As01 004

Proteins were extracted from 500 mg of whole primary foliage leaves with a double volume (1 ml) of sample buffer [62.5 mM Tris–HCl, pH 6.8, 10% (v/v) glycerol, 1% (w/v) SDS, 5% (v/v) ß-mercaptoethanol]. After heating the suspensions at 65 °C for 5 min, cellular debris was removed by centrifugation. Equal amounts of protein (10 µg) were subjected to SDS–PAGE, and separated proteins were transferred to nitrocellulose membranes (Carl Roth, Roti-NC) by electroblotting (Humbeck et al., 1994 (link)). After incubation with the primary antibody, immunoreactive bands were visualized using a peroxidase-coupled secondary antiserum with chemiluminescence detection (ECL Select Amersham, Pierce Thermo Scientific Waltham, MA, USA; Ultra TMA-6 Lumigen, Southfield, MI, USA). Primary antibodies were directed towards HvWHIRLY1 (Grabowski et al., 2008 (link), 2nd peptide), LHCB1 (Agrisera AS01004), LHCB4 (Humbeck and Krupinska, 2003 (link)), D1 (Agrisera AS01016), LHCA1 (Agrisera AS01005), FNR (AntiProt F01A-1), Cu/ZnSOD (Agrisera AS06170), and an antibody directed towards PSI (kindly provided by Poul Eric Jensen, University of Copenhagen, Denmark).

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.