INTRODUCTION
Photosynthesis, as measured by gas exchange, is typically assessed by the three canonical biochemical limitations of photosynthesis: the rubisco limitation, where carbon dioxide uptake is modeled assuming ribulose 1,5-bisphosphate (RuBP)-saturated rubisco kinetics; the RuBP regeneration limitation, where carbon dioxide uptake is modeled assuming a fixed rate of RuBP use as allowed by the production of electron transport products, ATP and NADPH; and the triose phosphate utilization (TPU) limitation, where carbon dioxide uptake is modeled as the rate of production of end products, freeing inorganic phosphate from organic phosphates (McClain & Sharkey 2019). The TPU limitation is unique among the three biochemical limitations in that it is limited by downstream processes, rather than just at rubisco. The dislocation of the limitation from rubisco means that regulatory mechanisms are engaged to slow down the rate of carbon assimilation (A ) so as not to outpace the rate of end-product synthesis. Energy-dependent quenching (qE ) is activated (Sharkey, Berry & Sage 1988) by elevated ΔpH across the thylakoid membrane, one component of proton-motive force (PMF) (Kramer & Crofts 1996). The elevated ΔpH results from kinetic and thermodynamic restrictions on the ATPase due to lowered levels of available inorganic phosphate (Sharkey & Vanderveer 1989). In addition, rubisco activation state decreases (Sharkey, Seemann & Berry 1986a; Socias, Medrano & Sharkey 1993), which may alleviate pressure on phosphate pools by limiting the maximum rate that carbon can be added to the organic phosphate pool. Because TPU limitation restricts the rate of photosynthesis rather than the availability of light, there is a potential for photodamage unless regulatory mechanisms are engaged (Powles 1984; Pammenter, Loreto & Sharkey 1993; Li, Müller-Moulé, Gilmore & Niyogi 2002).
These regulatory mechanisms are the only aspects of TPU limitation typically observed in steady-state gas exchange. While TPU limitation results in, and can be assessed through, gas exchange as O2- and CO2-insensitive photosynthesis (Sharkey 1985) or reverse sensitivity to O2 (Viil, Laisk, Oja & Pärnik 1977) or CO2 (Jolliffe & Tregunna 1973), it is easier to assess by the decline in electron transport rate associated with qE when CO2 is increased or O2 is decreased. The appearance of transient effects on photosynthesis associated with TPU limitation (Ogawa 1982; Walker, Sivak, Prinsley & Cheesbrough 1983) lead us to believe that, in the steady state, the rate of photosynthesis is not set by TPU, but instead, the rate is set by regulatory mechanisms that match the rates of carbon input to and carbon output from the organic phosphate pool.
The nitrogen required for rubisco and photosynthetic electron transport far exceed those required for TPU and subsequent end product synthesis (Evans & Clarke 2019). When TPU occurs, rubisco is deactivated andqE is increased reducing the efficiency of nitrogen use in both carbon metabolism and electron transport. Because TPU capacity is relatively cheap and entering TPU limitation forces deactivation of systems which use much more nitrogen, an ideal plant would never experience TPU limitation under physiological conditions. However, TPU limitation is commonly seen when the photosynthetic rate is only a few percent higher than what the plant experiences in ambient conditions (Yang, Preiser, Li, Weise & Sharkey 2016). There are a few possible reasons why excess TPU capacity would be detrimental. A precise balance of phosphate flux could control stromal inorganic phosphate concentration, affecting the partitioning of carbon into starch (Preiss 1982; Escobar-Gutiérrez & Gaudillère 1997). If TPU capacity were in excess, it could also limit the ability to build up a PMF across the thylakoid membrane because there would be plentiful phosphate available to the ATPase, preventing any kinetic or thermodynamic restriction to proton flow. The elevated ΔpH and consequent low luminal pH can activate energy-dependent quenching mechanisms that dissipate light energy to safeguard the photosystems.
If TPU capacity is inexpensive in terms of nitrogen cost, but is typically just above ambient photosynthetic rates, we would expect that TPU capacity is plastic. It has been found that TPU capacity is flexible, and in many cases changes in response to environmental conditions. Plants grown at low temperature can develop additional sucrose synthesis enzymes (Cornic & Louason 1980; Guy, Huber & Huber 1992; Holaday, Martindale, Alred, Brooks & Leegood 1992) which alleviates cold-induced TPU limitation (Sage & Sharkey 1987). Plants with reduced access to CO2 have reduced TPU capacity to match their lowered photosynthetic rate (von Caemmerer & Farquhar 1984; Sharkey & Vassey 1989). It has therefore been shown that TPU capacity can both increase and decrease in response to environmental conditions. This is reflected in environmental surveys, and plants have rarely been found to be TPU limited under ambient conditions in the field (Sage & Sharkey 1987; Ellsworth, Crous, Lambers & Cooke 2015). For this reason, TPU limitation is often not included in global models of photosynthesis (Lombardozzi et al. 2018; Rogers et al. 2020).
Ideally, if a plant is TPU limited, it will increase its TPU capacity to maximize the overall rate of photosynthesis, but it is also possible that rubisco capacity and electron transport capacity will be decreased to match TPU capacity. We tested the acclimation of plants to TPU limitation by exposure to elevated CO2 to determine whether plants eventually stop being TPU limited, and if they achieve this by increasing their TPU capacity. In addition, we established a timeline of the regulatory features surrounding TPU limitation, from how the plant handles the initial influx of energy until the plant engages slower regulatory features, such as rubisco deactivation and energy-dependent quenching.