**9. The Acclimation of Energy Balancing Networks to Long-Term Change in Energy Demand**

As mentioned above, plants must cope in the long term to changing ATP and NADPH demand to achieve energy balance. It is unclear whether the same mechanisms balance energy mismatches under long time scales as occur under shorter time scales. Furthermore, it is unknown to what degree energy balancing networks poise to a given demand and how this poise acclimates to changing demands. The acclimation of the energy balancing network can be investigated experimentally either via transition experiments or by examining mutants with an altered network capacity that forces flux through alternative facets of the network. We will first discuss the potential for acclimation of supply-side processes to changing energy demand before outlining how metabolic demand itself may acclimate to changes in energy balancing requirements.

According to our model of the two-component supply-side energy balancing system, malate valve activity should scale with excess ATP demand over long-term transitions to optimally use absorbed light. The largest driver of excess ATP demand for any condition is increased light, and so this model predicts that malate valve activity should increase with light. Indeed there appears to be a light and dark malate valve cycle, with the dark cycle relying on plastidic NAD-MDH and the light shuttle using plastidic NADP-MDH [58]. The switch to the NADP-MDH cycle is mediated through the light-dependent Fd-Trx system [114,115]. This activation (at least for NADP-MDH in isolated Pea chloroplasts) occurs within 10–20 min, and so activation of this component is likely integrated in our short-term measurements [116]. Activation occurs even more rapidly during a high-light transition [117]. This is an effective regulatory strategy at short time scales, since it activates malate valve activity when there is a surplus of NADPH, as when the C3 cycle is not consuming it fast enough and reduces it when there is too much NADP<sup>+</sup> [58]. It is also likely that as the malate valve is reaching full capacity, CEF plays a role in vivo during very short time scales, at least in C3 plants [118].

Other factors increase malate valve capacity over longer time scales. For example, after transfer to sustained high light, NADP-MDH expression and protein levels increase, suggesting increased capacity of the malate valve following hours of exposure to the new condition [119]. Interestingly, the same response is not observed when photoperiod increases, suggesting that a photosynthetic steady-state solution must be found and that the effect is not cumulative.

Interestingly, chloroplastic *nadh-mdh* mutants show no phenotype, even under stress conditions, potentially due to additional compensatory redox strategies [75,113]. It is important to note that the malate valve shuttles reducing power not only between the chloroplast and mitochondria, but also the peroxisome during photorespiration [120]. In contrast to chloroplastic *nadh-mdh* mutants, mitochondrial *nadh-mdh* mutants lacking both MDH isoforms (*mmdh1mmdh2*) show lowered photosynthetic rates and growth rates [121]. These decreases were likely due to impaired shuttling of reducing power from the mitochondrion to the peroxisome via the malate valve to provide the reducing power for hydroxypyruvate reduction in photorespiration, a viewpoint supported using 13C flux analysis of *mmdh1* [122]. Indeed *mmdh1mmdh2* show reduced photorespiratory capacity, but the reduced growth and photosynthesis is not explained strictly by decreased availability of reductant to photorespiration since mutants lacking the peroxisomal MDH isoforms show an even more subtle phenotype than *mmdh1mmdh2* [123,124]. This work with mitochondrial MDH indicates that the malate valve is not strictly required for energy balancing, but it is important for optimal photosynthesis and long-term growth.

The capacity for CEF itself may also increase over longer time scales to allow for increased energy balancing demand. Notably, NDH and FQR content change under different growth conditions [51]. The ratio of PSI and PSII re-proportions when plants are grown under light regimes with outputs that favor PSI or PSII. After hours or days, this results in changes to the actual stoichiometry of PSI and PSII photosystems, in green algae [125–127] and plants [128]. This re-proportioning also occurs days following transition between different light qualities, which can increase the capacity for CEF [129]. At short time scales, repartitioning of light energy between PSI and PSII occurs when reduced PQ builds up and triggers the phosphorylation of the PSII light harvesting complex. These then migrate to PSI to balance out energy capture [130]. While these state transitions occur in response to long-term differences in energy supply (changes in light regimes), it is not clear if this happens in response to long-term changes in energy demand. Such a change would predict that as conditions decrease in the ratio of photorespiration, there should be a decrease in demand for CEF and, therefore, a decrease in the PSI/PSII ratio.

Measurements of PSI/PSII from plants grown under conditions of different ATP/NADPH demand did not indicate that the capacity for CEF change with energy demand via changes in photosystem stoichiometry. Specifically, there was no difference in PSI/PSII in aspen trees exposed to elevated CO2 (560 PPM) over a single season following 5 years of elevated high CO2 treatment [131]. However, this increase in CO2 is not expected to change the demand for CEF by all this much (~1% of LEF

change). Additionally, micro-array work in soybeans exposed to 550 PPM also show no difference in photosystem expression, but interestingly show an increased expression of a mitochondrial ATP/ADP antiporter [132]. Overall, these findings do not point clearly to the acclimation of the capacity of CEF in response to changing energy demand, but the treatments resulted in relatively modest changes in energy demand and CEF was not evaluated specifically. There is clearly room for more work examining this question specifically.
