Conclusions
Several remarkable results lead us to propose that multiple electron acceptors can protect PSII against photodamage under combined high light and elevated temperatures. One remarkable observation from our work was the nearly temperature-independent responses of the light reactions. Most strikingly, the redox state of QA, often taken as a measure of the regulatory balance of the light reactions, tended to become more oxidized as temperature increased (Figure 1E). These results imply that, even as assimilation was significantly reduced by temperature, the light reactions were stimulated (rather than inhibited) in a light intensity dependent manner. Under fluctuating light conditions, increasing temperature increased ɸ II, decreased both q I and q Ecomponents of NPQ, and resulted in a less reduced QA, suggesting that HT decreased the accumulation of states that would normally result in photodamage (Figure 6). These effects were more pronounced following acclimation to HT and fluctuating/high light. Separate experiments suggest that the rates of photodamage are decreased and the rates of PSII repair increased under HT.
The small impact of temperature on the light reactions can in large part be explained by the fact that the sinks for electrons from LEF remained highly active. When A decreased, electrons flowed to other electron acceptors. A large fraction of these acceptors can be accounted for by increased photorespiration, but an additional fraction appears to be sent to a light intensity dependent process, possibly involving O2 reduction by a process with a relatively lowK m for O2, or other metabolic electron sinks.
Similarly, the regulation (or control) of the light reactions through the pmf was less engaged at higher temperatures, with increased ATP synthase activity (g H+), and decreased pmf (Figure 1). This combination of increased LEF and high g H+ are consistent with increases in sinks for both electrons and ATP, i.e. there did appear to be a depletion of sinks or substrates for ATP synthase. These results pose an interesting question: how is the supply of outputs of ATP and NADPH from the light reactions maintained in balance with metabolic demand despite the diversion of energy to alternative processes? In particular, photorespiration requires additional ATP/NADPH compared to the CBB cycle, and one might expect that ATP supplementation would be required under high photorespiratory conditions at HT. However, CEF (Figure 1F–G), which is thought to supplement ATP under such conditions (Strand, Fisher, & Kramer, 2017), actually decreased under elevated temperatures in the young leaves, suggesting that other processes allowed for ATP/NADPH balance. One possibility is alternative electron flow to O2, as suggested by comparisons of ETR calculated from the sum of the velocity for carboxylation and oxygenation (ETRGE, Figure 4) and chlorophyll fluorescence (ETR), which should have increased ATP production without net changes in NADPH (Miyake, 2010). On the other hand, in mature leaves, CEF increased in HL, consistent with the increased photorespiration accounting for a large fraction of electrons under HT (Figure 4). In either case, we show clear evidence that photorespiration is an essential electron sink that appears to help maintain open PSII redox sites under high light and temperature stresses.
A second remarkable observation is the light intensity-and temperature-dependent down-regulation or inhibition of rubisco activity at low O2 in (Figure 5, Supplementary Figure 5 and 6). This effect can most easily be explained by deactivation of rubisco itself, though our data cannot rule out other indirect effects that result in apparent loss of rubisco activity estimated by gas exchange. Nevertheless, the effect was mostly seen in HL, but was more pronounced at HT and under low CO2 conditions, and was rapidly reversible upon increasing CO2, suggesting that it serves a regulatory role.
Overall, cowpea appears to have mechanisms that allow the light reactions to maintain high activity and low propensity for ROS generation, through a combination of highly active alternative energy sinks, including photorespiration and other, yet undefined, electron sinks. We speculate that breeding or engineering crops to increase the capacity of these sinks could lead to more robust crop productivity under field conditions where acute combined high light and temperature stresses occur.
Acknowledgements
The authors would like to thank Drs Philip Roberts and Bao Lam Huynh for providing cowpea seeds and along with Drs. Jeffrey Ehlers, Wayne Loescher and Irvin Widders for useful discussions and David Hall for helping with the DEPI experiments.