Skip to main content
. 2016 Aug 10;7:12377. doi: 10.1038/ncomms12377

Figure 4. RVE1 regulates GA biosynthesis by directly modulating GA3ox2 expression.

Figure 4

(a) Relative expression levels of GA3ox1 and GA3ox2 in Col and RVE1-OX grown under phyB-on conditions. (b) Relative expression levels of GA3ox2 in 35S:RVE1-GR transgenic seeds incubated in darkness for 24 h without (Mock, 0.05% dimethy; sulfoxide (DMSO)) or with 5 μM DEX and/or 50 μM cycloheximide (CHX). Asterisks denote statistically significant difference (P<0.01, Student's t-test). (c) Transient transcriptional assay of GA3ox2p:LUC by RVE1 overexpression or empty vector control in Arabidopsis protoplasts. (d) ChIP assay of GA3ox2 DNA precipitated in 35S:Myc-RVE1 by Myc antibody or serum control. Post-harvested 35S:Myc-RVE1 seeds were incubated under phyB-on condition. The top diagram indicates the genomic structure of GA3ox2. Black boxes indicate exons. P1 to P4 fragments show PCR regions in the ChIP assay and E1 to E4 sites are putative EE-expand motifs. (e) EMSA assay. The oligos were synthesized by fusing three copies of the E3 or E4 motifs and their flanking sequences. The oligos labelled with biotin were used for probes and incubated with or without RVE1 recombinant proteins. Excess amounts (× 50) of unlabelled wild-type (WT) or mutant oligos were added as the competitors. Arrow denotes shifted bands of protein–DNA complexes. (f) Seed dormancy phenotype of Col, RVE1-OX and RVE1-OX/GA3ox2-OX transgenic seeds grown under white light for 4 days. (g) Light-mediated seed germination phenotype of Col, RVE1-OX and RVE1-OX/GA3ox2-OX transgenic seeds grown under phyB-on conditions. (h) Measurement of GA1 and GA4 contents of freshly harvested Col, rve1 and RVE1-OX seeds imbibed in darkness for 24 h. Data are means±s.d., n=3. n.d., not detected. Asterisks indicate significant differences from Col using Student's t-test (P<0.01). (i) Germination phenotype of RVE1-OX in the absence or presence of exogenous GA3 (10 μM). Seed coats were removed to facilitate GA3 absorption and embryos were incubated in darkness for 2 days. (j) Germination percentage of Col, ga1 and rve1/ga1 in the absence or presence of various concentrations of exogenous GA3. Freshly harvested seeds were incubated under white light for 3 days. For all column figures, mean±s.d., n=3.