Fig. 2: dwf5 destabilizes the SPL9 protein and sequesters its function instead of affecting miR156 and SPL gene expression. | Nature Communications

Fig. 2: dwf5 destabilizes the SPL9 protein and sequesters its function instead of affecting miR156 and SPL gene expression.

From: Coordinated regulation of vegetative phase change by brassinosteroids and the age pathway in Arabidopsis

Fig. 2

a, b The expression levels of miR156, SPL3, SPL9, SPL13 (a), MIR172B, miR172, TOE1 and TOE2 (b) in 12-day-old WT and dwf5 in short days. Data are means ± SD from a representative experiment with two technical replicates for each sample. Asterisk denotes significant difference from WT using Student’s t-test at P < 0.01. c dwf5 reduces the SPL9 protein level. The SPL9 protein in pSPL9::3×FLAG-rSPL9 and pSPL9::3×FLAG-rSPL9 dwf5 transgenic lines was detected by Western blotting using an anti-FLAG antibody. The Rubisco protein was stained with ponceau as the protein loading control, WT was served as the negative control. Numbers between two blots denote the relative normalized value for each sample. d dwf5 sequesters SPL9 function to activate the expression of MIR172B. 15-day-old pSPL9::GR-rSPL9 and pSPL9::GR-rSPL9 dwf5 plants grown on 1/2 MS medium in short days were treated with MOCK or dexamethasone (DEX) for 3 h, and total RNA was isolated for qRT-PCR analysis. Data are means ± SD from a representative experiment with three technical replicates for each sample. Asterisk denotes significant difference using one-way ANOVA; *p < 0.05; **P < 0.01; ***P < 0.001. e Destabilization of the SPL9 protein in dwf5 is proteasome-dependent.15-day-old pSPL9::3×FLAG-rSPL9 and pSPL9::3×FLAG-rSPL9 dwf5 plants were treated with 50 µM MG132 and/or 100 mM CHX for 1 h in short days. The total protein was extracted and detected by Western blotting using an anti-FLAG and anti-ACTIN antibody, respectively. f BL treatment of dwf5 increases SPL9 accumulation. 15-day-old pSPL9::3×FLAG-rSPL9 dwf5 plants in short days were treated with MOCK or 1 µM BL for 1 h, and total protein was extracted and detected by Western blotting using an anti-FLAG and anti-ACTIN antibody, respectively. Numbers between two blots denote the relative normalized value for each sample. The intensity of each sample was first normalized to the Rubisco band or the corresponding ACTIN band (c, e, f), then the resultant value was normalized again to the value of FLAG-rSPL9 (c), FLAG-rSPL9 DMSO (e), and FLAG-rSPL9 dwf5 0 h (f). Band intensity was determined using Image J. All experiments were repeated 3 times biologically.

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