Fig. 5: Both GC1 and gc1-a interact with and promote the degradation of SbpPLAII-1.
From: Natural variation in Glume Coverage 1 causes naked grains in sorghum
a GC1-nLuc and gc1-a-nLuc were co-transformed into tobacco leaves along with cLuc-tagged SbpPLAII-1 proteins by LCI assays. The typical sorghum G protein γ subunit of type A (SbGγA) and the empty cLuc were used as negative controls. Protein detection in this assay is shown in Fig. S10A. b In vitro pull‐down assays show both GST-tagged GC1 and GST-tagged gc1-a physically interact with MBP-tagged SbpPLAII-1. The MBP‐tagged SbpPLAII-1 protein pulled down with GST-GC1 or GST-gc1-a was detected by anti‐MBP antibody. The combination of GST and MBP-SbpPLAII-1 was used as a negative control. c GFP-tagged GC1 and GFP-tagged gc1-a both interact with Flag-tagged SbpPLAII-1 in co-immunoprecipitation assays. Each protein was independently extracted from tobacco leaves. The SbpPLAII-1-Flag protein co‐precipitated with GFP-GC1 or GFP-gc1-a was detected by anti‐Flag antibody. The combination of GFP and SbpPLAII-1-Flag was used as a negative control. d Glume architecture and glume cell morphology by longitudinal paraffin-section in the wild type Ci846 and SbpPLAII-1-OE millet lines at flowering stage. Top left bar = 2 mm. Bottom left bar = 500 μm. Right bar = 50 μm. Statistics Data are mean ± s.e.m. n = 10 biological replicates. P-values were determined by two-tailed unpaired t-test. *Significant probability level at P < 0.05. **Significant probability level at P < 0.01. ***Significant probability level at P < 0.001. e Gene expression of Cyclin-CDK related genes in SbpPLAII-1-OE millet plants. Gene expression detection of SiCYCA2;3, SiCYCB2;2 and SiCYCB2;2 (the homologs to SbCYCA2;3, SbCYCB2;2 and SbCYCB2;2, respectively) in the early young panicles of SbpPLAII-1-OE millet plants by qPCR assays. Three biological repeats were performed. P-values were determined by multiple two-tailed unpaired t-test. f GFP-tagged GC1 and gc1-a proteins can promote the degradation of Flag-tagged SbpPLAII-1. Total proteins of GFP, GFP-GC1, GFP-gc1-a, and Flag-SbpPLAII-1 were individually extracted from tobacco leaves. A gradient of concentrations (1/10, 3/10, 5/10, and 10/10 ratio of 100 μL volume) of GFP-GC1 or GFP-gc1-a protein was incubated with Flag-SbpPLAII-1 protein (10 μL volume). Each reaction was topped up by cell lysis from wild type tobacco leaves. GFP protein was used in control reaction. See in “Methods” section. Source data are provided as a Source Data file.
