously influence redox poise, photoprotection and the energetic output of photosynthesis (Allen, 2002; Alric, 2015). The relative importance of these functions is likely context dependent. Quantifying FLV activity therefore requires not only demonstrating O₂ or NO (nitric oxide) reduction (Burlacot, Richaud, et al., 2020; Burlacot et al., 2022; Bag et al., 2023) but integrating that flux with LEF, pmf formation, ATP demand and carbon metabolism. However, measuring O₂ reduction alone does not reveal whether this flux primarily supports photoprotection, energetic balance or compensation for restricted carbon metabolism. Other O₂-consuming pathways further complicate this interpretation. Together, these examples illustrate the central problem: identifying where electrons flow does not necessarily explain what physiological function that flux serves. Resolving function requires integration of pathway activity with energetic, redox and metabolic flux measurements.

6. A Framework for Quantitative Photosynthetic Bioenergetics

A realistic future objective is to construct integrated chloroplast energy map in which photochemical measurements, metabolite pools and flux measurements are analysed as complementary layers rather than independent datasets. Such an approach could proceed through a sequential experimental framework.

First, define the photochemical state. Chlorophyll fluorescence would quantify PSII operating efficiency, excitation pressure and energy dissipation; P700 measurements would resolve PSI redox poise; and ECS would provide information on proton-motive-force formation and proton flux.

Second, quantify major carbon and oxygen fluxes. Gas exchange would establish net CO₂ assimilation, while oxygen-resolved measurements such as MIMS could quantify O₂ evolution and oxygen-dependent alternative electron pathways.

Third, determine energetic and metabolic state. Measurements of ATP/ADP, NADPH/NADP+ and central carbon metabolites would establish whether changes in photochemistry are accompanied by corresponding changes in energy, redox and carbon pools.

Fourth,determine turnover rather than abundance alone.¹³CO₂ or ¹³C-bicarbonate pulse labelling combined with LC-MS and metabolic-flux analysis could reveal whether observed changes result from altered production, consumption, export or recycling.

Finally, integrate these layers into a common energy model. Such models should seek to account for electron flow through LEF and alternative pathways, proton-motive-force generation, ATP synthesis and consumption, NADPH production and utilisation, carbon fixation and allocation, and exchange of reducing equivalents with other cellular compartments. The objective is therefore not to replace fluorescence, P700 or ECS with omics. Rather, each measurement addresses a different level of the photosynthetic energy system. Photochemical measurements define what the photosynthetic machinery is doing; metabolomics describes the metabolic state associated with that activity; and fluxomics begins to reveal where the resulting energy and carbon actually go.

7. Outlook: From Descriptive Photosynthesis to Predictive Bioenergetics

Photosynthetic biophysics has provided exceptionally powerful tools for resolving the behaviour of PSII, PSI and the thylakoid proton circuit. Metabolomics has simultaneously revealed extensive reorganisation of carbon, energy and redox metabolism during environmental acclimation. Yet these areas are still frequently interpreted separately. The next major advance should therefore be to connect them quantitatively. Fluorescence, P700 and ECS can define the photochemical state of photosynthesis, while metabolomics can establish whether downstream metabolism maintains ATP, NADPH, carbon and redox homeostasis. Flux-based approaches are required to determine how rapidly electrons, ATP, reductant and carbon move through competing pathways.

Measurements must increasingly be performed under comparable physiological conditions and, ideally, on the same biological material. Temporal resolution will be essential because photosynthetic regulation can change within seconds whereas metabolite pools and long-term acclimation operate over very different timescales. Cellular compartmentation must also be addressed, particularly when chloroplast and mitochondrial metabolism exchange carbon and reducing equivalents. Finally, energetic models will require careful accounting of what is measured directly and what is inferred from stoichiometric assumptions.

The goal should be to progress beyond identifying whether a photosynthetic pathway is activated. Instead, future studies should ask: how much electron flux enters the pathway, how does it alter proton and ATP production, where is the resulting reductant consumed or exported, and what physiological function does this redistribution ultimately support?

Integrating photosynthetic biophysics, metabolomics and fluxomics offers a route from descriptive stress physiology toward predictive photosynthetic bioenergetics. Such quantitative energy budgets could reveal why apparently similar photochemical phenotypes produce very different metabolic outcomes, explain compensatory responses to photosynthetic engineering, and ultimately provide a more rational basis for improving photosynthetic resilience and productivity.