An Integrated Framework for Carbon Recycling and Energy Economy in Hypoxic Plant Tissues: Roles of PEPC, Rubisco and Pyrophosphate-Driven Metabolism

1. Introduction

Plant metabolism is commonly described within a conceptual framework that assumes tissues operate under fully aerobic conditions, in which oxygen supply does not limit mitochondrial respiration and carbon dioxide readily diffuses to and from the atmosphere. However, this assumption is unlikely to apply to a large proportion of plant tissues. In many organs, internal gas exchange is constrained by tissue structure, diffusion path length, and limited connectivity with the external atmosphere. As a result, oxygen availability within plant tissues is frequently restricted, and hypoxia should be considered a common physiological condition rather than an exceptional stress.

Bulky and metabolically active organs such as stems, fruits, seeds, storage roots, and woody tissues are particularly prone to diffusion limitation. In these systems, oxygen must diffuse over relatively long distances to reach respiring cells, while continuous metabolic activity consumes oxygen along the diffusion pathway. At the same time, carbon dioxide produced by respiration accumulates internally because its efflux is similarly restricted. Consequently, steep internal gradients in both O₂ and CO₂ are commonly established (Armstrong 1980; Geigenberger 2003; Ho et al. 2010; van Dongen et al. 2003; Verboven et al. 2008). These tissues therefore operate within a coupled environment characterised by low oxygen and elevated carbon dioxide concentrations.

A key consequence of this diffusion-limited environment is that it simultaneously constrains energy production and alters carbon availability. Reduced oxygen supply limits mitochondrial oxidative phosphorylation, leading to decreased ATP production and changes in adenylate balance, redox state, and metabolic flux. Under these conditions, glycolytic flux must increase to sustain cellular energy demand, often supported by fermentative pathways that regenerate NAD⁺. This response is typically interpreted as an energetic limitation, in which reduced ATP yield necessitates increased substrate consumption to maintain metabolic activity (Drew 1997; Geigenberger 2003).

In parallel, the accumulation of respiratory CO₂ creates conditions that favour internal carbon recycling. Dissolved CO₂ equilibrates with bicarbonate, providing substrate for carboxylation reactions catalysed by enzymes such as phosphoenolpyruvate carboxylase (PEPC). In addition, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is present in many non-photosynthetic tissues, where it can contribute to the reassimilation of internally released CO₂ (Moroney et al. 2001; Schwender et al. 2004). These processes indicate that respiratory carbon loss may be partially mitigated under diffusion-limited conditions.

A further layer of metabolic adjustment involves the use of pyrophosphate (PP_i)-dependent reactions, which can substitute for ATP-dependent steps in central metabolism. Enzymes such as PP_i-dependent phosphofructokinase and vacuolar H⁺-translocating pyrophosphatases enable metabolic flux to be maintained with a reduced ATP requirement, particularly when oxidative phosphorylation is constrained (Igamberdiev and Kleczkowski 2021; Stitt 1998). This alternative energy economy has the potential to buffer the energetic cost of hypoxia by lowering the ATP demand per unit of metabolic flux.

Despite substantial progress in understanding these processes individually, they have largely been considered in isolation. Hypoxia research has focused primarily on energy limitation and fermentative metabolism, whereas studies of non-foliar carbon fixation have emphasised CO₂ reassimilation and anaplerotic pathways. The integrative consequences of diffusion limitation—namely the simultaneous occurrence of ATP limitation and enhanced internal carbon availability—have received comparatively little attention.

Here, we propose that PP_i-dependent metabolism, fermentative pathways, and internal CO₂ recycling via PEPC and Rubisco function as components of an integrated metabolic system that supports continued metabolic activity under diffusion-limited conditions. Within this framework, reduced ATP yield increases substrate demand, but carbon loss is not necessarily proportional, leading to a decoupling between energy demand and net CO₂ release. This perspective provides a unified conceptual basis for understanding how plant tissues maintain carbon-use efficiency, sink strength, and metabolic function in environments where gas exchange is inherently restricted.

In this review, we first examine the physical basis of diffusion limitation and the development of internal O₂ and CO₂ gradients in plant tissues. We then consider the metabolic consequences of oxygen limitation, including changes in ATP production and glycolytic flux. This is followed by an analysis of PP_i-dependent metabolism as an alternative energy system, and the role of internal CO₂ accumulation in supporting carbon recycling through PEPC and Rubisco. Finally, we integrate these processes into a conceptual framework that links energy economy and carbon balance in diffusion-limited tissues, and discuss the implications for plant growth, storage, and productivity.

2. Diffusion Limitation and Internal Oxygen–Carbon Dioxide Gradients

2.1. Restricted Gas Exchange Is a Common Feature of Plant Tissues

Gas exchange in plants is governed by diffusion and is therefore strongly influenced by tissue structure, diffusion path length, and the availability of external interfaces. In well-aerated tissues such as leaves, stomata provide direct connections to the atmosphere, allowing relatively rapid exchange of O₂ and CO₂. Oxygen diffuses inward from both leaf surfaces, while respiratory CO₂ diffuses outward, resulting in relatively shallow internal gradients (Figure 1A).

In contrast, many plant organs—including stems, fruits, seeds, storage roots, tubers, and woody tissues—lack such direct exchange pathways and are characterised by long diffusion distances, dense cellular organisation, and limited intercellular air spaces (Geigenberger 2003; van Dongen et al. 2003). In these tissues, oxygen must diffuse through multiple cell layers or be supplied indirectly via vascular transport, while CO₂ must diffuse outward through the same constrained pathways.

As a consequence, internal gas composition is determined by the balance between diffusion and metabolic activity. Continuous respiratory consumption of oxygen leads to declining O₂ concentrations with increasing distance from the site of supply, while ongoing CO₂ production generates opposing gradients. These gradients are spatially structured, combining longitudinal components along vascular pathways with radial gradients extending into surrounding parenchyma (Figure 1B). Under these conditions, metabolically active regions of bulky tissues frequently operate under chronically reduced oxygen availability.

Importantly, these internal gradients are not exceptional or restricted to stress conditions such as flooding. Rather, they arise as a direct consequence of organ structure and function. In this context, hypoxia should be regarded as a common physiological state in many plant tissues, particularly those characterised by high metabolic activity and restricted gas exchange.

2.2. Diffusion Limitation Creates a Coupled O₂–CO₂ Environment

A central consequence of restricted gas exchange is that oxygen limitation and carbon dioxide accumulation occur simultaneously. While the effects of declining O₂ have been widely studied, the parallel accumulation of CO₂ has received comparatively less attention.

If respiratory oxygen consumption exceeds the rate of diffusive supply, internal O₂ concentration declines along diffusion pathways. At the same time, respiratory CO₂ production continues, and when its rate of generation exceeds diffusive loss, CO₂ accumulates within the tissue. These processes are intrinsically coupled: the same structural constraints that limit oxygen entry also restrict carbon dioxide exit.

Diffusion-limited tissues should therefore be viewed as systems characterised by both low O₂ and elevated CO₂, rather than by oxygen deficiency alone. This dual constraint has important metabolic implications. Reduced oxygen availability constrains mitochondrial ATP production, while elevated CO₂ increases the local availability of dissolved inorganic carbon, potentially favouring carboxylation reactions.

Although these outcomes follow directly from diffusion–reaction principles, they have largely been treated independently in the literature. Hypoxia research has focused primarily on energy limitation, redox balance, and survival responses, whereas studies of internal carbon fixation have emphasised PEPC activity, bicarbonate utilisation, and, in some systems, Rubisco-mediated reassimilation. The metabolic consequences of these two features occurring together remain insufficiently integrated.

2.3. Developmental and Functional Significance of Internal Gradients

Internal gradients in O₂ and CO₂ are not merely passive consequences of tissue structure but influence metabolic strategy and developmental processes. In developing seeds, fruits, and storage tissues, restricted gas exchange coincides with high biosynthetic demand, creating conditions in which both energy supply and carbon conservation become limiting factors (Schwender et al. 2004; ?).

In these systems, the accumulation of internal CO₂ provides a substrate pool for reassimilation, while reduced oxygen availability constrains ATP production. This combination creates a metabolic context in which energy economy and carbon economy are intrinsically linked.

Experimental evidence from developing seeds and other heterotrophic tissues demonstrates that internally generated CO₂ can be reassimilated, improving carbon conversion efficiency during biosynthesis (Rangan et al. 2024; Schwender et al. 2004). These findings indicate that diffusion limitation does not simply impose constraints on metabolism, but also creates opportunities for metabolic integration.

2.4. A Conceptual Gap in Current Understanding

Taken together, current evidence supports three key propositions: (i) diffusion limitation is a common feature of many plant tissues; (ii) this limitation leads to the coupled occurrence of low O₂ and elevated CO₂; and (iii) plants possess metabolic systems capable of responding to both conditions, including fermentative pathways, PP_i-dependent reactions, and CO₂ reassimilation via PEPC and Rubisco.

What remains largely missing is a unified framework that treats these processes as components of a coordinated metabolic strategy. Existing studies have typically addressed energy limitation and carbon recycling separately, without explicitly considering how they interact under diffusion-limited conditions.

A central consequence of this separation is that the relationship between energy demand and carbon balance remains poorly defined. Reduced oxygen availability decreases the efficiency of ATP generation, implying that a greater quantity of respiratory substrate is required to sustain metabolic activity. At the same time, internal CO₂ accumulation creates the potential for partial reassimilation of respiratory carbon. The net outcome depends on the balance between these opposing processes, yet this balance has rarely been quantified in a unified manner.

This issue is particularly relevant when considering the partitioning of respiration into growth-associated and maintenance-associated components. Growth respiration is directly linked to biosynthetic processes and biomass accumulation, whereas maintenance respiration reflects the energetic cost of sustaining existing cellular structures and metabolic function. Under diffusion-limited conditions, constraints on ATP production are expected to differentially affect these components, with potential consequences for both biomass accumulation and carbon-use efficiency.

Here, we argue that diffusion limitation necessitates an integrated analysis in which carbon flux, energy demand, and respiratory partitioning are considered simultaneously. Such an approach allows the effects of reduced ATP yield, altered metabolic pathways, and internal carbon recycling to be evaluated within a common framework. In subsequent sections, we develop this perspective by linking gas diffusion constraints to quantitative descriptions of substrate demand, respiratory partitioning, and carbon-use efficiency in plant tissues.

3. Metabolic Consequences of Oxygen Limitation

Reduced oxygen availability alters several key aspects of plant metabolism. A primary effect is the restriction of flux through the mitochondrial electron transport chain, which limits ATP production via oxidative phosphorylation (Drew 1997; Geigenberger 2003; Gupta et al. 2009). As a consequence, the energetic yield per unit of respiratory substrate declines, altering the balance between energy supply and metabolic demand (Plaxton and Podestá 2006; Stitt 1998; van Dongen and Licausi 2015).

To compensate for reduced ATP generation, cells typically increase glycolytic flux. Glycolysis can proceed in the absence of oxygen but produces substantially less ATP per molecule of substrate than mitochondrial respiration. Under conditions of severe oxygen limitation, fermentative pathways are activated to regenerate NAD⁺, thereby sustaining glycolytic flux (Bailey-Serres and Voesenek 2008; Drew 1997; Geigenberger 2003, 2011; ?). These adjustments allow continued metabolic activity but are associated with reduced energetic efficiency and increased substrate consumption per unit of ATP generated.

Changes in oxygen availability also influence decarboxylating reactions within central metabolism. Several steps in the tricarboxylic acid cycle involve the release of CO₂, and restriction of mitochondrial flux modifies the balance between carbon oxidation and biosynthetic processes (Geigenberger 2003). In parallel, shifts in metabolic pathway utilisation may alter the relative contributions of biosynthesis and respiratory carbon loss.

Despite these adjustments, respiration continues to generate CO₂. In diffusion-limited tissues, this CO₂ may accumulate internally, increasing the local concentration of dissolved inorganic carbon and creating the potential for reassimilation through carboxylation reactions (Schwender et al. 2004). This introduces an additional layer of complexity, as carbon loss through decarboxylation may be partially offset by internal recycling.

From a systems perspective, these responses highlight a central consequence of oxygen limitation: a reduction in the efficiency with which substrate is converted into ATP, coupled with continued carbon flux through decarboxylating pathways. This combination implies that a greater quantity of substrate is required to sustain metabolic processes, with potential implications for the partitioning of respiration between growth-associated and maintenance-associated functions. Processes associated with maintenance, which are continuous and less flexible, may become proportionally more significant as energetic constraints intensify, whereas growth-related fluxes may be more strongly constrained by reduced ATP availability.

These considerations emphasise the need to evaluate oxygen limitation not only in terms of metabolic pathway changes, but also in terms of its impact on substrate demand, respiratory partitioning, and carbon-use efficiency. In the following sections, these relationships are examined in the context of alternative energy systems and internal carbon recycling.

4. Fermentation and Redox Balancing

Under conditions of severe oxygen limitation, fermentative metabolism becomes essential for sustaining glycolytic flux by regenerating cytosolic NAD⁺ (Drew 1997; Geigenberger 2003). In plant tissues, pyruvate may initially be reduced to lactate via lactate dehydrogenase; however, this pathway is typically transient. As cytosolic pH declines, metabolism shifts towards ethanolic fermentation, in which pyruvate is decarboxylated to acetaldehyde and subsequently reduced to ethanol, thereby maintaining redox balance while permitting continued glycolysis (Drew 1997; Felle 2005).

Fermentation is often described as a low-efficiency emergency pathway due to the limited ATP yield per mole of substrate. However, in diffusion-limited tissues this characterisation is incomplete. Reduced ATP generation via oxidative phosphorylation necessitates an increase in glycolytic flux to sustain cellular energy demand. In a response dominated by ethanolic fermentation, this increased flux is inherently coupled to carbon loss through decarboxylation, leading to reduced carbon-use efficiency.

Importantly, fermentative metabolism represents only one possible configuration of metabolic adjustment under oxygen limitation. Alternative pathways contribute to redox balancing while partially conserving carbon, particularly through the diversion and recycling of pyruvate and related intermediates. One prominent route is the conversion of pyruvate to alanine via alanine aminotransferase. This reaction does not involve decarboxylation and therefore conserves carbon while providing a temporary sink for pyruvate and nitrogen. Accumulation of alanine is a consistent feature of hypoxic plant tissues and is considered a key mechanism for maintaining metabolic stability under oxygen limitation (Miyashita et al. 2007; Rocha et al. 2010).

In parallel, organic acid metabolism provides additional flexibility in redox and carbon management. Pyruvate and phosphoenolpyruvate can be channelled into malate synthesis via anaplerotic reactions, including phosphoenolpyruvate carboxylase (PEPC). Malate can serve as a temporary carbon store and redox buffer, and its interconversion with other tricarboxylic acid cycle intermediates allows redistribution of reducing equivalents between cellular compartments (??). Under hypoxia, partial operation of the tricarboxylic acid cycle and associated shuttle systems can therefore contribute to maintaining redox balance without complete oxidation of substrates.

Sucrose metabolism is also closely integrated into these responses. In storage tissues, sucrose breakdown via sucrose synthase is generally favoured over invertase under oxygen limitation, as it conserves energy by coupling cleavage to UDP formation rather than ATP-dependent phosphorylation (Koch 2004; Sturm and Tang 1999). The resulting UDP-glucose can be recycled through reversible reactions involving UDP-glucose pyrophosphorylase, linking sucrose metabolism to the cellular PP_i pool. This facilitates continued carbon flux through glycolysis while reducing ATP demand. In addition, the reversible nature of several steps in sucrose metabolism allows partial re-synthesis of sucrose, thereby contributing to carbon retention under conditions where net export or utilisation is constrained.

The utilisation of inorganic pyrophosphate (PP_i) as an alternative phosphoryl donor further reinforces these adjustments. PP_i-dependent enzymes, such as pyrophosphate:fructose-6-phosphate 1-phosphotransferase, can substitute for ATP-dependent steps in glycolysis, reducing the overall ATP requirement for maintaining metabolic flux (Plaxton 1996; Stitt et al. 2010). By lowering ATP demand, these pathways decrease the glycolytic flux required to sustain cellular energy balance, thereby indirectly reducing carbon loss through fermentation.

Fermentation also interacts with cellular pH, proton balance, and the distribution of reducing equivalents. The coordinated operation of alanine synthesis, organic acid metabolism, and sucrose cycling contributes to stabilising cytosolic pH and redox state. In addition, CO₂ released through decarboxylating reactions may accumulate in diffusion-limited tissues and become available for reassimilation via carboxylation reactions, further enhancing carbon retention (Schwender et al. 2004; ?).

Collectively, these processes demonstrate that fermentative metabolism cannot be considered in isolation. Instead, it forms part of an integrated metabolic network in which PP_i-dependent reactions, amino acid synthesis, organic acid interconversions, and sucrose metabolism collectively determine the balance between energy production, redox stability, and carbon conservation under oxygen limitation.

5. Pyrophosphate-Dependent Metabolism Under Energy Constraint

The metabolic adjustments described above, including the accumulation of alanine, malate and sucrose, primarily reflect changes in carbon partitioning under oxygen limitation. However, these changes cannot be understood independently of the underlying energetic constraint. Reduced oxygen availability decreases ATP yield, increasing the substrate demand required to sustain metabolic flux.

Consequently, the observed shifts in carbon metabolism and the engagement of alternative pathways must be considered alongside mechanisms that reduce ATP demand. One such mechanism is the utilisation of inorganic pyrophosphate (PP_i) as an alternative phosphoryl donor. In this context, PP_i-dependent metabolism does not represent a separate response, but rather an integral component of a coordinated adjustment that links energy conservation with carbon retention

Another important adaptation to energy limitation involves the use of pyrophosphate (PP_i) as an alternative energy source. Several plant enzymes are capable of using PP_i in place of ATP to drive metabolic reactions. This offers an attractive framework for understanding how metabolism may continue under oxygen limitation while reducing ATP demand (Geigenberger 2003; Igamberdiev and Kleczkowski 2021; Lee and Jeon 2020; Stitt 1998).

One example is PP_i-dependent phosphofructokinase, which catalyzes:

$$\mathrm{F}6\mathrm{P}+{\mathrm{PP}}_{\mathrm{i}}\to \mathrm{F}1,6\mathrm{BP}+{\mathrm{P}}_{\mathrm{i}}$$

(1)

This reaction provides a glycolytic step that does not require ATP. Similarly, vacuolar proton-pumping pyrophosphatases use PP_i to generate proton gradients across membranes. Together these systems can lower the ATP cost of metabolism during periods when oxidative phosphorylation is restricted by limited oxygen availability.

Effective ATP Yield Under Hypoxia

Under conditions where oxidative phosphorylation is partially constrained, the effective ATP yield per unit metabolic flux can be expressed as a weighted combination of oxidative and non-oxidative pathways:

$$Y_{\mathrm{eff}}=f_{\mathrm{ox}}{Y}_{\mathrm{ox}}+\left(1-f_{\mathrm{ox}}\right)Y_{\mathrm{ana}}$$

(2)

where:

• $Y_{\mathrm{eff}}$ is the effective ATP yield per unit glycolytic flux under hypoxia,
• $f_{\mathrm{ox}}$ is the fraction of ATP derived from oxidative phosphorylation (dimensionless, 0–1),
• $Y_{\mathrm{ox}}$ is the ATP yield associated with oxidative metabolism,
• $Y_{\mathrm{ana}}$ is the ATP yield associated with anaerobic ATP production (e.g. glycolysis-linked substrate-level phosphorylation).

As oxygen availability declines, $f_{\mathrm{ox}}$ decreases and $Y_{\mathrm{eff}}$ approaches $Y_{\mathrm{ana}}$, resulting in a reduction in energetic efficiency.

Glucose Demand Under Hypoxia

To maintain a given metabolic ATP demand, a reduction in ATP yield must be compensated by increased substrate flux. The relative glucose demand under hypoxia can therefore be expressed as:

$$G_{\mathrm{hyp}}=G_{\mathrm{aer}}\left({\displaystyle \frac{Y_{\mathrm{ox}}}{Y_{\mathrm{eff}}}}\right)$$

(3)

where:

• $G_{\mathrm{hyp}}$ is the glucose demand under hypoxic conditions,
• $G_{\mathrm{aer}}$ is the glucose demand under fully aerobic conditions.

This formulation captures the increasing substrate requirement as ATP yield declines.

Damping of Glucose Demand by PP_i-Dependent Metabolism

Pyrophosphate-dependent reactions reduce the requirement for ATP by substituting PP_i in key metabolic steps. This effect can be represented conceptually as a damping of glucose demand:

$$G_{\mathrm{hyp},{\mathrm{PP}}_{\mathrm{i}}}=G_{\mathrm{hyp}}\left(1-d_{{\mathrm{PP}}_{\mathrm{i}}}\right)$$

(4)

where:

• $G_{\mathrm{hyp},{\mathrm{PP}}_{\mathrm{i}}}$ is the glucose demand under hypoxia with PP_i-dependent metabolism,
• $d_{{\mathrm{PP}}_{\mathrm{i}}}$ is a dimensionless damping coefficient representing the extent to which PP_i metabolism reduces ATP demand (0–1).

Importantly, $d_{{\mathrm{PP}}_{\mathrm{i}}}$ is expected to increase under stronger energetic limitation, reflecting a greater relative contribution of PP_i-dependent reactions.

This perspective shifts the role of PP_i metabolism from that of an auxiliary pathway to that of an integral component of metabolic regulation in diffusion-limited tissues. The consequences of this shift can be understood by explicitly separating the effects of hypoxia on ATP yield, substrate demand and carbon loss, as illustrated conceptually in Figure 2.

This energetic buffering provides a mechanistic basis for the retention of carbon within central metabolism, thereby supporting the accumulation of metabolites such as alanine, malate and sucrose under conditions where fermentative carbon loss would otherwise predominate.

5.1. Origin, Turnover and Steady-State Levels of PP_i in Plant Cells

Inorganic pyrophosphate (PP_i) is a ubiquitous by-product of biosynthetic metabolism, generated during a wide range of anabolic reactions including nucleic acid synthesis, protein synthesis, and the activation of sugars and other metabolites. Many of these reactions involve the cleavage of ATP to AMP and PP_i, resulting in the continuous production of PP_i in metabolically active cells (Heinonen 2001; Stitt 1998).

In most non-plant systems, PP_i is rapidly hydrolysed by soluble inorganic pyrophosphatases, maintaining cytosolic PP_i concentrations at very low levels and effectively rendering PP_i a thermodynamic sink (Heinonen 2001). This rapid hydrolysis is often considered essential to drive biosynthetic reactions forward by removing one of the reaction products.

In plant cells, however, the metabolism of PP_i differs in important respects. Cytosolic PP_i concentrations are maintained at levels that are sufficiently high to support PP_i-dependent enzymatic reactions (Stitt 1998). In addition to soluble pyrophosphatases, plants possess membrane-bound H⁺-translocating pyrophosphatases (V-PPases) that utilize PP_i as an energy source to drive proton transport across endomembranes, particularly the tonoplast (Maeshima 2000). This provides a direct mechanism by which PP_i can be conserved and utilized, rather than dissipated.

The steady-state level of PP_i in plant cells therefore reflects a balance between its production in biosynthetic reactions, its hydrolysis by pyrophosphatases, and its utilization in PP_i-dependent processes. Under conditions where ATP availability is constrained, such as hypoxia or high metabolic demand, the relative importance of PP_i-dependent reactions may increase, allowing continued metabolic flux while reducing the reliance on ATP.

These features distinguish plant PPi metabolism from that of many other organisms and support the concept of a PPi-based energy economy. Under diffusion-limited conditions, this system provides a mechanism to reduce ATP demand while maintaining metabolic flux, thereby linking energy status directly to carbon economy. In this context, PPi-dependent reactions are not simply substitutes for ATP-dependent processes, but integral components of a coordinated metabolic strategy that balances ATP availability, oxygen supply, and carbon conservation.

While PPi-dependent metabolism reduces the energetic cost of maintaining metabolic flux, it does not by itself determine the fate of carbon within the system. Under diffusion limitation, the accumulation of internal CO₂ creates additional opportunities for carbon retention through reassimilation pathways, which are considered in the following section.

6. Internal CO₂ Accumulation and Refixation

Respiratory metabolism continuously produces CO₂ through oxidative decarboxylation reactions. In tissues where gas diffusion is limited, CO₂ concentrations within the intercellular spaces and cells can increase substantially above atmospheric levels (Geigenberger 2003; Schwender et al. 2004).

Carbon dioxide dissolved in the aqueous phase rapidly equilibrates with bicarbonate through the action of carbonic anhydrase. This conversion creates the possibility for metabolic refixation of respiratory CO₂ through carboxylation reactions that utilize bicarbonate as a substrate (DiMario et al. 2017; Moroney et al. 2001).

The accumulation of internal CO₂ therefore represents both a metabolic challenge and an opportunity within the broader context of oxygen limitation and constrained ATP supply. Elevated CO₂ levels may influence cellular pH and enzyme activities, but they also provide a substrate pool that could potentially be reassimilated into organic metabolites (Kurkdjian and Guern 1989; Raven 1985).

The decoupling between glucose consumption and CO₂ release under hypoxic conditions (Figure 3) provides a mechanistic basis for this opportunity. Where carbon is retained through metabolic recycling rather than lost via fermentation, internally generated CO₂ may accumulate to high concentrations while remaining available for reassimilation. This situation contrasts with classical views of fermentation as a pathway associated with substantial carbon loss. Instead, it supports a model in which hypoxia promotes a shift towards internal carbon retention and recycling, thereby increasing the potential importance of anaplerotic carboxylation and Rubisco-mediated refixation in non-photosynthetic tissues.

6.1. Internal CO₂ Accumulation in Diffusion-Limited Tissues

The principles outlined above can be generalised across a range of diffusion-limited tissues. Respiratory metabolism continuously produces CO₂, and in plant tissues where gas exchange is restricted this CO₂ may accumulate internally to concentrations substantially above those of the external atmosphere. This phenomenon is not confined to specialised storage organs, but arises whenever the rate of respiratory CO₂ production exceeds the capacity for diffusive transport to the surrounding environment. Such conditions are common in a wide range of plant tissues, including stems, roots, fruits, seeds and other bulky or densely structured organs (Geigenberger 2003; van Dongen et al. 2003).

The internal atmosphere of plant tissues is determined by the balance between metabolic gas exchange and physical diffusion. Where diffusion path lengths are long, intercellular air spaces are limited or poorly connected, or external barriers to gas exchange are present, steep gradients in both O₂ and CO₂ can develop. These gradients are an inherent consequence of diffusion–reaction coupling and do not require extreme environmental conditions (Geigenberger 2003).

Woody stems provide a clear demonstration of this principle. Measurements across multiple species indicate that internal CO₂ concentrations within stems commonly reach several percent and may exceed 10% in some cases. The radial movement of gases is restricted by bark, phloem and xylem tissues, while ongoing respiration in living cells contributes to substantial internal CO₂ accumulation.

A similar situation occurs in fruits, tubers and seeds, where dense tissue structure and high respiratory activity combine to limit gas exchange. Developing oilseeds, for example, represent a particularly informative system because internal CO₂ accumulation has been linked directly to carbon reassimilation by Rubisco, improving carbon conversion efficiency during storage product synthesis (Schwender et al. 2004).

These observations indicate that internal CO₂ accumulation should be regarded as a normal feature of many metabolically active plant tissues rather than an exceptional stress response. The extent to which accumulated CO₂ is lost, buffered, transported or reassimilated is likely to depend on tissue structure, developmental stage and metabolic state.

6.2. CO₂–Bicarbonate Equilibrium and Carbonic Anhydrase

From a metabolic perspective, this equilibrium is important because it determines the availability of bicarbonate for carboxylation reactions that contribute to carbon retention. Once CO₂ accumulates within plant tissues, its physiological significance depends not only on its gaseous concentration but also on its behaviour in the aqueous phase. Dissolved CO₂ is in reversible equilibrium with bicarbonate and protons:

$$\mathrm{C}{\mathrm{O}}_2+{\mathrm{H}}_2\mathrm{O}\rightleftharpoons \mathrm{H}\mathrm{C}\mathrm{O}{3}^{-}+{\mathrm{H}}^{+}$$

(5)

This reaction establishes a dynamic pool of dissolved inorganic carbon, comprising CO₂, ${\text{HCO}}_3^{-}$ and, to a lesser extent, ${\text{CO}}_3^{2-}$. Under physiological pH conditions typical of plant cells, bicarbonate represents a major component of this pool, linking respiratory CO₂ production directly to both proton generation and the availability of carboxylation substrates (Raven 1985; Smith and Raven 1979).

Although the interconversion of CO₂ and bicarbonate can occur spontaneously, the reaction is strongly accelerated by carbonic anhydrase, a widely distributed enzyme present in multiple cellular compartments, including the cytosol, plastids and mitochondria (DiMario et al. 2017; Moroney et al. 2001). The presence of carbonic anhydrase ensures that equilibrium between CO₂ and ${\text{HCO}}_3^{-}$ is achieved rapidly relative to metabolic fluxes, effectively coupling respiratory CO₂ production to intracellular acid–base chemistry and metabolic processes (Badger and Price 1994; Fabre et al. 2007).

The distribution of carbonic anhydrase across cellular compartments suggests that CO₂/${\text{HCO}}_3^{-}$ interconversion is not confined to a single location but may occur throughout the cell. This has important implications for the spatial dynamics of dissolved inorganic carbon, particularly in tissues where internal CO₂ concentrations are elevated. Under such conditions, localised increases in CO₂ may be rapidly translated into changes in bicarbonate concentration and proton availability within different compartments (DiMario et al. 2017; Moroney et al. 2001; ?).

The equilibrium between CO₂ and bicarbonate is strongly influenced by pH, such that shifts in cytosolic or organellar pH will alter the relative proportions of CO₂ and ${\text{HCO}}_3^{-}$. Conversely, increases in CO₂ concentration will tend to drive proton production through hydration reactions, potentially contributing to changes in intracellular pH depending on buffering capacity (Kurkdjian and Guern 1989; Raven 1985). The CO₂–bicarbonate system therefore represents a bidirectional link between carbon metabolism and acid–base regulation.

From a metabolic perspective, the generation of bicarbonate is particularly significant because it provides substrate for carboxylation reactions. Phosphoenolpyruvate carboxylase, which utilises ${\text{HCO}}_3^{-}$ rather than CO₂ as a substrate, is widely expressed in plant tissues and plays a central role in anaplerotic carbon fixation (Chollet et al. 1996; O’Leary 1982). Elevated internal CO₂ concentrations may therefore increase the availability of bicarbonate for PEPC-mediated reactions, linking respiratory CO₂ production directly to carbon reassimilation pathways.

In addition to PEPC, bicarbonate may influence other metabolic processes either as a substrate or through indirect effects on enzyme activity and proton balance. The extent to which these processes contribute to overall carbon economy in diffusion-limited tissues remains an open question, but the presence of a substantial and dynamic dissolved inorganic carbon pool provides a mechanistic basis for potential recycling of respiratory CO₂ (Geigenberger 2003; Schwender et al. 2004).

Together, these considerations indicate that internal CO₂ accumulation cannot be viewed solely in terms of gaseous diffusion, but must be understood within the context of aqueous chemistry, enzyme-catalysed equilibration and metabolic utilisation of dissolved inorganic carbon. This framework provides the foundation for examining how CO₂ accumulation may influence intracellular pH and metabolic regulation in the following section.

6.3. Consequences for Intracellular pH and Buffering

The consequences of CO₂ accumulation for intracellular pH must be interpreted in the context of hypoxia-induced metabolic reconfiguration, where changes in carbon flux, proton balance and energy status occur simultaneously.

The interconversion of CO₂ and bicarbonate is intrinsically linked to proton production and consumption, and therefore to intracellular pH regulation. As a consequence, accumulation of CO₂ within plant tissues has the potential to influence cellular acid–base balance, particularly in environments where diffusion constraints and metabolic activity coincide. However, the extent to which internal CO₂ accumulation translates into measurable changes in cytosolic pH depends on the buffering capacity of the cell and the integration of multiple metabolic processes (Kurkdjian and Guern 1989; Raven 1985).

Cytosolic pH in plant cells is typically maintained within a relatively narrow range, often close to neutrality, despite substantial fluctuations in metabolic activity and environmental conditions. This stability reflects the operation of several buffering and regulatory systems, including phosphate buffers, organic acids, protein charge groups and active proton transport across cellular membranes (Felle 2001; Smith and Raven 1979). The vacuole, in particular, plays a central role as a dynamic reservoir for protons and organic acids, allowing adjustments in cytosolic pH through controlled exchange across the tonoplast (Martinoia et al. 2007).

Under conditions where internal CO₂ concentrations are elevated, hydration of CO₂ may contribute to proton generation, potentially influencing cytosolic pH. At the same time, ongoing metabolic processes such as glycolysis, mitochondrial respiration and, under more severe oxygen limitation, fermentative pathways also contribute to proton production or consumption. The resulting cytosolic pH therefore reflects the balance between multiple interacting processes rather than the effect of CO₂ alone (Geigenberger 2003; Gout et al. 2001).

Experimental evidence indicates that cytosolic pH can shift under conditions of hypoxia or metabolic perturbation, although these changes are often moderate and transient due to effective buffering. In root tissues and other actively metabolising systems, decreases in cytosolic pH have been observed during oxygen limitation, frequently accompanied by adjustments in metabolic fluxes and ion transport (Drew 1997; Felle 2005). Similar responses may occur in other diffusion-limited tissues where respiratory CO₂ accumulation and restricted oxidative phosphorylation coincide.

Organic acids, particularly malate, play a dual role in this context. In addition to serving as intermediates in central carbon metabolism, they contribute to cellular buffering capacity and can participate in proton-coupled transport processes. The interconversion between organic acids and their conjugate bases provides a mechanism for stabilising cytosolic pH while simultaneously linking acid–base balance to carbon metabolism (Igamberdiev and Eprintsev 2016; Raven and Smith 1976).

The interaction between CO₂ accumulation, bicarbonate formation and intracellular pH regulation is therefore best understood as part of an integrated system in which carbon metabolism and proton balance are closely coupled. Rather than acting as an isolated driver of acidification, CO₂ contributes to a broader network of processes that collectively determine cytosolic pH and its stability.

These considerations are particularly relevant for diffusion-limited tissues, where elevated internal CO₂, altered respiratory metabolism and changes in energy status occur simultaneously. In such environments, even modest shifts in cytosolic pH may have significant consequences for enzyme activity, metabolite partitioning and transport processes. At the same time, the buffering capacity of the cell constrains the magnitude of these changes, allowing metabolic function to be maintained within viable limits.

Understanding how intracellular pH is regulated under these conditions provides an essential foundation for analysing the metabolic consequences of CO₂ accumulation. In particular, the sensitivity of key enzymes and pathways to pH and bicarbonate availability will determine how carbon flux is redistributed in diffusion-limited tissues.

6.4. PEPC-Mediated Carboxylation in Heterotrophic Tissues

In this context, PEPC-mediated fixation provides a direct link between internally generated CO₂ and the accumulation of organic acids such as malate, thereby integrating carbon reassimilation with the alternative metabolic pathways described in the preceding section.

Phosphoenolpyruvate carboxylase catalyzes the irreversible fixation of bicarbonate into oxaloacetate using phosphoenolpyruvate as substrate:

$$\mathrm{PEP}+{\mathrm{HCO}}_3^{-}\to \mathrm{Oxaloacetate}+{\mathrm{P}}_{\mathrm{i}}$$

(6)

This reaction is widespread in plant metabolism and occurs in many non-photosynthetic tissues. The oxaloacetate produced can be converted to malate or aspartate, providing intermediates for several metabolic processes (Chollet et al. 1996; O’Leary 1982).Plants may express different forms of PEPC in a tissue specific way (Millsteed et al. 2025; ?). The forms in the photosynthetic leaves of C4 plants may support accumulation of higher levels of C4 product due to reduced affinity for the product (malate) resulting from a single amino acid substitution in the PEPC Paulus et al. (2013). The patterns of cell or tissue specific expression of different PEPC genes in relation to hypoxia needs to investigated.

In heterotrophic tissues PEPC is often associated with anaplerotic replenishment of tricarboxylic acid cycle intermediates, particularly during periods of active nitrogen assimilation and amino acid synthesis. However, PEPC-mediated fixation alone does not necessarily lead to long-term carbon retention. If the resulting C₄ acids are subsequently decarboxylated, the carbon may ultimately be released again as CO₂ (Chollet et al. 1996; O’Leary 1982).

Consequently, additional pathways may be required to convert fixed carbon into metabolites that can contribute to carbohydrate storage. This is one reason why PEPC is most compelling when considered alongside transport, compartmentation and downstream reassimilation pathways rather than as a stand-alone carbon-conserving mechanism Ting et al. (2017); Walker et al. (2011); Zhi et al. (2021).

6.5. Rubisco in Non-Green Tissues

The operation of this pathway under hypoxia implies that carbon reassimilation can occur even when ATP availability is constrained, particularly if supported by PP_i-dependent metabolism and reduced energetic costs elsewhere in the network.

Although Rubisco is traditionally associated with photosynthetic carbon fixation in chloroplasts of leaf tissues, the enzyme is also present in many non-green plant organs. Developing seeds, fruit tissues and stems may contain Rubisco even when chlorophyll content is low (Henry et al. 2020; Schwender et al. 2004; ?).

In these tissues Rubisco does not necessarily function in classical photosynthesis driven by light reactions. Instead, ATP and reducing equivalents required for the Calvin cycle may be supplied by respiratory metabolism or by local plastid metabolism (Schwender et al. 2004).

Rubisco-mediated carboxylation produces two molecules of 3-phosphoglycerate:

$$\mathrm{RuBP}+{\mathrm{CO}}_2\to 23\mathrm{PGA}$$

(7)

These intermediates can re-enter central carbon metabolism and contribute to synthesis of sugars or starch. In this way Rubisco may function as part of a carbon recycling system that reassimilates internally released CO₂ and improves carbon conversion efficiency in heterotrophic tissues (Schwender et al. 2004).

7. Contribution of Photosynthesis in Outer Tissues

In some cases, highly photosynthetic tissues may be found in outer parts of the plant to which light can penetrate Henry et al. (2020).These tissues may suffer from limited potential for gas exchange with the atmosphere due to barriers to water loss. Examples include seeds and stems with an outer photosynthetic layer surrounding large amounts of highly hypoxic tissues. The photosynthetic pericarp of seeds is surrounded by a seed coat that limits gas exchange. Tree trunks are often green due to similar surface photosynthesis. This provides an opportunity to alleviate some of the hypoxia and re-fix some of the carbon in these organs. Photosynthesis in the wheat pericarp peaks at mid seed development when respiration to support biosynthesis of seed storage proteins and carbohydrates peaks in the endosperm. PEPC is highly active in the endosperm and Rubisco in the pericarp at this time Millsteed et al. (2025). However, the deep tissues in these organs are likely to remain highly limited in O2 and have excess CO2. A significant proportion of the photosynthate from the leaves entering the developing seed is respired to support the synthesis of the starch, proteins and lipids stored in the seed. Without effective re-fixation this would result in a significant loss of carbon and yield in a crop. The extent of contribution of photosynthesis in these systems to the metabolic processes described here remains to be established.

Taken together, these processes indicate that internal CO₂ accumulation, bicarbonate equilibration and carboxylation reactions are integral components of a coordinated metabolic response to oxygen limitation. Rather than representing isolated biochemical pathways, PEPC- and Rubisco-mediated reactions provide mechanisms for retaining carbon within central metabolism under conditions where fermentative loss might otherwise dominate. When considered alongside PP_i-dependent energy conservation and alternative carbon sinks such as alanine and sucrose, these processes support a model in which hypoxia promotes a shift towards integrated carbon recycling and energetic efficiency in diffusion-limited tissues.

8. Integration of Metabolic Pathways in Diffusion-Limited Tissues

8.1. A Unified Framework for Energy Limitation and Carbon Balance

The preceding sections highlight two fundamental consequences of diffusion limitation in plant tissues: a reduction in oxygen availability, which constrains ATP production, and an accumulation of respiratory carbon dioxide, which increases the availability of inorganic carbon. These two features are intrinsically linked but have rarely been considered together.

This framework indicates that metabolism in diffusion-limited tissues is best understood as an integrated system in which energy limitation and carbon availability are simultaneously modified. Within this framework, fermentative pathways, PPi-dependent reactions, and CO₂ reassimilation via PEPC and Rubisco do not operate as independent responses, but as interacting components of a coordinated metabolic strategy.

This perspective shifts the interpretation of hypoxia from a purely limiting condition to a context in which alternative energy economies and carbon recycling pathways become central to maintaining metabolic function.

8.2. Role of PPi-Dependent Metabolism in Energy Buffering

Pyrophosphate-dependent metabolism plays a central role in this decoupling by reducing ATP demand. By substituting PPi for ATP in key metabolic steps, PPi-dependent enzymes effectively lower the energetic cost of maintaining metabolic flux under oxygen limitation.

This reduction in ATP requirement dampens the increase in substrate demand that would otherwise be necessary to compensate for reduced ATP yield. In doing so, PPi-dependent metabolism partially offsets the energetic penalty of hypoxia and contributes to maintaining metabolic continuity.

Importantly, the contribution of PPi-dependent reactions is expected to increase as ATP availability declines, making this pathway particularly relevant under more severe diffusion limitation.

8.3. Internal CO₂ Recycling and Carbon Retention

At the same time, the accumulation of internal CO₂ provides a substrate pool for carbon recycling. Through the action of carbonic anhydrase, CO₂ is rapidly converted to bicarbonate, which can be fixed by PEPC into oxaloacetate and subsequently into organic acids.

These intermediates may serve as temporary carbon reservoirs or be further metabolized through pathways that lead to reassimilation via Rubisco or incorporation into biosynthetic processes. In this way, internally generated CO₂ can be partially retained within the metabolic network rather than being lost to the atmosphere.

The extent to which this recycling contributes to overall carbon balance will depend on the relative rates of fixation, decarboxylation and transport, as well as the spatial organisation of metabolic processes within tissues and cells.

8.4. Integrated Consequences for Metabolism and Sink Strength

The combined effects of reduced ATP demand and enhanced carbon retention define a distinct metabolic mode characteristic of diffusion-limited tissues. In this mode, metabolic flux is maintained through increased glycolytic activity and alternative energy pathways, while carbon loss is constrained through recycling mechanisms.

This integrated system provides a potential explanation for how metabolically active tissues can sustain growth, storage and biosynthesis under conditions that would otherwise appear energetically unfavourable. By decoupling energy demand from carbon loss, plants may maintain higher carbon-use efficiency and sustain sink strength even when oxygen availability is limited.

This perspective also suggests that diffusion limitation should not be viewed solely as a constraint on metabolism, but as a condition that favours the emergence of coordinated energy and carbon management strategies.

8.5. Conceptual Synthesis

The relationships described above are illustrated in Figure 3, which separates the effects of hypoxia on ATP yield, substrate demand and carbon loss. This framework highlights three key features of diffusion-limited metabolism: (i) declining ATP yield increases substrate demand, (ii) PPi-dependent metabolism dampens this increase by reducing ATP requirements, and (iii) carbon recycling pathways reduce net CO₂ loss relative to substrate flux.

Together, these processes define a metabolic system in which energy limitation and carbon conservation are tightly integrated. Recognizing this integration provides a new conceptual basis for understanding metabolism in plant tissues where gas exchange is inherently restricted. This integrated perspective is further distilled into a conceptual diagram in Figure 4, which summarizes how diffusion limitation simultaneously reshapes energy demand and carbon balance in plant tissues.

9. Conclusions and Future Directions

Diffusion limitation is an inherent feature of many plant tissues and results in a coupled internal environment characterised by reduced oxygen availability and elevated carbon dioxide. This dual constraint fundamentally alters the relationship between energy metabolism and carbon balance by simultaneously limiting mitochondrial ATP production and increasing the local availability of respired carbon.

The evidence synthesised in this review indicates that plant metabolism is not passively constrained under these conditions but is actively reconfigured. Fermentative metabolism, pyrophosphate (PP_i)-dependent reactions, and internal CO₂ recycling via PEPC and, in specific contexts, Rubisco, should therefore be viewed not as independent responses but as components of an integrated metabolic system. Within this framework, reduced ATP yield under hypoxia increases substrate demand, but carbon loss is not necessarily proportional. Instead, PP_i-dependent metabolism reduces ATP requirements, while internal CO₂ reassimilation promotes carbon retention.

A central outcome of this integration is the partial decoupling between substrate consumption and net CO₂ release. Under reduced oxygen availability, declining ATP yield from oxidative phosphorylation necessitates increased glycolytic flux to sustain cellular energy demand (Drew 1997; Geigenberger 2003). In a fermentation-dominated response, this increased substrate flux is accompanied by proportional carbon loss through decarboxylation and ethanol production, maintaining a tight coupling between energy demand and CO₂ release (Drew 1997). However, this coupling is not obligatory. When metabolic configurations favour carbon retention—through reduced decarboxylation, diversion of carbon into organic acid pools, or reassimilation of internally generated CO₂—net carbon loss can be substantially lower than predicted from substrate demand alone (Schwender et al. 2004). In such cases, substrate consumption increases to meet energetic requirements, while CO₂ release remains constrained.

This decoupling provides a mechanistic basis for maintaining carbon-use efficiency in tissues where gas exchange is restricted, allowing continued metabolic activity, growth, and storage despite energetic constraints. The conceptual relationships emerging from this synthesis are summarised in Figure 4, which integrates the effects of oxygen limitation, PP_i-dependent energy buffering, and carbon recycling into a unified framework. This perspective shifts the interpretation of hypoxia from a purely limiting condition to one that favours coordinated metabolic adaptation, in which energy economy and carbon conservation are jointly optimised.

Several key questions remain unresolved. Quantitative estimates of the contribution of PP_i-dependent reactions to whole-tissue energy balance are limited, and the extent to which internal CO₂ reassimilation contributes to long-term carbon retention remains poorly constrained. In addition, the spatial organisation of these processes within tissues, cells, and organelles is not well understood. A critical limitation of current knowledge is that most studies rely on bulk tissue measurements that obscure spatial heterogeneity in gas availability and metabolic activity.

Recent advances in spatially resolved and cell-type-specific approaches provide an opportunity to address these limitations. Techniques such as spatial transcriptomics, single-cell RNA sequencing, and imaging-based metabolite profiling enable the integration of gene expression, metabolic activity, and tissue structure within their native spatial context (Long et al. 2021; Tao et al. 2026; ?; ?). These approaches are particularly well suited to diffusion-limited systems, where steep gradients in O₂ and CO₂ occur over short distances.

In this context, spatially resolved analysis offers a promising route for testing and refining the framework proposed here. Gradients in oxygen and carbon dioxide are inherently spatial, varying across vascular pathways, storage parenchyma, and subcellular compartments. Resolving how enzyme activity, metabolite pools, and fluxes are distributed across these gradients will be essential for understanding how metabolic regulation is achieved in vivo. In particular, coordinated analysis of glycolytic flux, PP_i-dependent metabolism, and carboxylation pathways may reveal how energy economy and carbon recycling are partitioned across tissues and cell types.

Integration of spatial transcriptomic and metabolomic data with gas profiling and quantitative flux analysis has the potential to move beyond bulk measurements toward a mechanistic understanding of diffusion-limited metabolism. Such approaches may ultimately enable identification of spatially organised metabolic networks that underpin sink strength, carbon-use efficiency, and biomass accumulation. These insights will be essential for evaluating the extent to which coordinated energy conservation and carbon recycling can be exploited to improve productivity in crop systems.

9.1. Testable Predictions Emerging from the Framework

The integrated framework proposed here generates several experimentally testable predictions that distinguish it from classical models of hypoxic metabolism.

Prediction 1: Decoupling of substrate consumption and CO₂ release under hypoxia. If carbon recycling and PP_i-dependent metabolism are active, increases in substrate flux required to sustain ATP demand under reduced oxygen availability will not be matched by proportional increases in net CO₂ efflux. This can be tested by simultaneous measurements of substrate utilisation and CO₂ release across controlled oxygen gradients.

Prediction 2: Spatial coordination of PP_i-dependent metabolism and carboxylation pathways. The framework predicts that expression and activity of PP_i-dependent enzymes (e.g. PFP, V-PPase) and carboxylation pathways (PEPC, Rubisco where present) will be spatially correlated with regions of low oxygen and elevated CO₂ within tissues. Spatially resolved transcriptomic, proteomic, and metabolite profiling should reveal co-localisation of these processes along oxygen gradients.

Prediction 3: Enhanced retention of carbon in organic acid and amino acid pools under hypoxia. Under diffusion limitation, metabolic flux is predicted to favour accumulation of intermediates such as malate and alanine as temporary carbon reservoirs. Quantitative flux analysis should demonstrate increased partitioning of carbon into these pools relative to ethanol and CO₂ under conditions where PP_i-dependent metabolism is active.

Prediction 4: Reduced ATP cost per unit metabolic flux in PP_i-engaged systems. Systems with active PP_i-dependent metabolism should exhibit a lower ATP requirement per unit of glycolytic flux compared with systems relying solely on ATP-dependent pathways. This can be assessed through combined measurements of adenylate energy charge, PP_i levels, and metabolic flux.

Testing these predictions will be essential for evaluating the extent to which coordinated energy conservation and carbon recycling contribute to metabolic efficiency in diffusion-limited plant tissues.

10. Financial Support

RH was supported by funds from the ARC Research Hub for Engineering Plants to Replace Fossil Carbon (Grant IH230100006).