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.