*4.5. RNA Extraction and RT-PCR*

Isolation of total RNA from treated samples was performed using an RNA extraction kit (Promega, Madison, WI, USA). The cDNA synthesis was performed with total RNA (2 µg) reverse transcribed using All-In-One RT MasterMix (Applied Biological Materials, Zhenjiang, China). RT-PCR analysis was conducted using 2 × T5 Super PCR Mix (Tsingke, Beijing, China) and Taq Master MixTaq mix (Vazyme Biotech, Nanjing, China). All primers used in this study are listed in Supplementary Table S2. Quantification for gel intensity was carried out using Image J software (https://imagej.nih.gov/ij/).

## **5. Conclusions**

In this study, we identified a total of 143 BSK proteins from 17 plant species. The phylogenetic analysis showed that the expansion of the BSK genes originated from embryophytes. A further comparative study revealed that most of the BSK genes in *Arabidopsis* were constitutively expressed and responded to some hormones or abiotic stresses. We also found some interesting post-transcriptional regulation patterns in *BSK5*, *BSK7*, and *BSK9*. Our results will further provide clues for the functional analysis of the important functions of BSK family genes in plants.

## **Supplementary Materials:** The following are available online at http://www.mdpi.com/1422-0067/20/5/1138/s1.

**Author Contributions:** Z.L. and J.L. conceived and designed the experiments; Z.L. and J.S. conducted the experiments analyzed the data; Z.L. wrote the manuscript and J.L. reviewed the manuscript.

**Funding:** This work was supported by the National Natural Science Foundation of China (31801277).

**Conflicts of Interest:** The authors declare no conflict of interest.
