Leaf dorsoventrality candidate gene CpARF4 has conserved expression pattern but divergent tasiR‐ARF regulation in the water fern Ceratopteris pteridoides

PREMISE Leaves are traditionally classified into microphylls and megaphylls, and recently have been regarded as independently originating in lycophytes, ferns, and seed plants. The developmental genetics of leaf dorsoventrality, a synapomorphy in vascular plants, has been extensively studied in flowering plants. AUXIN RESPONSE FACTOR4 ( ARF4 ) genes are key to leaf abaxial identity in flowering plants, but whether they exist in ferns is still an open question. METHODS ARF4 genes from Ceratopteris pteridoides , Cyrtomium guizhouense , and Parathelypteris nipponica were mined from transcriptomes and investigated in terms of evolutionary phylogeny and sequence motifs, with a focus on the tasiR‐ARF binding site. In situ hybridization was used to localize expression of CpARF4 in Ceratopteris pteridoides . 5′RNA ligase‐mediated‐RACE was employed to verify whether CpARF4 transcripts were sliced by tasiR‐ARF. RESULTS ARF4 genes exist in ferns, and this lineage originates from a gene duplication in the common ancestor of ferns and seed plants. ARF4 genes are of a single copy in the ferns studied here, and they contain divergent and, at most, one tasiR‐ARF binding site. CpARF4 is expressed in the abaxial but not the adaxial domain of leaf primordia at various developmental stages. Transcript slicing guided by tasiR‐ARF is active in C. pteridoides , but CpARF4 probably has not been affected by it. CONCLUSIONS Fern ARF4 genes differ in copy number and tasiR‐ARF regulation relative to flowering plants, though they can be similarly expressed in the abaxial domain of leaves, revealing a key role for ARF4 genes in the evolution of leaf dorsoventrality of vascular plants.