Identification and characterization of ARFs in Orchidaceae species
The ARF gene family comprises important transcription factors involved in various plant morphogenetic processes (Korasick et al. 2014). Different numbers of ARF genes have been identified in different plant species, for example, 23 genes in Arabidopsis (Okushima et al. 2005), 31 genes in maize (Xing et al. 2011), and 25 genes in rice (Wang et al. 2007). In this study, we identified a total of 112 ARF genes in five Orchidaceae species, comprising 16 in Phalaenopsis aphrodite, 34 in Phalaenopsis equestris, 24 in Vanilla planifolia, 17 in Apostasia shenzhenica, and 21 in Dendrobium catenatum. Thus, P. equestris contained the highest number of ARF genes, indicating that gene amplification during evolution of the ARF family was more pronounced in P. equestris than in the other Orchidaceae species studied. The 112 ARF genes were grouped into seven subfamilies. Subfamilies 4 and 7 contained the highest number of genes, whereas subfamily 2 contained the fewest genes and was detected only in A. shenzhenica. Thus, subfamily 2 was indicated to be unique to the Orchidaceae.
A typical ARF protein contains the DBD, MR, and CTD domains. Among these domains, DBD is required for specific binding to the AuxRE (TGTCTC) motif in the promoter of the target genes (Chandler 2016). The amino acid composition of the MR determines whether the ARF acts as a transcriptional activator or repressor, and CTD participates in homologous and heterologous interactions among ARF proteins (Shen et al. 2015). Most of the 112 ARF genes in the orchid species contained all three domains. Interestingly, compared with the ARF genes of other subfamilies, those belonging to subfamily 3 lacked the CTD domain, which was consistent with the structure of AtARF3. A recent study showed that AtARF3 does not bind to functional elements in the classical TIR1/AFB signaling pathways and is independent of TIR1/AFB receptors (Kubeš and Napier 2019). This suggests the possibility that subfamily 3 ARF genes in orchids do not play a role in auxin response. In addition, multiple alignment of ARF amino acid sequences indicated that the members of subfamily 4, which are rich in glutamine residues in the MR, may function as transcriptional activators in the orchid IAA response, whereas the remaining ARF proteins, which are rich in threonine, serine, and proline, potentially act as transcriptional repressors in the IAA response. The ratio of transcriptional activators to transcriptional repressors among the identified ARF genes was approximately 0.37, which is consistent with the results of a previous study of ARF genes in Dendrobium (Chen et al. 2017)
Evolutionary analysis of Orchidaceae ARF family
To explore the evolution of the ARF family in Orchidaceae, we selected representative amino acid sequences for ARF proteins in five model plants and the five orchid species to construct a phylogenetic tree. Compared with the model plants, the orchid species lacked one class of ARF genes that included AtARF17 in Arabidopsis and its lineal homologs in other model plants. AtARF17 plays an important role in synthesis of the pollen wall in Arabidopsis. Mutation of AtARF17 leads to thinning of the pollen wall, abnormal lignin biosynthesis in the anther exine, anther non-dehiscence, and, ultimately, male sterility (Wang et al. 2017; Yang et al. 2013). According to a previous study, AtARF17 is temporarily overexpressed during tapetum development, and abnormal expression of AtARF17 inhibits tapetum development, which interferes with pollen development (Xu et al. 2019). The Orchidaceae exhibits an evolutionary trend towards forming cohesive pollen. This trend is usually accompanied by gradual loss of the exine. In the most advanced orchids, such as Dendrobium, the non-exinate pollen is aggregated into clusters, and only the periphery of the pollen cluster is covered by the exine (Brown and Lemmon 1994; Fitzgerald et al. 1994; Zavada 1983). We speculate that this unique phenomenon may be associated with the loss of AtARF17 homologs during evolution. The lack of ARF17 homologs results in loss of the inner and outer wall of the pollen cluster, which improves the adhesion between pollen grains and leads to the formation of pollinia or pollinaria, thus resulting in the unique pollination mechanism of orchids.
Analysis of transcriptome data for Orchid ARF family
The information content of an organism is recorded in its genomic DNA and expressed through transcription (Martin et al. 2013; Tang and Tang 2019). In this study, publicly available transcriptome data for different tissues of five Orchidaceae species were used to compare the expression levels of ARF genes in different tissues and developmental stages. Because these data were generated in different experimental projects, the number and identity of tissues analyzed differed among the species.
Although there were some differences in the expression patterns of the ARF subfamilies in different orchid species, some commonalities were observed. For example, subfamily 4 ARF genes, the only putative transcriptional activators detected among the five orchid species, were highly expressed during flower development, indicating that members of subfamily 4 may play an important role in the establishment of flower morphology in orchids. The expression of ARF genes in developing seeds and roots in most orchid species was generally lower than that in other tissues, which may be associated with the high sensitivity of the root tissue to IAA. Members of subfamily 2, a class unique to A. shenzhenica, were highly expressed in stems and leaves. Interestingly, although subfamily 3 comprised several ARF genes, they were specifically expressed in the pollen and style of the five orchid species. Although there is a lack of pollen and style transcriptome data in orchids, the present results suggested that subfamily 3 ARF genes play an important role in pollen and style development. The specific functions of the above-mentioned genes require further investigation.
Summary
The ARF gene family is an important group of auxin-responsive transcription factor-encoding genes in plants, but information on this gene family has been lacking for the Orchidaceae. In this study, five orchid species were selected for genome-wide identification of ARF genes. A total of 112 ARF genes were identified and grouped into seven subfamilies by phylogenetic analysis. Amino acid sequence analysis predicted that the ARF genes in subfamily 4 are transcriptional activators, whereas the other genes are transcriptional repressors. Compared with the ARF family of model plants, the Orchidaceae species lack ARF genes that regulate synthesis of the pollen exine, which might explain the absence of the outer pollen wall in the orchid pollen mass. Comparative transcriptome analysis of the five orchid species suggested that ARF genes in subfamily 4 regulate the establishment of orchid flower morphology and promote flower development, whereas those of subfamily 3 are involved in pollen development. Overall, this study elucidates the complete ARF gene family in selected orchid species. The information generated will facilitate further research on the genetics of reproductive organ development in orchids and the role of IAA in this process.