Figure 5
VQ1 and VQ10 proteins altered DXS aggregation and oligomerization patterns in chloroplasts. (A) Nicotiana benthamiana plants were transiently transformed by agroinfiltration with plasmids harboring 35S:DXS-GFP (DXS), 35S:3xHA-VQ1 (HA-VQ1), and 35S:3xHA-VQ10 (HA-VQ10) as indicated plus p19 plasmid to avoid posttranscriptional silencing. Three days after agroinfiltration, total protein extracts were prepared from leaves co-transformed with the indicated combination of plasmids and analyzed by Western blot with a-GFP antibody to detect DXS. The GFP signals from the bands corresponding to oligomers (o, solid triangle) and monomers (m, clear triangle) are shown in plants under the increasing DTT concentrations indicated. The bottom panel shows de Ponceau-stained Rubisco (R, solid dot) to assess for equal loading. The right panel shows the quantification by ImageQuant TL software as the mean of the oligomer/monomer ratio ± standard error (SE) of four independent biological replicates for each condition. (B) CoIP of DXS and VQ10 under low reducing (0.05 mM DTT) and strong reducing (5 mM DTT) conditions. IP was performed with anti-GFP magnetic beads and the levels of DXS-GFP and HA-VQ10 analyzed by western blot with the indicated antibodies. The positions of DXS monomer (m) and oligomers (o) in the upper panels and VQ10 monomer (m), dimer (d), and oligomers (o) in the lower panels are marked at the right side. Molecular mass markers in kDa are shown at the left side of panels. (C) CoIP of DXS-GFP and either wild type HA-VQ10 or mutated HA-VQ10(C58S) proteins was performed at 5 mM DTT.
