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.