Rubisco assay
The Rubisco content in a leaf bade was determined as described by Makino
et al. (1986). Rubisco was separated by SDS-PAGE and stained with
Commassie Brilliant Blue R-250 (CBB-R). Subsequently, the Rubisco
protein bound to CBB-R was cut out from the SDS-PAGE gel and extracted
with formamide at 50 oC for 5 h. The absorption of the
extract was measured at 595 nm, and the Rubisco content was determined.
Rubisco activation was determined as described by Suganami et al.
(2018). A leaf blade was illuminated at an irradiance of 1,100 µE
m-2 s-1 and 25 oC
under ambient air conditions (40 Pa CO2, 21 kPa
O2) for at least 1 h, and the illuminated leaf was
immediately frozen in liquid N2. The frozen leaf was
homogenized in the Rubisco extraction buffer [100 mM
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) / NaOH (pH
8.0), 20 mM MgCl2, and 10 mM dithiothreitol (DTT)]
with quartz sand on ice and centrifuged (19,000×g , 10 s at 4oC) to obtain a solution containing Rubisco. Rubisco
activation was determined by multiplying the initial activity with the
maximum activity, and the carbamylation potential was determined by
multiplying the total activity with the maximum activity.