FBW2 is required for a proper abscisic acid (ABA) response. (A) The percent germination of WT, fbw2-4, ago1-25 and FBW2ox seeds in the presence of increasing concentrations of ABA (± standard deviation). (B) The effect of ABA on root elongation, reported as the ratio of root length in the presence of 2.5 μm ABA to root length in the absence of ABA (± standard deviation).

Mutations in FBW2 suppress sqn and ago1 by increasing ARGONAUTE1 (AGO1). (A) Eighteen-day-old rosettes of WT, sqn-1, sqn-1 fbw2-1, and two independently isolated sqn-1 lines containing an AGO1::FLAG-AGO1 transgene. sqn-1 AGO1::FLAG-AGO1 transgenics resemble sqn fbw2 mutants. (B, C) Western blot of protein extracts from 14-day-old rosettes or floral buds probed with an anti-AGO1 antibody. Ponceau staining and a nonspecific band (*) were used as a loading control. (-) indicates samples that were slightly under-loaded.

Mutations in FBW2 enhance the se-1, hst-3, hyl1-2 and hen1-6 mutant phenotypes. Twenty-one-day-old plants singly and doubly mutant for fbw2-1 and hst-3 (A and B) se-1 (C and D), or hyl1-2 (E and F), and 28-day-old hen1-6 and hen1-6 fbw2-4 plants (G and H). hyl1-3 fbw2-1 and se-1 fbw2-1 double mutants have phenotypes similar to hyl1-3 and se-1 plants transformed with ARGONAUTE1::FLAG-AGO1 (I and J) or WT plants containing the miR168 insensitive 2m-AGO1 construct (K). (L) Western blot of protein extracts from 14 day-old seedlings probed with an anti-AGO1 antibody. Ponceau staining and a nonspecific band (*) were used as a loading control.

Mutations in FBW2 suppress hypomorphic ago1 mutations. (A) Eighteen-day-old rosettes of ago1 mutants and ago1 fbw2-1 double mutants. The shapes of leaves 1-4 are also illustrated. (B) The number of juvenile, adult and cauline leaves in ago1 and ago1 fbw2-1 double mutants (± standard deviation). (C) 14-day-old fbw2-1, ago1-36 and ago1-36 fbw2-1 double mutants grown on MS media. (D) Relative abundance of microRNA (miRNA) targets in various genotypes as measured by quantitative-real time polymerase chain reaction in 14-day-old (top) or 20-day-old (bottom) rosettes. Target genes were normalized to EIF4. Actin was used as a non-target control (± standard deviation). (E) Small RNA blots of low-molecular-weight RNA isolated from 14-day-old or 20-day-old rosettes probed with oligonucleotides complementary to specific miRNAs. Methionyl transfer RNA was used as a loading control.

Loss-of-function mutations of FBW2 suppress the phenotype of sqn-1. (A) An 18-day-old rosettes of wild-type (WT), fbw2-1, sqn-1, sqn-1 fbw2-1, sqn-1 fbw2-4 and sqn-1 fbw2-1 plants containing a genomic pFBW2::FBW2-FLAG construct. Rosettes are same magnification. (B) Leaf shape of successive juvenile (light grey) adult (dark grey) and cauline leaves (black) in WT, fbw2-1, sqn-1 and sqn-1 fbw2-1 plants. Juvenile leaves were defined by the absence of abaxial trichomes (n = 24; ± standard deviation). (C) Relative abundance of miRNA targets in various genotypes as measured by quantitative-real time polymerase chain reaction. Target genes were normalized to EIF4. Actin was used as a non-target control (± standard deviation). (D) The genomic structure of FBW2 and the amino acid changes produced by fbw2-1, fbw2-2 and fbw2-3. The locations of the T-DNA insertions in fbw2-4 (SALK_144548) and fbw2-5 (SALK_071588) are also illustrated.

Over expressing FBW2 decreases ARGONAUTE1 (AGO1) protein levels. (A) Primary transformants containing a genomic 35S::FBW2 (FBW2ox) transgene. The percentage of plants displaying each phenotype is indicated (n = 345 plants). (B) L1 plants with, and without, the FBW2ox construct. FBW2ox restores the expression of the 35S::GUS transgene present in L1. (C) Western blot of protein extracts from 14-day-old seedlings probed with an anti-AGO1 antibody. AGO1 is reduced in plants containing FBW2ox. Actin was used as a loading control. (D) The proteasome inhibitor MG132 has no effect on AGO1 protein levels. Western blots of protein isolated from plants treated as indicated were probed with anti-AGO1 antibody. The lower anti-AGO1 blot is a longer exposure. The increased abundance of ubiquitinated proteins in MG132-treated plants indicates that the treatment was effective. Ponceau staining and a nonspecific band (*) were used as a loading control. (E) Abundance of miRNA targets in various genotypes as measured by quantitative-real time polymerase chain reaction. The abundance of these transcripts is significantly greater in FBW2ox sqn-1 than in the parental lines, suggesting that sqn-1 and FBW2ox operate independently to reduce AGO1 activity. Results were normalized to EIF4 (± standard deviation).