Genome-Wide Analysis of LIM Family Genes in Foxtail Millet (Setaria italica L.) and Characterization of the Role of SiWLIM2b in Drought Tolerance

LIM proteins have been found to play important roles in many life activities, including the regulation of gene expression, construction of the cytoskeleton, signal transduction and metabolic regulation. Because of their important roles in many aspects of plant development, LIM genes have been studied in many plant species. However, the LIM gene family has not yet been characterized in foxtail millet. In this study, we analyzed the whole genome of foxtail millet and identified 10 LIM genes. All LIM gene promoters contain MYB and MYC cis-acting elements that are related to drought stress. Based on the presence of multiple abiotic stress-related cis-elements in the promoter of SiWLIM2b, we chose this gene for further study. We analyzed SiWLIM2b expression under abiotic stress and hormone treatments using qRT-PCR. We found that SiWLIM2b was induced by various abiotic stresses and hormones. Under drought conditions, transgenic rice of SiWLIM2b-overexpression had a higher survival rate, higher relative water content and less cell damage than wild type (WT) rice. These results indicate that overexpression of the foxtail millet SiWLIM2b gene enhances drought tolerance in transgenic rice, and the SiWLIM2b gene can potentially be used for molecular breeding of crops with increased resistance to abiotic stress.


Introduction
LIM domain proteins are widely found in eukaryotes [1] and have been found to play important roles in many aspects of development, including the regulation of gene expression, cytoskeleton assembly, signal transduction and the regulation of metabolism [2][3][4][5][6]. In animals, LIM proteins have also been found to play a role in cell adhesion and cell movement, participating in tumorigenesis and metastasis [7]. The LIM domain was first discovered in the three homeodomain proteins LIN11 (lineage 11), ISl-1 (insulin I) and MEC-3 (Mechanotransduction 3) [8][9][10][11], and was named based on the first letters of these protein names [11]. The LIM domain consists of a double zinc finger sequence, In recent years, studies have shown that LIM genes up-regulated expression respond to abiotic stresses, including drought, high salinity and hormones, suggesting that LIM genes may play a role in plant resistance to abiotic stress [22,23,30]. The functions of LIM family genes in cytoskeleton formation, signal transduction and the phenylpropane secondary metabolic pathway [15,21] also suggest that this family is important for abiotic stress because these processes are also important for stress responses. For example, the cytoskeleton participates in various life activities in cells and also plays a key regulatory role in the resistance of plants to various stress responses [31,32]. Plant antitoxins synthesized via the phenylpropane metabolic pathway are important for resistance to stress as well as for growth and development [33,34]. Because of their potential importance in the response to abiotic stress, LIM genes have been the focus of studies in many plants.
Drought stress is one of the main factors limiting global agricultural production [35]. Foxtail millet (Setaria italica L.) is an important food and feed crop in arid regionsof the world [36] and has strong resistance to various abiotic stresses, especially drought and low nutrition. Therefore, foxtail millet is used as a model monocotyledon crop for abiotic stress resistance research [35]. However, the mechanism of abiotic stress resistance in foxtail millet is still largely unclear [37]. Although the LIM gene family potentially plays an important role in abiotic stress response, this family has not yet been characterized in foxtail millet. Here we took advantage of the foxtail millet genome sequence [38,39] to identify all the members of the LIM gene family. Based on the complexity of LIM protein function, we identified all LIM genes in foxtail millet. We identified 10 SiLIM genes and classified these genes into two main groups. Quantitative real-time PCR (qRT-PCR) analysis showed that a LIM-like gene, SiWLIM2b, was induced by various stresses, and overexpression of SiWLIM2b increased the drought resistance of transgenic rice. These results provide evidence for the functions of a valuable novel LIM-like gene, SiWLIM2b, which can potentially be used for molecular breeding of resistance to abiotic stresses in graminaceous crops.

Phylogenetic Analysis and Chromosomal Distribution of 10 LIM-Like Genes
By searching the foxtail millet genome using the LIM domain (PF00412) as a keyword, we identifieda total of 10 putative LIM genes. The amino acid lengths of the LIMs ranged from 199 to 1441 residues, the predicted protein molecular weights ranged from 22.02 kDa to 154.76 kDa, and the isoelectric points ranged from 4.64 to 8.98 (Supplementary Table S1). To explore the phylogenetic relationships between the LIM genes, a phylogenetic tree was constructed using the LIM domain proteins from maize, rice, Arabidopsis and foxtail millet ( Figure 1A). Phylogenetic analysis showed that foxtail millet LIMs could be divided into the DA1&DAR (four proteins) and the CPR-like (six proteins) subfamilies ( Figure 1A). The DA1&DAR-like LIM proteins were further divided into Class I and Class II [23], and the CPR-like subfamily was further divided into the WLIM1, WLIM2, βLIM1 and PLIM2 subgroups ( Figure 1A) [22]. We found that the SiLIM genes were more closely related to genes from maize and rice than to genes from Arabidopsis ( Figure 1A).
To visualize the genome distribution of the LIM genes in foxtail millet, the MapGene2Chrom tool [40] was used to map the chromosomal locations. The SiLIMs were mapped to chromosomes 1, 3, 4, 7 and 9 ( Figure 1B), with most genes located on chromosome 9 (Seita.9G164800, Seita.9G201000, Seita.9G459200 and Seita.9G458000) ( Figure 1B). Thus, the LIM genes are unevenly distributed in the foxtail millet genome. The precise locations of the 10 LIM genes are listed in Supplementary Table S1. proteins in foxtail millet, Arabidopsis, maize and rice. The phylogenetic tree was constructed using the maximum likelihood method in Molecular Evolutionary Genetics Analysis 7.0 software based on the comparison of LIM amino acid sequences. Dark green, light green, pink, purple, dark blue and red represent the DA1&DAR class I, DA1&DAR class II, PLIM2, WLIM2, βLIM1 and WLIM1 subgroups, respectively. Yellow represents the foxtail millet LIM genes, and the red five-pointed star indicates the SiWLIM2b (Seita.9G458000) gene. The species acronym is shown before each LIM protein name: Seita, Setaria italica; At, Arabidopsis thaliana; Os, Oryza sativa; Zm, Zea mays. (B) Distribution of SiLIM genes in the foxtail millet genome. The SiLIM genes are located on chromosomes 1, 3, 4, 7 and 9. The SiWLIM2b gene is located on chromosome 9 and is marked in red. Chromosomal distances are given in Mb.

Functional Domains and Gene Structure Analysis of the 10 LIM-Like Genes
To further analyze the functional domains of SiLIM genes, we used the SMART website to obtain the conserved domains in the foxtail millet LIM proteins. All LIM proteins in the CRP subfamily contain two LIM domains, and members of the DA1&DAR subfamily contain a conserved LIM domain and two to three conserved UIM domains (Figure 2A). proteins in foxtail millet, Arabidopsis, maize and rice. The phylogenetic tree was constructed using the maximum likelihood method in Molecular Evolutionary Genetics Analysis 7.0 software based on the comparison of LIM amino acid sequences. Dark green, light green, pink, purple, dark blue and red represent the DA1&DAR class I, DA1&DAR class II, PLIM2, WLIM2, βLIM1 and WLIM1 subgroups, respectively. Yellow represents the foxtail millet LIM genes, and the red five-pointed star indicates the SiWLIM2b (Seita.9G458000) gene. The species acronym is shown before each LIM protein name: Seita, Setaria italica; At, Arabidopsis thaliana; Os, Oryza sativa; Zm, Zea mays. (B) Distribution of SiLIM genes in the foxtail millet genome. The SiLIM genes are located on chromosomes 1, 3, 4, 7 and 9. The SiWLIM2b gene is located on chromosome 9 and is marked in red. Chromosomal distances are given in Mb.

Functional Domains and Gene Structure Analysis of the 10 LIM-Like Genes
To further analyze the functional domains of SiLIM genes, we used the SMART website to obtain the conserved domains in the foxtail millet LIM proteins. All LIM proteins in the CRP subfamily contain two LIM domains, and members of the DA1&DAR subfamily contain a conserved LIM domain and two to three conserved UIM domains (Figure 2A). Exon-intron structural divergence within families plays a pivotal role in the evolution of multigene families [41]. We used the Gene Structure Display Server (GSDS) to construct a sketch of the exon-intron structure of each SiLIM gene. The gene structures are consistent with the classification of the proteins based on the phylogenetic tree. We found that the genes in each subfamily generally had similar exon-intron structures. The six LIM genes belonging to the CPR-like subfamily contain four introns, and two of the LIM genes in the DA1&DAR subfamily contain 10 introns. The other two of the LIM genes in the DA1&DAR subfamily genes contain 11 introns ( Figure 2B). The number of exons in the 10 LIM genes ranged from 5 to 12 ( Figure 2B), and we found examples of exon loss or acquisition during the evolution of the LIM genes in foxtail millet. The gain or loss of exons indicates that the function of these genes may change.

Promoter Analysis and Tissue-Specific Expression of the LIM Genes in Foxtail Millet
Cis-regulatory elements play an important role in regulating tissue-specific gene expression [42], and also participate in drought response, pathogen defense and cell wall metabolism [43]. Analysis of the foxtail millet LIM gene promoters revealed that there are many cis-acting elements associated with the response to hormones and abiotic stresses ( Figure 3A) and that all of SiLIM gene promoters contain MYB and MYC elements, which are related to drought and ABA response ( Figure 3A) [44][45][46]. Most SiLIM gene promoters have a light-responsive element (Sp1, TCCC-motif, or G-box) and the abscisic acid response element (ABRE, MBS) ( Figure 3A). In addition, some foxtail millet LIM gene promoters contain hormone response elements, including gibberell in response elements (Pbox, GARE-motif), the salicylic acid response element (TCA-element), methyl jasmonate response elements (CGTCA-motif, TGACG-motif) and the low-temperature responsive element (LTR) (Figure Exon-intron structural divergence within families plays a pivotal role in the evolution of multi-gene families [41]. We used the Gene Structure Display Server (GSDS) to construct a sketch of the exon-intron structure of each SiLIM gene. The gene structures are consistent with the classification of the proteins based on the phylogenetic tree. We found that the genes in each subfamily generally had similar exon-intron structures. The six LIM genes belonging to the CPR-like subfamily contain four introns, and two of the LIM genes in the DA1&DAR subfamily contain 10 introns. The other two of the LIM genes in the DA1&DAR subfamily genes contain 11 introns ( Figure 2B). The number of exons in the 10 LIM genes ranged from 5 to 12 ( Figure 2B), and we found examples of exon loss or acquisition during the evolution of the LIM genes in foxtail millet. The gain or loss of exons indicates that the function of these genes may change.

Promoter Analysis and Tissue-Specific Expression of the LIM Genes in Foxtail Millet
Cis-regulatory elements play an important role in regulating tissue-specific gene expression [42], and also participate in drought response, pathogen defense and cell wall metabolism [43]. Analysis of the foxtail millet LIM gene promoters revealed that there are many cis-acting elements associated with the response to hormones and abiotic stresses ( Figure 3A) and that all of SiLIM gene promoters contain MYB and MYC elements, which are related to drought and ABA response ( Figure 3A) [44][45][46]. Most SiLIM gene promoters have a light-responsive element (Sp1, TCCC-motif, or G-box) and the abscisic acid response element (ABRE, MBS) ( Figure 3A). In addition, some foxtail millet LIM gene promoters contain hormone response elements, including gibberell in response elements (P-box, GARE-motif), the salicylic acid response element (TCA-element), methyl jasmonate response elements (CGTCA-motif, TGACG-motif) and the low-temperature responsive element (LTR) ( Figure 3A).
There are differences of cis-elements in LIM genes from different subgroups. Among the LIM genes of CRP-like subgroup of foxtail millet, 83.3% of these genes contain auxin response elements, 83.3% of these genes contain GA response elements and 66.6% of these genes contain SA response elements. Among the LIM genes of DA1&DAR subgroup of foxtail millet, 50% of these genes contain auxin response elements, 75% of these genes contain GA response elements and 25% of these genes contain SA response elements, which were significantly lower than the CRP-like subgroup. In the CRP subgroup of LIM genes, 33.3% of these genes contain LTR elements, 66.6% of these genes contain JA response elements, while in the DA1-DAR subgroup of LIM genes, 75% of these genes contain LTR elements and 75% of these genes contain JA response elements, which is significantly higher than that in the CRP subgroup. More details about the cis-elements in SiLIM family gene promoters are shown in Supplementary Table S2.
). There are differences of cis-elements in LIM genes from different subgroups. Among the LIM genes of CRP-like subgroup of foxtail millet, 83.3% of these genes contain auxin response elements, 83.3% of these genes contain GA response elements and 66.6% of these genes contain SA response elements. Among the LIM genes of DA1&DAR subgroup of foxtail millet, 50% of these genes contain auxin response elements, 75%of these genes contain GA response elements and 25% of these genes contain SA response elements, which were significantly lower than the CRP-like subgroup. In the CRP subgroup of LIM genes, 33.3% of these genes contain LTR elements, 66.6% of these genes contain JA response elements, while in the DA1-DAR subgroup of LIM genes, 75% of these genes contain LTR elements and 75% of these genes contain JA response elements, which is significantly higher than that in the CRP subgroup. More details about the cis-elements in SiLIM family gene promoters are shown in Supplementary Table S2.  We analyzed the expression profiles of the LIM genes in foxtail millet roots, stems, leaves and tassel inflorescences using the RNA-seq data in the European Nucleotide Archive database [47,48] (Figure 3B). This analysis showed that Seita.1G250500 was specifically expressed in tassel inflorescences and that Seita.3G353200 was mainly expressed in the root and not expressed in the leaf. The remaining genes were expressed in all four tissues. Seita.3G375500, Seita.7G179500, Seita.9G201000 and Seita.9G459200 were most highly expressed in leaves. The Seita.9G458000 (SiWLIM2b) gene had the highest and lowest expression levels in roots and leaves, respectively. Seita.4G104700 and Seita.9G164800 were most highly expressed in stems and Seita.4G050800 was most highly expressed in roots ( Figure 3B). This expression profiling would facilitate combinatorial usage of SiLIMs in plant cell processes of different plant tissues, whereas SiLIMs expressed in all four tissues may participate in a broad set of plant cell processes. The seita.9G458000 was more highly expressed in roots and tassels than other nine LIM genes, which indicated that seita.9G458000 plays a more important role in roots and tassel inflorescences.

Analysis of SiWLIM2b Expression under Various Treatments
By comparing and analyzing the cis-elements in the promoter region of LIM genes in foxtail millet, we found that the promoter region of seita.9G458000 contained the most cis-elements related to abiotic stress (27), while the promoter region of seita.9G201000 contained the least cis-elements related to abiotic stress (14). Therefore, we selected the WLIM2 subfamily gene SiWLIM2b ( Figure 1A) for further study. In order to explore the possible role of SiWLIM2b in foxtail millet, we used qRT-PCR to analyze the expression of the SiWLIM2b gene in response to different abiotic stresses and exogenous hormones. We found that SiWLIM2b was up-regulated in response tosalicylic acid (SA), methyl jasmonate (MeJA), gibberellic acid (GA), abscisic acid (ABA), high Ca 2+ (120 ppm), and high salt (150 mM NaCl) ( Figure 4A,B). SiWLIM2b was significantly down-regulated after 2 h of simulated drought (15% PEG) or high pH (8.0) ( Figure 4A,B). Under nitrogen deficiency (ND), SiWLIM2b was slightly up-regulated ( Figure 4B).

Subcellular Location of the SiWLIM2b Protein
In order to identify the subcellular localization of the SiWLIM2b protein, the full-length coding sequence (CDS) of the SiWLIM2b gene was cloned into an expression vector containing the GFP tag (16318hGFP) with expression of the SiWLIM2b-GFP fusion protein driven by the CaMV35S promoter. The plasmid SiWLIM2b-GFP was co-transformed into tobacco protoplasts with the nucleus marker AT2G03340-mCherry [49,50]. The SiWLIM2b-GFP fusion protein was mainly distributed in both the cytoplasm and nucleus, and GFP expressed from an empty 35S::GFP expression vector, which served as a control, was mainly distributed in the plasma membrane, cytoplasm and nucleus ( Figure 4C). We speculated that the SiWLIM2b protein may act as a transcription factor and may shuttle between the cytoplasm and nucleus.

Overexpression of SiWLIM2b Enhances Drought Resistancein Transgenic Rice
Because the SiWLIM2b gene is regulated by drought stress, we wanted to further analyze the functions of SiWLIM2b in drought resistance. Phenotypic analysis was performed using homozygous T3 seeds of transgenic rice lines expressing SiWLIM2b. We analyzed the tolerance of SiWLIM2b transgenic rice under drought stress in a greenhouse and found that three independent transgenic rice lines grew better than wild type (WT) under drought stress ( Figure 5A). Three transgenic lines were analyzed. At least 25 seedlings from each line were measured. qRT-PCR showed that the SiWLIM2b gene was more highly expressed in the three transgenic rice lines than in WT (p < 0.01) ( Figure 5B). After drought treatment, WT plants withered and died earlier than the transgenic rice plants, and the survival rate of the transgenic rice lines was higher than that of WT (p < 0.01) ( Figure 5C). Relative water content analysis showed there was no significant difference between the transgenic lines and WT under normal conditions, whereas under drought conditions, the three transgenic rice lines had significantly higher relative water contents than WT (p < 0.01) ( Figure 5D). We measured the malondialdehyde (MDA) content of transgenic rice and WT, and found that there was no significant difference in the MDA content under normal conditions, whereas under drought treatment, the MDA content of the three transgenic rice lines was significantly lower than that of WT (p < 0.01) ( Figure 5E). The lower MDA content indicates that the transgenic lines had a lower degree of membrane damage and higher drought resistance than the WT.

Analysis of SiWLIM2b Expression under Various Treatments
By comparing and analyzing the cis-elements in the promoter region of LIM genes in foxtail millet, we found that the promoter region of seita.9G458000 contained the most cis-elements related to abiotic stress (27), while the promoter region of seita.9G201000 contained the least cis-elements related to abiotic stress (14). Therefore, we selected the WLIM2 subfamily gene SiWLIM2b ( Figure  1A) for further study. In order to explore the possible role of SiWLIM2b in foxtail millet, we used qRT-PCR to analyze the expression of the SiWLIM2b gene in response to different abiotic stresses and exogenous hormones. We found that SiWLIM2b was up-regulated in response tosalicylic acid (SA), methyl jasmonate (MeJA), gibberellic acid (GA), abscisic acid (ABA), high Ca 2+ (120 ppm), and high salt (150 mM NaCl) ( Figure 4A,B). SiWLIM2b was significantly down-regulated after 2h of simulated drought (15% PEG) or high pH (8.0) (Figures 4A,B). Under nitrogen deficiency (ND), SiWLIM2b was slightly up-regulated ( Figures 4B).  LIM genes have been shown to regulate the phenylpropanoid secondary metabolic pathway [5,6,21,29]. Therefore, we analyzed the expression of three genes encoding key rate-limiting enzymes in the phenylpropane pathway: PAL (phenylalanine ammonia-lyase), 4CL2 (4-coumarate coenzyme A (CoA) ligase 2) and C3H (coumarate 3-hydroxylase) [51]. The qRT-PCR data showed that these phenylpropane synthesis-related genes were more highly expressed in the transgenic rice lines than in WT under both normal growth conditions and under drought treatment ( Figure 5F). In addition, the expression levels of the three genes were significantly higher under drought than under normal conditions. LIM genes have been shown to regulate the phenylpropanoid secondary metabolic pathway [5,6,21,29]. Therefore, we analyzed the expression of three genes encoding key rate-limiting enzymes in the phenylpropane pathway: PAL (phenylalanine ammonia-lyase), 4CL2 (4-coumarate coenzyme A (CoA) ligase 2) and C3H (coumarate 3-hydroxylase) [51]. The qRT-PCR data showed that these phenylpropane synthesis-related genes were more highly expressed in the transgenic rice lines than in WT under both normal growth conditions and under drought treatment ( Figure 5F). In addition, the expression levels of the three genes were significantly higher under drought than under normal conditions.

Discussion
Plants encounter various biotic and abiotic stresses in the stage of growth and development, as one of the members of plant protein family, LIM proteins are heavily studied. Previous research showed that plant LIM proteins are mainly involved in actin cytoskeleton remodeling and the transcriptional regulation of gene expression. Han et al. found that cotton WLIM1a played a role in developing cotton fibers [21]. Recent studies have showed that LIM gene expression in plants was induced by various biotic and abiotic stresses. Although the LIM proteins family has been studied extensively in many crops, knowledge about foxtail millet stress tolerance was limited. Here, a genome-wide analysis of the LIM family was performed in foxtail millet and 10 LIM genes were identified. Among the 10 LIM genes, SiWLIM2b was screened to study its function in stress responses and this study will provide some information towards further study of the LIM gene in other species.

Discussion
Plants encounter various biotic and abiotic stresses in the stage of growth and development, as one of the members of plant protein family, LIM proteins are heavily studied. Previous research showed that plant LIM proteins are mainly involved in actin cytoskeleton remodeling and the transcriptional regulation of gene expression. Han et al. found that cotton WLIM1a played a role in developing cotton fibers [21]. Recent studies have showed that LIM gene expression in plants was induced by various biotic and abiotic stresses. Although the LIM proteins family has been studied extensively in many crops, knowledge about foxtail millet stress tolerance was limited. Here, a genome-wide analysis of the LIM family was performed in foxtail millet and 10 LIM genes were identified. Among the 10 LIM genes, SiWLIM2b was screened to study its function in stress responses and this study will provide some information towards further study of the LIM gene in other species.
An accurate evolution history is the first step to understanding the evolution relationship of genes, and may provide some useful information to study the pathway and function they are involved in or regulated by [52]. To understand the relationship of foxtail millet LIM proteins with other LIM proteins in different species, a phylogenetic tree of the LIM domain proteins from rice, maize, Arabidopsis and foxtail millet was constructed. The result showed that foxtail millet LIM proteins shared genes more closely related with rice and maize LIM proteins than with Arabidopsis LIM proteins, which indicated that the LIM gene family differentiated after the divergence of monocotyledons and dicotyledons. These results are consistent with the present understanding of plant evolutionary history [53]. Zhang, G. Y. et al. revealed five major duplications between chromosomes 2 and 9, 4 and 1, 7 and 1, 6 and 2, and 5 and 3 [38]. In this study, chromosomal distributionanalysis of the LIM gene family of foxtail millet showed that the LIM genesof foxtail millet was only distributed on chromosomes 1, 3, 4, 7 and 9. This may indicate that the formation of LIM genes family of foxtail millet occurred after the chromosome duplication events.
The function of a gene is related to its structure, for example, introns can increase transcription levels by affecting the transcription rate, nuclear output and transcript stability [54]. Both the functional domain analysis and exon-intron structure analysis showed that LIM genes were conserved in the same subgroup, which indicated that LIM genes in the same subgroup have some functional similarities. Both of the functional domains and exon-intron structures of DA1&DAR subfamily genes is more complex than that of CRP subfamily genes, which may suggest that substantial differentiations might have occurred during the evolution of this gene family. The DA1&DAR protein has some roles that are different to CRP-like protein. For example, the DA1 family also plays a role in regulating the degradation of abnormal proteins [55,56] and in the cell cycle [57]. DA1 was also found to regulate the size of seeds and organs in Arabidopsis [58,59].
Cis-regulatory elements in the promoter regions of genes play significant roles in plant stress responses [60]. Hence, we analyzed the cis-acting elements in promoters of the SiLIM genes and found elements related to GA, auxin, MeJA, and SA response. These elements play an important role in many physiological processes, such as plant development, senescence and maturation, secondary metabolism and response to various environmental stresses [37,61,62]. For example, the P-box and GARE-motifsare associated with the GA signal transduction pathway, and the CGTCA-motif and TGACG-motif are associated with the response to MeJA [37,61,62]. In addition, we found the ABRE in the promoters of SiLIM genes, and confirmed that the bZIP transcription factors AREB1, AREB2, and ABF3 interact with the ABRE motifs in the DREB2A promoter to co-regulate plant responses to osmotic stress [63]. All promoters contained more than one element related to drought and ABA response, such as MYC and MYB elements [44]. Based on this evidence, we speculate that the SiLIM genes are probably involved in abiotic stress responses.
The LIM genes have been found to be induced by various stresses and hormones in other plants. The expression levels of PbLIMs were significantly induced by SA, ABA and MeJA in pear [30]. The LIM genes of tomato were induced by ABA, cold, drought, NaCl and heat treatment [22]. The LIM genes of Brassica rapa were induced by cold, ABA and pH (pH5, pH7 and pH9) treatments [64]. Similar results were found in this study. QPCR analysis showed that the SiWLIM2b gene was induced by various stresses (PEG, Ca 2+ , ND, NaCl, pH) and exogenous hormones (ABA, SA, MeJA, GA). These studies may illustrate that the functions of LIM genes in different species appear to be similar. We chose the SiWLIM2b gene for further study and found that SiWLIM2b was induced by drought. In order to further study the role of SiWLIM2b in drought resistance, we studied the phenotype of transgenic rice heterologously by expressing the SiWLIM2b gene under drought conditions. We found that the SiWLIM2b gene can improve drought resistance when expressed in transgenic rice ( Figure 5). MDA can inhibit the activity of cell protective enzymes and reduce the antioxidant content [65,66]. When the enzymes and membrane systems of plant tissues are destroyed, the MDA content is greatly increased. Therefore, MDA reflects the antioxidant capacity of plant tissues, and can also serve as a measure of plant resistance to external stresses [67]. Determination of the MDA content of transgenic lines and WT under drought stress showed that under drought stress transgenic lines had much less membrane damage and higher drought resistance than the kitaake (WT) [65,67]. These results indicate that the SiWLIM2b gene is involved in drought response and could be used as a new candidate gene in molecular breeding for crop stress resistance.
A recent study reported that the function of LIM proteins is closely related to its subcellular localization and LIM proteins with nuclear localized or co-localized in the cytoplasm and nucleus act in regulation of gene expression [6,14,21]. In this study, we found that the SiWLIM2b protein mainly accumulated in both the cytoplasm and nucleus, suggesting that it may function in the regulation of gene expression. In our study, the expression of three genes encoding key rate-limiting enzymes in the phenylpropane pathway was up-regulated in the transgenic rice lines. This suggests that SiWLIM2b gene may be involved in the phenylpropane pathway. It was confirmed that the synthesis substances of the phenylpropanol pathway greatly enhance the tolerance to drought in plants [24]. Wei et al. confirmed that the TaMyb1D gene in transgenic tobacco regulated the expression of related genes in the phenylpropanol metabolism pathway, and over-expression of TaMyb1D enhanced drought resistance of transgenic tobacco [68]. Similar results were found in our study, three phenylpropane synthesis-related genes were up-regulated expression in transgenic rice seedlings after drought treatment. This suggests that over-expression of the SiWLIM2b gene may enhance drought resistance of transgenic rice by participating in the secondary metabolic pathway of phenylpropane. However, we found that SiWLIM2b did not have transcriptional activation activity (Supplemental Figure S1). We speculate that SiWLIM2b may also regulate the expression of genes by interacting with other transcription factors [28]. Further experiments are required to identify the relationship between the SiWLIM2b gene and the phenylpropanoid pathway and drought resistance.

Plant Materials
Foxtail millet seeds were grown in nutrient soil (1:1 mix of nutrient soil and vermiculite) at 28 • C for three weeks, and then subjected tovarious abiotic stresses, namely drought (15% PEG), high salinity (150 mM NaCl), high pH (8.0) and high Ca 2+ (120 ppm), or treatment with exogenous hormones, namely ABA (20 µM), SA (20 µM), GA (20 µM) and MeJA (20 µM). Seedlings were sampled at different times after being transferred to a solution containing each substance. In order to subject foxtail millet seedlings to low nitrogen conditions (ND treatment), foxtail millet seeds were germinated at 28 • C for 2 days, then transferred into Hoagland nutrient solution and grown to the four-leaf stage. Seedlings were then transferred into nitrogen-free nutrient solution and sampled after 0, 1, 4, 8, 10, 12 and 24 h. All samples were frozen in liquid nitrogen and stored at −80 • C.

Identification and Phylogenetic Analysis of LIMFamily Genes
The Hidden Markov Model (HMM) of the LIM domain (PF00412) was obtained from Pfam 31.0 (https://en.wikipedia.org/wiki/Pfam, accessed date: 11 August 2018). The putative LIM genes were identified in the foxtail millet genomic database (Phytozome v12.1, https://phytozome.jgi.doe.gov/pz/ portal.html#, accessed date: 11 August 2018) using 'LIM' and 'PF00412' as key wordswith a threshold E values ≤ 1.0. Each putative SiLIM gene sequence was checked using SMART (http://smart.emblheidelberg.de/, accessed date: 11 August 2018) to confirm the presence of the LIM domain. The protein sequences of the Arabidopsis LIM genes were acquired from TAIR (https://www.arabidopsis.org/, accessed date: 11 August 2018) and those of the rice and maize LIM genes were acquired from the Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html#, accessed date: 11 August 2018). In order to investigate the evolutionary relationships among the LIM domain proteins in different plant species, aphylogenetic tree was constructed [69]. All Arabidopsis, rice, maize and foxtail millet LIM protein sequences were imported using MEGA7.0 [70], and multiple sequence alignments were performed using ClustalW with the default multiple alignment parameters. The phylogenetic tree was constructed using the maximum likelihood method. Information of LIM indifferent species (Arabidopsis, rice, maize) is listed in Supplemental Table S3). The percentage of similarity compared with the sequences of LIM proteins in foxtail millet and the LIM proteins of maize, rice and arabidopsis thaliana on the same evolutionary branch is listed in Supplemental Table S4.
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).

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 (log 2 RPKM values) was made using EVOLVIEW. The expression profiling data are shown in Supplementary Table S5.

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.

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.

Phenotypic Analysis of Transgenic Rice in a Greenhouse
The SiWLIM2b complete open reading frame (ORF) was amplified by PCR usingthe primers F1 (BamH I) and R1 (Sac I). The amplified PCR fragment was cloned into vector pMWB014 (driven by the ubiquitin promoter) digested with BamH I and Sac I. The pMWB014-SiWLIM2b was confirmed by sequencing and transformed into the kitaake rice (WT) cultivar by Agrobacterium-mediated transformation. The transformed callus was cultured on medium with 1.0 mg/L glufosinate. Six transgenic rice lines were obtained by using primers F2 (designed according to the CDS of SiWLIM2b gene) and R2 (designed according to NOS terminator sequence) for PCR verification. The resulting transgenic rice was cultured to the T3 generation. We selected three transgenic lines in the T3 generation (OE1135, OE1136, and OE1144) for subsequent functional analysis. To determine whether transgenic rice seedlings could resist drought stress, we conducted a pot experiment in the greenhouse. Rice seeds were soaked with 2.5% sodium hypochlorite (NaClO) for 30 min, then rinsed with tap water 5-6 times. Sterilized seeds were germinated in a 28-degree incubator, and the water was changed every 12 h. After germination, the seeds wereplanted in nutrient soil and grown to the three-leaf stage under a 16-h/8-h photoperiod at 30 • C. Rice seedlings were then subjected to drought treatment for 12 days and survival was scored by dividing the number of plants alive after drought treatment for 12 days by the total number of plants surveyed, using one green leaf as the standard for survival. Relative water content [77,78] and MDA content [66] were determined in transgenic and control rice after 5 days of drought treatment. All experiments were set up with three independent biological replicates.

Differential Expression of Phenylpropane Secondary Metabolic Pathway Genes in Transgenic and Control Rice Plants
Expression of phenylpropane secondary metabolic pathway-related genes in control and transgenic rice was analyzed by qRT-PCR. Rice were grown to the four-leaf stage in Hoagland nutrient solution, then treated with 6% PEG for three days. RNA was extracted and reverse transcribed into cDNA, which was used as template for qRT-PCR. Primers used in qRT-PCR are listed in Supplemental  Table S4.

Conclusions
A total of 10 LIM protein-coding genes were identified in foxtail millet via a genome-wide analysis. Analysis of evolutionary relationships indicated that SiLIM genes could be divided into two large subgroups. The chromosome positions, gene structures, functional domains and promoters of the SiLIM genes were also analyzed. The SiWLIM2b gene was found to respond to various abiotic stresses and hormone treatments, and overexpression of the SiWLIM2b gene in rice improved drought tolerance.