*4.3. Chromosome Locations and Gene Structures of LIM Family Genes and Identification of Functional Domains and Cis-Acting Elements*

Foxtail millet chromosome lengths were obtained from the file Setaria\_italica.JGIv2.0.38.gff3, and the position of each *LIM* gene on the foxtail millet chromosomes was obtained from Phytozome

(https://phytozome.jgi.doe.gov/pz/portal.html#, accessed date: 11 August 2018). All *SiLIM* genes were mapped onto the nine foxtail millet chromosomes according their physical position (bp) using MapGene2Chromosome v2 (http://mg2c.iask.in/mg2c\_v2.0/, accessed date: 12 August 2018). The exon-intron structures of the *LIM* genes were determined by comparing the CDS with the genomic sequences using the GSDS (http://gsds.cbi.pku.edu.cn/, accessed date: 12 August 2018) [71].

The SMART online tool (http://smart.embl-heidelberg.de/, accessed date: 11 August 2018) was used to analyze the functional domains in the SiLIM proteins, and functional domain sketches were drawn using the ExPAsy-PROSITE website (https://prosite.expasy.org/, accessed date: 13 August 2018). The molecular weights and the oretical isoelectric points of the SiWLIMs were calculated using the ExPASy online tool (https://web.expasy.org/protparam/, accessed date: 13 August 2018).

To obtain the promoter sequences we downloaded approximately 2.0 Kb of sequence upstream of each *SiLIM* coding sequence from the Phytozome database. *Cis*-acting elements were analyzed using the plant *cis*-acting element database PlantCARE (http://bioinformatics.psb.ugent.be/webtools/ plantcare/html/, accessed date: 15 August 2018) [72]. Heat map showing the number of *cis*-acting elements in the promoters were made using EVOLVIEW (http://www.evolgenius.info/evolview/ #mytrees//, accessed date: 7 September 2018) [73].

## *4.4. Tissue-Specific Expression Profiling Using RNA-Seq Data*

Illumina RNA-seq data for four tissues namely roots, stems, leaves and tassel inflorescences were downloaded from the European Nucleotide Archive database [SRX128223 (root); SRX12825 (stem); SRX128224 (leaf); SRX128226 (spica)] [47,48]. Four tissues were obtained from young seedlings of Zhang-gu, which grew in nutrient soil for 40 days in a greenhouse [38]. RNA-seq data were mapped onto the gene sequences of foxtail millet using CLC Genomics Workbench V4.7.1 tooland normalized by FPKM (reads per kilobase per million). A heat map showing tissue-specific expression profiles (log2RPKM values) was made using EVOLVIEW. The expression profiling data are shown in Supplementary Table S5.

## *4.5. Total RNA Extraction and qRT-PCR Analysis*

Total RNA was extracted from foxtail millet using the Plant Total RNA Kit (Zhuangmeng, Beijing, China) according to the manufacturer's manual. The extracted RNA was detected by electrophoresis on a 1% agarose gel, and the first strand of cDNA was synthesized using the EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech, Beijing, China). The relative expression level of the *SiWLIM2b* gene under various stresses was analyzed by qRT-PCR. The Primer3 website (http://bioinfo.ut.ee/primer3-0.4.0/, accessed date: 4 November 2017) was used to design primers for qRT-PCR [74,75], and the 2−∆∆Ct method was used to calculate the relative expression level ofthe *SiWLIM2b* gene. The actin gene (Genbank number: AF288226) of foxtail millet was used as an internal control. QRT-PCR was performed for three biological replicates, with three technical replicates per biological replicate. All primer sequences used are shown in Supplementary Table S6.

## *4.6. Subcellular Localization of SiWLIM2b*

We constructed a vector expressing a GFP-tagged fusion protein for subcellular localization analysis of the SiWLIM2b protein. A cDNA containing the coding sequence of *SiWLIM2b* was cloned into the 16318h GFP vector and expressed under the control of the CaMV35s promoter. The recombinant plasmids were confirmed by sequencing. The full-length cDNA coding sequences of AT2G03340 which were located in the nucleus [49] were cloned into the mCherry ORF (WRKY25-RFP) under the control of the CaMV 35S promoter [50]. The plasmid SiWLIM2b-GFP and AT2G03340-mcherry were co-transformed into tobacco protoplasts using the PEG-mediated method and observed under a confocal microscope 16h after transformation [76]. The 35S::GFP vector was transformed as the control.
