In Arabidopsis leaf primordia, the expression of HD-Zip III, which promotes tissue differentiation on the adaxial side of the leaf primordia, is repressed by miRNA165/166 (miR165/166). Small RNAs, including miRNAs, can move from cell to cell. In this study, HD-Zip III expression was strikingly repressed by miR165/166 in the epidermis and parenchyma cells on the abaxial side of the leaf primordia compared with those on the adaxial side. We also found that the MIR165A locus, which was expressed in the abaxial epidermis, was sufficient to establish the rigid repression pattern of HD-Zip III expression in the leaf primordia. Ectopic expression analyses of MIR165A showed that the abaxial-biased miR165 activity in the leaf primordia was formed neither by a polarized distribution of factors affecting miR165 activity nor by a physical boundary inhibiting the cell-to-cell movement of miRNA between the adaxial and abaxial sides. We revealed that cis-acting factors, including the promoter, backbone, and mature miRNA sequence of MIR165A, are necessary for the abaxial-biased activity of miR165 in the leaf primordia. We also found that the abaxial-determining genes YABBYs are trans-acting factors that are necessary for the miR165 activity pattern, resulting in the rigid determination of the adaxial-abaxial boundary in leaf primordia. Thus, we proposed a molecular mechanism in which the abaxial-biased patterning of miR165 activity is confined.
Significance Statement In this paper, we propose a molecular mechanism for determination of activity pattern of miR165/166 in leaf primordia, which depends on both mature miRNA sequence and MIRNA backbone. We consider that our finding will provide an insight into the roles of MIRNA transcript sequences in the mechanisms for determination of miRNA distribution to confine small RNA-meditated gene expression in plants.