Figure 4.

Regulation of ARF16 by miR160.

(A) and (B) Pro_ARF16:GFP-ARF16 root, showing ARF16 expression in distal stem cells and root cap cells and the localization of the GFP-ARF16 fusion protein (green) in the nucleus.

(C) The GFP signal provided by Pro_ARF16:GFP-ARF16 was also present in distal cells of the emerging lateral root.

(D) and (E) Pro_MIR160c:GUS root, showing miR160 expression in the vascular bundle of primary (D) and emerging lateral (E) roots.

(F) Pro_ARF16:GUS-mARF16 root, showing miR160-uncoupled ARF16 (mARF16) expression in both the root cap region and the vascular bundle.

(G) and (H) In developing embryos, ARF16 (Pro_ARF16:GFP-ARF16) was expressed in the basal-most region (root cap region) at the heart (G) and bent cotyledon (H) stages.

(I) and (J) miR160 (Pro_MIR160c:GUS) was detected in the heart-stage embryo (I), and this miRNA then spread throughout the embryo except for the embryonic root cap region (J).

Bars = 50 μm in (A) to (J).

(K) Expression of Pro_MIR160c:GUS, GUS-ARF16, and GUS-mARF16 in leaves of 20-d-old plants. Numbers in the middle panel refer to the order of leaves produced from the plant. Bars = 1.0 cm.

(L) RNA gel blot analysis of ARFs and miR160 expression in leaves. Lanes 1 to 7 correspond to the order of developing leaves as shown in (K). Full-length transcripts of the ARF genes were detected only in younger leaves, whereas miR160 was highly accumulated in older leaves. Arrows and asterisks indicate the full-length and 3′ cleaved products of each mRNA.