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.

中文翻译：

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叶背腹候选基因 CpARF4 在水蕨 Ceratopteris pteridoides 中具有保守的表达模式但不同的 tasiR-ARF 调节

前提叶传统上分为小叶植物和大叶植物，最近被认为独立起源于石松植物、蕨类植物和种子植物。叶背腹性的发育遗传学是维管植物中的一种突触，已在开花植物中得到广泛研究。AUXIN RESPONSE FACTOR4 (ARF4) 基因是开花植物叶片背面特性的关键，但它们是否存在于蕨类植物中仍然是一个悬而未决的问题。方法从转录组中挖掘来自 Ceratopteris pteridoides、Cyrtomium guizhouense 和 Parathelypteris nipponica 的 ARF4 基因，并在进化系统发育和序列基序方面进行研究，重点是 tasiR-ARF 结合位点。原位杂交用于定位 CpARF4 在 Ceratopteris pteridoides 中的表达。5' 采用 RNA 连接酶介导的 RACE 来验证 CpARF4 转录物是否被 tasiR-ARF 切片。结果 ARF4 基因存在于蕨类植物中，该谱系起源于蕨类植物和种子植物共同祖先中的基因重复。在这里研究的蕨类植物中，ARF4 基因是单拷贝的，它们包含不同的，最多只有一个 tasiR-ARF 结合位点。CpARF4 在不同发育阶段的叶原基的背面而非正面区域中表达。由 tasiR-ARF 引导的转录切片在 C. pteridoides 中很活跃，但 CpARF4 可能不受其影响。结论 相对于开花植物，蕨类 ARF4 基因在拷贝数和 tasiR-ARF 调控方面存在差异，尽管它们可以在叶子的背面区域类似地表达，