Analysis of D1 and Rubisco protein contents
To extract D1 protein, fresh stems or leaves (0.3 g) were homogenized in
2 mL of radioimmunoprecipitation assay (RIPA) buffer containing 1 mM
PMSF and the proper amount of protease inhibitor in a precooled mortar,
kept in an ice bath for 2 h, and then centrifuged at 12,000×g for 10 min
at 4°C. The supernatant containing D1 protein was temporarily stored at
4°C. To extract Rubisco, fresh stems or leaves (0.1 g) were homogenized
in 1.5 ml of 60 mM Tris-HCl (pH 7.8) buffer consisting of 5% PVP (w/v),
0.1% NaCl (w/v) and 2% glycerol (v/v) in an ice bath and subsequently
centrifuged at 12,000×g for 10 min at 4°C (Zhang et al., 2016). Rubisco
proteins were distributed in the supernatant. The proper quantity (0.1
mL) of the extract of D1 protein or Rubisco and an equal volume of
protein loading buffer were mixed together, incubated at 100°C for 5 min
and then stored at 4°C for further analysis. An aliquot of each sample
(20 μl) was loaded into one well of an SDS-PAGE gel prepared in advance
(Zhang et al., 2016), and the proteins were separated by SDS-PAGE with a
Mini-PROTEAN 3 system (Bio-Rad, USA). The separated proteins in the gels
were blotted onto PVDF membranes, and the membranes were stained with
Ponceau S to confirm equal protein transfer. The membranes were then
washed three times (5 min each) in TBST (50 mM Tris [pH 7.5], 150 mM
NaCl, 0.1% Tween 20) and subsequently blocked with 5% (w/v) nonfat
powdered milk in TBST for 1.5 h. The membranes containing then D1
protein and the large subunit of Rubisco were incubated together with
PsbA global antibodies (1:10,000 dilution) (Agrisera, Sweden) and
anti-Rubisco antibodies (1:1,000 dilution) (Bioss, Beijing, China)
overnight at 4°C. The blots were washed with TBST three times and then
incubated with goat anti-rabbit HRP-conjugated secondary antibodies
(1:3,000 dilution) at room temperature for 50 min. The blots were
subsequently washed three times in TBST, and protein visualization was
performed using a Tanon 5200 enhanced chemiluminescence (ECL) detection
system (Tanon, Shanghai, China).