**1. Introduction**

As sessile organisms, plants need to proceed in a coordinated manner to adapt to the constantly changing environment and react to stress conditions for growth and development. Phytohormones play a crucial role during these processes. Among them, brassinosteroids (BRs) are generally known as important plant hormones that play fundamental roles in various cellular, physiological, and developmental processes during plant life cycle [1,2].

To date, the BR signaling pathway has been well established and a number of the intracellular components of this pathway have been identified by genetic, genomic and proteomic studies. In the current model of the BR signaling pathway, the BR signal is perceived by BRASSINOSTEROID INSENSITIVE 1 (BRI1), a membrane-localized receptor kinase [3,4]. The direct binding of the BR ligand with BRI1 induces its association BRI1 with its co-receptor BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1), enhancing the signaling output through reciprocal BRI1 transphosphorylation, inactivation of a glycogen synthase kinase-3 BRASSINOSTEROID INSENSITIVE 2 (BIN2) and activation of phosphatase BRI1 SUPPRESSOR 1 (BSU1), as well as the downstream transcription factors BRASSINAZOLE-RESISTANT 1 (BZR1) and BRI1-EMS-SUPPRESSOR 1 (BES1) [5–8]. Moreover, the transcription factor gene family, in turn, controls the expression of numerous target genes, which control various cellular, physiological, and developmental processes [6,9].

In the initial steps of BR signaling transduction, three receptor-like cytoplasmic kinases named BRASSINOSTEROID-SIGNALING KINASEs (BSKs) from *Arabidopsis* have been identified as BR-responsive proteins, including *BSK1*, *BSK2*, and *BSK3* [10]. *BSK1* and *BSK3* interact with BRI1 in vivo and are phosphorylated by BRI1 in vitro [10]. The phosphorylated BSK proteins further activate downstream phosphatase BSU1 for BR signaling transduction [6,11]. In *Arabidopsis*, there are 12 BSK proteins with putative kinase catalytic domain at the N-terminus and tetratricopeptide repeats (TPRs) at the C-terminus. However, the available results indicate that not all members are involved in BR signaling only. *BSK4*, *BSK6*, *BSK7*, and *BSK8* were reported to play a partial overlapping role in plant growth as well as in BR signaling with *BSK3* [12]. In contrast, *BSK3* was found as the only BSK member involved in BR-mediated plant root growth in a recent study [13]. Unexpectedly, the YODA mitogen-activated protein kinase pathway is activated by SHORT SUSPENSOR (SSP/BSK12) during embryogenesis, which has not been shown to be regulated by BRs [14]. In addition, the loss-of-function mutant *bsk5* is sensitive to salt stress and abscisic acid (ABA) hormone [15]. Silencing OsBSK1-2 inhibits flagellin- and chitin-triggered immune responses in rice [16]. Moreover, the *Arabidopsis BSK1* directly interacts with the immune receptor FLAGELLIN SENSING2 (FLS2) and further phosphorylates MAPKKK5 for the activation of pattern-triggered immunity (PTI) [17,18]. However, the detailed characterization of BSK family proteins and their functional importance in plants remains unclear.

In the present study, we screened the available genomes and identified a total of 143 BSK proteins from 17 plant species. We further performed a detailed analysis of their classification, phylogeny, and alternative splicing. Finally, we verified the expression profiles of the selected BSK genes in *Arabidopsis* by investigating their transcriptional levels upon exposure to abiotic stresses and hormones. Moreover, a novel post-transcription regulation pattern was found in several BSK genes, and potential significant functions of BSK genes were proposed. Our results provide important information about the evolution of the BSK gene family in plants and provide a basis for further studies of the functions of BSK family proteins.

## **2. Results**

#### *2.1. Identification and Characterization of the Brassinosteroid-Signaling Kinase (BSK) Genes in Plants*

In this study, a genome-wide analysis of the BSK gene family was performed on the basis of the completed genome sequences. Using the *Arabidopsis* Information Resource (TAIR), PlantGDB, Phytozome, and National Center for Biotechnology Information (NCBI) databases, we first retrieved the available BSK sequences from the currently sequenced genomes. A total of 17 plant genomes were analyzed to identify potential orthologous genes of BSK. These plants, representing the major clades of plants, included eight dicots (*Aquilegia coerulea*, *Arabidopsis thaliana*, *Brassica rapa*, *Glycine max*, *Gossypium raimondii*, *Medicago truncatula*, *Populus trichocarpa*, and *Solanum tuberosum*), five monocots (*Brachypodium distachyon*, *Oryza sativa*, *Setaria italica*, *Sorghum bicolor*, and *Zea mays*), and another four plant species (*Zostera marina*, *Ananas comosus*, *Physcomitrella patens*, and *Marchantia polymorpha*). The genome-wide analysis led to the identification of 143 BSK proteins in these plants. By investigating the extent of lineage-specific expansion of the BSK genes in plants, we found that the BSK gene originated in embryophytes (Figure 1A). Furthermore, almost all selected plants were found to have at least five BSK genes, with *B. rapa* having the highest number (21) of BSK genes (Figure 1B) and

*M. polymorpha* having only one BSK gene. This result indicated that the BSK genes were subjected to a *Int. J. Mol. Sci.*  large-scale expansion in higher plants. **2019**, *20*, x FOR PEER REVIEW 3 of 17

**Figure 1.** A comparative analysis of BSK genes in plants. (**A**) Evolution of core components of brassinosteroid (BR) signaling from aquatic plants to land plants indicated by dotted arrows. As representatives, component numbers of bryophyte, lycophyte and angiosperm were obtained from *Marchantia polymorpha*, *Physcomitrella patens*, *Selaginella moellendorffii*, *Arabidopsis thaliana*, and *Oryza sativa*, respectively. The numbers of BRI1-like and BSK proteins were indicated within blue and yellow circles, respectively. (**B**) The evolutionary relationships and the numerical details of the BSK family of each species. **Figure 1.** A comparative analysis of BSK genes in plants. (**A**) Evolution of core components of brassinosteroid (BR) signaling from aquatic plants to land plants indicated by dotted arrows. As representatives, component numbers of bryophyte, lycophyte and angiosperm were obtained from *Marchantia polymorpha*, *Physcomitrella patens*, *Selaginella moellendorffii*, *Arabidopsis thaliana*, and *Oryza sativa*, respectively. The numbers of BRI1-like and BSK proteins were indicated within blue and yellow circles, respectively. (**B**) The evolutionary relationships and the numerical details of the BSK family of each species.
