PMC

Pictures are shown to compare the stamen phenotype in JA or GA repeatedly treated ga1-3 gai-t6 rga-t2 rgl1-1 (Q3) and opr3 plants with respective untreated controls.

(A) Comparison of the stamen phenotype among ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1(penta), ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 myb21-t1 myb24-t1 (hepta), ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 myb21-t1 (hexa1) and ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 myb24-t1 (hexa2). (B) SEM of stamen filament epidermal cells in the penta, hepta, hexa1 and hexa2 mutants. Segments shown were all from the middle part of the filament. Some individual cells were outlined with black lines for easy visualization. (C) Comparison of stamen and pistil lengths among different genotypes. Filament and pistil lengths were measured from SEM pictures (n = 30). (D) Average number of epidermal cells per stamen filament in penta, hepta, hexa1 and hexa2. n: number of stamens used in counting.

(A) Schematic diagram shows the respective T-DNA insertions in the three MYB genes. Black box: exon; black line: intron; triangle: T-DNA insertion site. (B) RT-PCR analysis of MYB24 transcripts in myb24-t1 and MYB57 transcripts in myb57-t1 and northern analysis of MYB21 transcripts in myb21-t1. Total RNA for RT-PCR and northern analysis was extracted from the young flower buds. (C) Comparison of main shoots bearing siliques among different mutant lines as indicated. (D) Comparison of the stamen phenotype among different mutant lines as indicated. Genotypes for flowers a-h in (D) corresponds to that showed in (C).

At least three independent samples were used for RT-PCR analysis for each individual gene and a representative gel picture for each gene was shown here. Total 34 genes were identified as DELLA-repressed stamen-enriched genes (summarized in ) based on their relative more abundant expression in the stamen than in one or more of the rest of the floral organs. Primer pairs corresponding to these genes were listed in . Se, sepal; Pe, petal; St, stamen; Pi, pistil.

(A–B) Semi-quantitative analysis of LOX2, GA2ox1, GA3ox1 and GA20ox2 (A), DAD1 (in red line), MYB21, MYB24 and MYB57 (B) expression in the ga1-3 gai-t6 rga-t2 rgl1-1 (Q3) mutant flowers at 18, 48, 72 and 96 hrs after GA treatment. Data were averaged from 2–4 batches of independently treated samples and ACTII was used as the normalization control. The graph was drawn based on Log₁₀ scale of the ratio of the expression levels of GA treated versus untreated samples.

(A–B) Semi-quantitative analysis of MYB21, MYB24, MYB57 (A), GA2ox1, GA3ox1 and GA20ox2 (B) expression in the opr3 mutant flowers at 18, 48, 72 and 96 hrs after GA treatment. Data were averaged from 2–4 batches of independently treated samples and ACTII was used as the normalization control. The graph was drawn based on Log₁₀ scale of the ratio of the expression levels of GA treated versus untreated samples. (C–D) Semi-quantitative analysis of LOX2 (in red line), GA2ox1, GA3ox1 and GA20ox2 (C), MYB21, MYB24 and MYB57 (D) expression in the ga1-3 gai-t6 rga-t2 rgl1-1 (Q3) mutant flowers at 18, 48, 72 and 96 hrs after JA treatment. Data were averaged from 2–4 batches of independently treated samples and ACTII was used as the normalization control. The graph was drawn based on Log₁₀ scale of the ratio of the expression levels of JA treated versus untreated samples.

(A) RT-PCR analysis of MYB21 and OPR3 gene expression in WT, opr3 and opr3 MYB21OE-1. Total RNA was extracted from the young flower buds. ACTIN was used as the normalization control. (B) Comparison of the flowers at stage 14 in different genotypes. The flower in opr3 MYB21OE-1 shows elongated filament than that in opr3. (C and D) Comparison of seed set in different genotypes as shown (C) and of plant growth of WT (Col-0) (50 days old), opr3 (50 days old) and opr3 MYB21OE-1. The third plant from left was an opr3 MYB21OE-1 plant with primary shoot (50 days old) whereas the last plant was a 60-day-old opr3 MYB21OE-1 with axillary shoots after its primary influence has been removed earlier. White arrows highlight siliques with seed set, red arrows highlight sterile siliques.

(A) RT-PCR analysis of GA- and JA-biosynthesis genes in the young flower buds of La-er WT control, ga1-3, ga1-3 gai-t6 rga-t2 rgl1-1 (Q3), ga1-3 gai-t6 rga-t2 rgl1-1rgl2-1 (penta), opr3 (in WS background) and Ws WT control. (B) Northern analysis of MYB21, GA20ox2, LOX1, LOX2 and OPR3 in the young flower buds of the La-er WT control, ga1-3, Q3, penta, opr3 and Ws WT control. (C) Semi-quantitative analysis of DAD1 expression in ga1-3, Q3 and penta relative to that in WT (Laer), respectively. Data were averaged from three independent batches of samples and ACTII was used as the normalization control. The expression level of WT is set as 1. (D) Comparison of JA contents among WT (La-er), opr3, ga1-3 gai-t6 rga-t2 rgl1-1 (Q3) and ga1-3 gai-t6 rga-t2 rgl2-1 rgl1-1 (penta) (). For WT and the penta mutant JA contents were averaged from four repeats. For the Q3 mutant, JA was detected in three out of the four repeats. For opr3, JA was detected only in one out of the four repeats. FW, fresh weight.

(A) RT-PCR analysis shows that the expression of the three MYB genes in the young flower buds are greatly reduced in ga1-3 but restored to the WT level in ga1-3 gai-t6 rgat2 rgl1-1 rgl2-1 (penta). (B–C) RT-PCR analysis shows that the repressed expression of the three MYB genes in ga1-3 was restored in ga1-3 gai-t6 rga-t2 rgl2-1 (Q2) and ga1-3 rga-t2 rgl1-1 rgl2-1 (Q4) two quadruple mutants but not in ga1-3 gai-t6 rgl1-1 rgl2-1 (Q1) and ga1-3 gai-t6 rga-t2 rgl1-1 (Q3) two quadruple mutants (B). This restoration of MYB expression nicely correlates with the recovery of fertility in Q2 and Q4 (C). Total RNA used in RT-PCR analysis was extracted from the young flower buds. (D) RT-PCR analysis shows that the three MYB genes are floral specific genes. IF, inflorescence; CL, cauline leaves; RL, rosette leaves; IT, internodes; RT, roots; SL, siliques. (E) Amino acid alignment of MYB21, MYB24 and MYB57 proteins. The conserved R2 and R3 domains and the NYWSV/ME/DDlWP/S motif are highlighted in red, blue and green, respectively.