As01 003

Total proteins were isolated from whole seedlings (grown under long-day conditions with 100 µmol·m⁻²·s⁻¹ light intensity at 22 °C for 10 days as indicated). Proteins were separated by 4–12% SDS-PAGE gel. After the proteins were transferred electrophoretically onto the nitrocellulose filter membrane, the membrane was incubated with corresponding antibodies. Anti-ATPC1 (AS08312, Agrisera, Vännäs, Sweden), anti-GAPDH (K90002P, Solarbio, Beijing, China), anti-PSAD (PHY0056A, PhytoAB, San Jose, CA, USA), anti-PSBQ (PHY2346A, PhytoAB, San Jose, CA, USA), anti-LHCA1 (PHY0043A, PhytoAB, San Jose, CA, USA), anti-LHCA6 (PHY0470S, PhytoAB, San Jose, CA, USA), anti-LHCB2 (AS01003, Agrisera, Vännäs, Sweden), and anti-RuBisCo (AG5359, Beyotime, Shanghai, China) antibodies were used for immunoblot analysis. GAPDH was used as the reference protein to determine the amount of protein loaded.

The alterations in photosystem II were evaluated using an analysis of CP43, CP47, Lhcb1 and Lhcb2 polypeptides. Proteins of thylakoid membranes from the controls and treated tomato plants grown under different combinations of temperature and light intensity were analyzed in a Laemmli SDS–PAGE system. The polyacrylamide concentrations of the stacking and resolving gels were 4 and 12%, respectively, with 4 M urea added to the resolving gel. The samples were incubated with a sample buffer (3:1) in the dark for 1 h at room temperature. Equal volumes of thylakoid membranes, corresponding to 3 µg Chl were loaded in every line. The proteins were transferred from gel to nitrocellulose membranes, and proteins were probed with antibodies for CP43 (AS11 1787 at a dilution of 1:3000, Agrisera, Vännäs, Sweden), CP47 (AS04 038 at a dilution 1: 2000, Agrisera, Vännäs, Sweden), Lhcb1 (As01 004 at a dilution of 1:2000, Agrisera, Vännäs, Sweden) and Lhcb2 (AS01 003 at a dilution 1:5000, Agrisera, Vännäs, Sweden). The Alkaline Phosphatase Conjugate Substrate Kit (Bio-Rad, Hercules, CA, USA) with goat anti-rabbit (GAR) secondary antibodies was used for the development of the blocked membranes, which were quantified with ImageJ software. Immunoblotting was repeated 3 times with thylakoid membranes from two different experiments.