ARF protein sequences were obtained from the public genome database Phytozome.
PvARF39 in cluster VII is most closely related to the foxtail millet ARF protein Seita.8G135700.1, which indicates that the paralog of PvARF39 has been lost or mutated gradually during the evolutionary process of switchgrass genome.
Another phylogenetic tree was firstly constructed only using switchgrass ARF protein sequences.
ARF proteins generally contain a DNA-binding domain (DBD) in the amino (N)-terminal region, a central region that functions as an activation domain (AD) or a repression domain (RD) [12, 13], and a carboxyl (C)-terminal dimerization domain (CTD), which is a protein-protein interaction domain that mediates ARF homo- and heterodimerization and also the heterodimerization of ARF and Aux/IAA proteins, another category of auxin response regulators [12-16].
In previous studies, ARF proteins were split into three clades (clades A, B, and C) based on phylogenic relationships, which could be traced back to the origin of land plants .
To identify ARF proteins in switchgrass, the Hidden Markov Model (HMM) profile of the conserved ARF domain (Pfam06507) was used as a query to search against the publicly available switchgrass genome database (Phytozome v11) by BlastP and tBlastN program.
To profoundly characterize the phylogenetic relationships of ARF proteins among switchgrass and other land plants, we selected ARFs from another 15 species, which have public genome database in Phytozome, to construct a phylogenetic tree together with PvARFs.
As a key component of the auxin signaling pathway, ARF proteins play vital roles in auxin response.
Because ARF proteins are transcription factors that regulate the expression of auxin response genes, we determined the response of PvARF genes to NAA treatment.
The 22 ARF proteins encoded by 22 ARF genes in Arabidopsis genome each contain a conserved DNA binding domain near their N terminus and most also have a dimerization domain near their C terminus that can interact with a corresponding domain in Aux/IAA proteins (Ulmasov et al., 1999).
Auxin probably activates ARF proteins by regulating turnover of ARF-interacting proteins called Aux/IAAs (Dharmasiri & Estelle, 2004).
We need to understand how the large families of AUX/IAA and ARF proteins regulate downstream gene expression.