**1. Introduction**

Photosynthetic organisms must match energy supply from the light reactions with metabolic demands to enable safe, flexible and efficient photosynthesis. Because of the interdependency between energy supply and metabolic demand, it is valuable to consider this linked energy network and not as a series of separate metabolic processes. This energy balancing network integrates ATP and reductant supply and metabolic demand to allow plants to efficiently and safely harvest energy from the sun under dynamic conditions (Figure 1). From this perspective, we will first discuss the basic mechanisms of energy balancing before presenting the demand for energy balancing under a variety of conditions. We will then discuss how the structure and efficiencies of the energy balancing network are poised to provide and turnover ATP and reducing power under a variety of conditions before exploring how the energy balancing network responds to long-term changes in metabolic demand.

**Figure 1.** The energy balancing network configured for "high-light and low-efficiency" and "low-light and high-efficiency" conditions. Shown for each light intensity are the energy-producing supply processes of linear electron flux (LEF), cyclic electron flux around photosystem I (CEF) and the malate valve (MV), which produce reducing power (referred to generically as NAD(P)H, in red) and ATP (yellow). Metabolic demand comprises the primary ATP and NADPH consuming processes in C3 plants, the C3 cycle, photorespiration (PR), nitrate assimilation (N) and the remaining metabolic sinks for ATP and NAD(P)H (other). Numbers represent the amount of light energy absorbed by either LEF or CEF (in μmol photons m−<sup>2</sup> s<sup>−</sup>1) needed to supply and balance the needs of metabolism. The thickness of all lines is proportional to the fluxes modeled as part of Table 1 calculated using data from Miyake et al. 2005 [1].

#### **2. Energy Balancing is Essential for Safe and Optimal Photosynthetic Systems**

The light reactions of photosynthesis provide the chemical energy needed for plant metabolism. The core reactions of oxygenic photosynthesis involve a process called "linear electron flow" (LEF), in which light energy is used to extract electrons from water and transfer them to NADP<sup>+</sup> while generating ATP from ADP and Pi [1,2], as detailed in Figure 2. These core processes store energy in two forms; ATP and NADPH. Extracting electrons from water and transferring them to NADP+, energy is stored in the two redox half reactions 4H<sup>+</sup> + O2/H2O and NADP<sup>+</sup> + H+/NADPH. In addition, the transfer of electrons results in the formation of the proton motive force (*pmf*), an electrochemical gradient of protons across the thylakoid membrane, which is dissipated by the ATP synthase to fuel the formation of ATP from ADP and Pi. The pmf is the sum of two energetic components; an electric field component (Δψ) and the free energy stored in a chemical gradient of protons (ΔpH). Vectorial electron transfer from the lumenal to the stromal face of the thylakoid membrane, within photosystem II (PSII) and the cytochrome b6f complex by the Q-cycle mechanism (reviewed in [3]) and photosystem I (PSI) results in the formation of Δψ. Both Δψ and ΔpH are energetically equivalent drivers of the ATP synthase [4,5], but have very different impacts on photophysiological processes, as discussed below. One important feature of LEF is that it produces ATP and NADPH in a fixed stoichiometry, likely 2.6 ATP to 2 NADPH, or 1.28 ATP/NADPH [6].

**Figure 2.** Basic Z-scheme model for the electronic and protonic circuits of the light reactions of photosynthesis, and the *pmf* paradigm for regulation of the light reactions. Scheme of linear electron flow (LEF) in oxygenic photosynthesis, in which light energy is captured by light harvesting complexes associated with photosystem II (PSII) and photosystem I (PSI), which initiates electron flow (orange arrows) from PSII, through the cytochrome *b6f* complex, plastocyanin (PC), to PSI and ferredoxin (Fd) and finally to NADPH. Also shown is the formation of the LEF is coupled to proton flow (blue arrows) at PSII and the cytochrome *b6f* complex, storing energy in the thylakoid proton motive force (*pmf*). Transfer of electrons from the lumenal to the stromal side of the thylakoid forms a transmembrane electric field (Δψ, blue arrow), while proton uptake from the stroma and deposition in the lumen lead to the formation of a transthylakoid pH gradient (ΔpH, red arrow), which together drive the synthesis of ATP from ADP + Pi at the thylakoid ATP synthase, storing energy in ΔGATP. The acidification of the lumen (indicated by the H<sup>+</sup> in the red box) activates violaxanthin deepoxidase (VDE) which converts violaxanthin (V) to zeaxanthin (Z) and protonates the PsbS protein, which triggers the photoprotective dissipation of light energy by the qE (black arrow). Lumen pH also regulates electron flow (red box with '-') to PSI by slowing the rate of PQH2 oxidation at the cytochrome *b6f* complex.

The chloroplast must also balance the output of energy into the ATP and NADPH pools to perfectly match metabolic demands. The pool sizes of ATP and NADPH are small relative to the high fluxes of energy from the light reactions. Thus, any imbalance in the production and consumption of ATP or NADPH can rapidly lead to "metabolic congestion," depletion or buildup of metabolic intermediates, leading to the accumulation of high energy intermediates of the light reactions within seconds [7–10]. On the other hand, if too little ATP and NADPH are produced metabolic demand is energy limited, meaning that central metabolism is sub-optimal. The "correct" output of ATP and NADPH is a moving target since metabolic demand for ATP and NADPH changes dynamically based on environmental and physiological contexts (See below and [11]). The supply of ATP and NADPH must be matched with demand both in total capacity and stoichiometrically, and therefore plants have evolved mechanisms for regulating total energy output and fine tuning ATP/NADPH production ratios.

To regulate total energy production, chloroplasts partition light energy between photochemical processes which generate ATP and NADPH (LEF) and the energy dissipating process of "non-photochemical quenching" (NPQ) [12–17]. When metabolic demand for energy is less than current supply, the major form of NPQ, termed qE (for 'energy dependent' quenching), is triggered by acidification of the lumen (i.e., by the ΔpH component of *pmf*), through activation of violaxanthin deepoxidase, which catalyzes the conversion of violaxanthin to antheraxanthin and zeaxanthin [18], and through protonation of the antenna protein PsbS [19,20]. The ΔpH component of *pmf* also down-regulates electron flow by slowing plastoquinol (PQH2) oxidation by the cytochrome *b6f* complex, preventing accumulation of electrons on highly reducing components of PSI, a process called "photosynthetic control" (reviewed in [21,22]) and subsequent PSI photodamage. Lumen acidification is, in turn, modulated by several processes that respond to the physiological state of the cell [1]. When metabolic demand is low, the activity of the ATP synthase is also down-regulated to slow proton efflux, increasing *pmf* and down-regulation of the light reactions [1,23–27]. The fraction of *pmf* stored in the

ΔpH or Δψ is modulated to adjust its regulatory impact of a particular *pmf* [8,26,28]. The responses of qE to lumen pH may also be modulated by altering the expression of qE-related components [29–31]. Quantitatively, the dynamic range of NPQ is large, able to effectively partition from <5% to >80% of absorbed light energy to or away from energy production within tens of minutes. Importantly, even though increased light induces NPQ and decreases photochemical efficiency, the increase in total absorbed photons often more than compensates for this reduction and total LEF increases to safely produce sufficient NADPH to meet metabolic demand. Note that NPQ can only modulate total NADPH production from LEF with no change to the production stoichiometry of 1.28 ATP/NADPH.
