Detailed kinetics of photosynthetic processes in response to CO2 pulses
A step change in CO2 to levels that cause TPU limitation induced kinetics in the electron transport chain (Fig 4). There were several kinetic stages. At first, the elevated CO2allowed a faster use of electrons, and PSI became oxidized. The plant had not yet entered TPU limitation, as indicated by the high proton conductivity of the ATP synthase (gH+ ). The second phase (Fig. 4, blue), beginning 40 s after the step change in CO2 flow and persisting until 80 s after the beginning of CO2 flow, was characterized by the reduction of Qa [qL is a fluorescence-based measure that increases with increased oxidation of Qa(Kramer et al. 2004)]. The reduction of Qacaused an increase in φNPQ and a decrease inφII even though NPQ [measured using the NPQt parameter (Tietz, Hall, Cruz & Kramer 2017)] did not respond within this timeframe. The reduction of Qawas correlated with the reduction of PSI. The kinetic constant for reduction of PSI by cytochrome b6f(ket ), decreased, so we conclude that the reduction of PSI was not due to excess electrons being transported downstream. Therefore, the reduction of PSI must be due to an acceptor-side limitation of PSI. In the same stage, a decline ingH+ can be seen, decreasing by over 50%. The lowgH+ that was observed has been shown to be associated with TPU limitation (Kiirats, Cruz, Edwards & Kramer 2009; Yang et al. 2016). The third kinetic stage (Fig 4, green) began 80 s after the beginning of the CO2 step change and exhibited slower regulatory mechanisms. Proton-motive force increased up to this point, and continued to increase during this phase, which caused an increase in energy-dependent NPQt , and a decrease in ket . These mechanisms prevent electrons from reaching PSI, alleviating the over-reduction of PSI. After the PMF increased sufficiently, photosynthesis entered a new steady-state (Fig. 4, red).
The appearance of PSI acceptor-side limitations is supported by the observed response of PSI oxidation state to flashes of saturating light (Fig 5). Leaves were given a brief dark interval to allow reduction of PSI and then PSI was oxidized by a saturating flash. When tested in the middle of TPU-induced transients (Fig 5a), PSI did not remain oxidized by the saturating flash, and instead began re-reducing due to inability to pass electrons to NADP+. Tests made some time after the onset of TPU-limiting conditions showed less re-reduction (Fig. 5b), and with more time, re-reduction was much less prominent (Fig. 5c).