Fast onset kinetics in responses to TPU limitation are directed by electron build-up on Qa
When plants were subjected to TPU-limiting conditions, the most immediate effects were transient changes in the redox states of electron transport components. It is known that while TPU-limited, increasing CO2 levels cause a reduction inφII because, while A cannot increase, the rate of photorespiration will go down (Stitt 1986; Sharkey et al.1988; Stitt & Grosse 1988). This, combined with the common observations of elevated PMF and non-photochemical quenching during TPU limitation, indicates the importance of qE in dissipating absorbed light energy when electron transport capacity exceeds TPU capacity. However, qE does not activate instantaneously, with the xanthophyll cycle and PSBS recruitment to the reaction center operating on the minutes timescale (Li et al. 2002). Therefore, we could reasonably predict excess accumulation of electrons on electron transport intermediates and PSI electron acceptors. Reduction of Qa decreases the quantum efficiency of photochemistry because PSII cannot accept any more energy. The energy that would be going towards photochemistry is instead shunted to nonphotochemical quenching, resulting in an increased yield of nonphotochemical quenching. This means thatφNPQ increases even thoughNPQt changes on a slower timescale. Immediately after entering TPU limitation, electrons build up on the electron transport chain due to decreased electron sink strength, and the bulk of the excess energy is most immediately handled by controls within the electron transport chain.
Though the reduction of Qa reduces the yield of photochemistry, the reduction of PSI following the imposition of TPU limitation is more concerning. Acceptor-side limitation of PSI is highly stressful due to the accumulation of ROS (Li, Wakao, Fischer & Niyogi 2009) and the inability of PSI to repair itself (Sonoike 1996, 2011). Electron transfer to PSI from the cytochrome b6f complex is slowed by elevated PMF due to the requirement to oxidize plastoquinol (Kramer & Crofts 1993, 1996). We found, however, that PMF does not build up fast enough to adjust to the limiting demand from the Calvin-Benson cycle and regulate electron flow to PSI, and electrons do indeed accumulate on PSI. This is not due to an accelerated rate of PSI reduction through the cytochrome b6f complex (ket , Fig. 4), so it must instead be due to an acceptor side limitation of PSI. Increasing [CO2] under TPU limitation reduces the rate of photorespiration, and ifA cannot increase due to TPU limitation the overall rate of consumption of both ATP and NADPH decreases. The NADPH pool turnover (half-time 0.01 s-1) is faster than that of ATP (half-time 0.28 s-1, Arrivault et al. , 2009), so the reduced consumption of electron transport products will affect NADP+ availability first. Restriction of NADPH oxidation has been suggested previously as the cause of oscillations in TPU limitation (Furbank, Foyer & Walker 1987). The restriction of NADP+ flux can be seen in the re-reduction of PSI during a saturation flash at the point of greatest PSI reduction (Fig 5a). During this saturation flash, light is in excess of what is required to oxidize PSI, and the only limitation would be the electron carriers removing the electrons from PSI.
The accumulation of electrons on electron carriers of the electron transport chain is resolved by slower regulation. PMF increases, causing a decrease in ket and an increase inNPQt . As these slower control mechanisms take hold, the transients in the other parameters slow and then stop. This is one example of damped oscillations, commonly found associated with TPU limitation (Ogawa 1982; Sivak & Walker 1986, 1987). The oscillations are caused by perturbations in the electron requirements of the Calvin-Benson cycle forcing Qa- based control of electron transport; they are damped by the onset of PMF-based controls of electron transport. Some, but not all measurements of oscillations are consistent with the period and convergence rates in our measurements of oscillations. We therefore propose that electron carrier reduction as described here is responsible for some, but not all, observations of oscillations in TPU limitation.
It has been repeatedly observed that TPU limitation is rare to nonexistent in wild plants (Rogers et al. 2020), but can often be found in experiments using high levels of light with elevated CO2, decreased O2, and/or low temperature. The most stressful moments will be when the plant enters TPU limitation and electrons accumulate on PSI electron acceptors, and therefore would be most stressful when the experimental design would cause fluctuations in photosynthetic abilities. For example, when a plant is held at low temperature, fluctuating light levels would exacerbate the stressful TPU conditions. We believe this can be stressful for plants in FACE experiments, where absolute consistency in CO2 levels across the field and perfect mixing cannot be reasonably expected. Allen et al. (2020) discussed the difficulties in maintaining perfect mixing across a FACE field, and we believe that the stress of entering and exiting TPU due to fluctuating CO2 levels can be a drag on plant performance, potentially reducing the expected stimulation of growth in high CO2 FACE experiments (Long, Ainsworth, Leakey, Ort & No 2006).