Figure 5.

HEN1 Plays a Rate-Limiting Role for miRNA Accumulation in Etiolated Seedlings.

The 3-d-old etiolated T2 seedlings of XVE:HA-HEN1 were treated with 0 to 10 μM β-estradiol for 24 h in the dark.

(A) qRT-PCR analysis of the mRNA level of HEN1 relative to that of UBQ10. Data are mean ± sd from two biological replicates (each with three technical replicates).

(B) Immunoblot analysis of protein level of HA-HEN1 with the antibodies pig anti-HEN1 (α-HEN1), mouse anti-HA (α-HA), and mouse anti-tubulin (α-TUB). Numbers below the α-TUB blot indicate the HA-HEN1 protein levels in the α-HA blot relative to TUB signals. n.d., nondetectable.

(C) Small RNA analyses of miR156/157 and miR159/319. DNA oligos used for each blot are indicated to the left of each blot. The bar graph shows 5S rRNA- and tRNA-normalized miRNA fold increase in HA-HEN1-induced seedlings. Data are mean ± sd quantitated using ImageJ on three biological replicates, shown in Supplemental Figure 8. *Significantly different from the noninduced sample (0 μM) (P < 0.05, Student’s t test; n = 3).