High density optical measurements
To create the timeline of optical measurements after the imposition of
TPU limitation, plants were first acclimated at 400 ppm
CO2 and 1000 µmol
m-2 s-1 light in the chamber of the
modified 6800-12A clear-top chamber. A list of times from 10-200 s was
randomized by script, and for each time interval a second script was
run. This script controlled a flow controller to rapidly switch the
plant from 400 ppm CO2 to 1500 ppm CO2.
A measurement of electrochromic shift was made by dark interval
relaxation kinetics (DIRK) (Takizawa et al. 2007) after the
chosen time period. Ten s later, a measurement of PSI oxidation state
decay and reoxidation by saturation flash was made. Leaves were then
incubated at 400 ppm CO2 for 10 min. The process was
then repeated, but instead of a DIRK to measure ECS, a saturation flash
was given to assess PSII characteristics, including the quantum
efficiency of photosystem II (φII ) (Baker 2008)
and oxidation status of the quinone Qa, measured asqL (Kramer, Johnson, Kiirats & Edwards
2004). Leaves were again incubated at 400 ppm
CO2 for 10 min. This process was repeated for every time
interval in the list. This protocol was used so that the disruptive
saturating flash did not affect subsequent measurements in the
timecourse.