Fig. 4: Comparison of transcripts and protein activities of different haplotype of PH13.
From: PH13 improves soybean shade traits and enhances yield for high-density planting at high latitudes
Transcript levels of PH13H1 in W82 and PH13H3 in TL1 respectively, were measured using primers detecting the expression of exon 3 (a) and 3’UTR (b). c Transcript levels of PH13H1, GmCOP1a, and GmCOP1b in W82. The second trifoliate leaves of 20-day-old seedlings grown under long-day conditions were collected at 4-hour intervals for RT-qPCR analysis. Data are mean ± SD (n = 3 biologically independent replicates) calculated relative to GmActin. d Protein structure of each haplotype. aa, amino acids. e Auxotrophic assays showing the interactions between different PH13 haplotypes with GmCOP1s. Yeast cells transformed with indicated constructs were selected on -LW (lacking Leu and Trp) or -LWHA (lacking Leu, Trp, His and Ade) medium. AD, GAL4 activation domain; BD, GAL4 DNA-binding domain. f Co-Immunoprecipitation (Co-IP) assay showing the interaction between each PH13 haplotype with GmCOP1b in tobacco leaves. The indicated constructs were co-transformed into tobacco which were then incubated at 25 °C in dark for 12 h and grown under white light (WL, 80 μmol m−2 s−1) for 36 h. The immunoprecipitates were detected using anti-GFP (at a 1:2500 dilution) and anti-Flag (at a 1:2500 dilution) antibodies, respectively. Empty vector (35 S::YFP) was used as a negative control. Numbers at bottom represent the relative IP efficiency, calculated as (IP-PH13/Input-PH13)/(IP-GmCOP1b/Input-GmCOP1). A representative result of three independent replicates is shown. g Immunoblots showing STF1/2 protein levels in the NILH1 and NILH3 lines under diurnal conditions. The first trifoliate leaves of 15-day-old seedlings grown under long-day condition were collected at 4-hour intervals. The membrane was probed with the anti-STF1/2 antibody (at a 1:1000 dilution), stripped, and then probed with the anti-HSP70 antibody (at a 1:10,000 dilution). The asterisk indicates a non-specific band. h The relative expression level of STF1/2 proteins represented by REU (Relative Expression Unit) was calculated by the formula [STF1/2] / [HSP70], in which ‘STF1/2’ and ‘HSP70’ indicate the digitized band intensity of STF1/2 or HSP70 in each sample collected at respective time point. The REU of STF1/2 in NILH1 at ZT0 was arbitrarily set to 1. Data are shown as means ± SD of three biological replicates (Supplementary Fig. 18). Source data are provided as a Source Data file.
