Proper regeneration from in vitro cultured Arabidopsis thaliana requires the microRNA-directed action of an auxin response factor

MicroRNAs (miRNAs) are important for the regulation of gene expression, and are involved in many developmental processes. A set of miRNAs which were differentially expressed between cells of totipotent (C1) and non-totipotent (C2) Arabidopsis thaliana calli was identified, some of which were affected during callus formation or shoot regeneration. One of those down-regulated after 10 days incubation in shoot induction medium (SIM) was MIR160a, for which transcript abundance was lower in C1 than in C2. Over-expression of MIR160 compromised shoot regeneration from in vitro cultured A. thaliana cells, while the transgenic expression of a miR160-resistant form of ARF10 was associated with a high level of shoot regeneration. The latter transgenic line also showed an elevated expression level of shoot meristem-specific genes CLAVATA3, CUP-SHAPEDCOTYLEDON1 and -2, and WUSCHEL. ARF10 expression was concentrated at the initiation sites of shoots or leaves, while during the early phase of shoot regeneration, the accumulation of the ARF10 mRNA was lower in the wild type than in the mARF10 transgenics, in contrast to the pattern of miR160 expression. Thus, miR160 and ARF10 both appear to be components of the regulation of shoot regeneration in vitro.