*2.4. Cis-Element Analysis of Rice Trihelix Genes*

To understand the genetic functions, metabolic networks, and regulatory mechanisms of rice trihelix genes, the shared *cis*-elements in the promoter regions of the *OsMSL*s were analyzed. The 1500-bp upstream *OsMSL* sequence was obtained and identified as a hypothetical promoter. The potential shared *OsMSL cis*-element was scanned and screened out and its distribution and function were analyzed. Two dehydration-responsive- and three light-responsive *cis*-elements common to all *OsMSL*s were identified and labeled by different colors in the promoter sequence (Figure 7). *Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 14 of 29

**Figure 7.** Predicted *cis*-elements in the promoter regions of the rice trihelix genes. All promoter sequences (−1500 bp) were analyzed. The trihelix genes are shown on the left side of the figure. The scale bar at the bottom indicates the length of promoter sequence. Green bar (GB): GATABOX element; purple bar (A): ACGTATERD1 element; red bar (GC): GT1CONSENSUS element; gray bar (I): INRNTPSADB element; blue bar (M): GT1GMSCAM4 element. **Figure 7.** Predicted *cis*-elements in the promoter regions of the rice trihelix genes. All promoter sequences (−1500 bp) were analyzed. The trihelix genes are shown on the left side of the figure. The scale bar at the bottom indicates the length of promoter sequence. Green bar (GB): GATABOX element; purple bar (A): ACGTATERD1 element; red bar (GC): GT1CONSENSUS element; gray bar (I): INRNTPSADB element; blue bar (M): GT1GMSCAM4 element.

*2.5. Expression Profiles of Trihelix Genes in Rice Tissues and Developmental Stages* The expression profiles of the various rice tissues including the root, stem, leaf, and sheath of four-leaf rice seedlings were investigated (Figure 8). As shown in Figure 8A, all 43 transcripts expressed in all tissues but their expression levels varied greatly in each tissue. Particularly, there are more highly expressing trihelix genes in the leaves and sheaths than the other organs. There are almost no genes with low expression levels and most of the genes remained at high expression levels in the leaves. In contrast, comparatively fewer genes with extremely high expression exist in the stems and none of them express at an extremely high level in the roots. As shown in Figure 7, A (ACGTATERD1) and M (GT1GMSCAM4) element are two dehydration-responsive elements and M is a core element. Therefore, *OsMSL*s probably participate in dehydration (including drought and salt) stress responses. GB (GATABOX element), GC (GT1CONSENSUS), and I (INRNTPSADB) are three light-responsive elements. They indicate that the *OsMSL*s family potentially consists of light-inducible/repressible genes. Light responsiveness is typical of the GT factor (now known as the trihelix family gene) and was confirmed in our *cis*-element study. To verify whether *OsMSL*s are regulated by light under both normal- and stress conditions, a dark treatment was added to the *OsMSL* expression analysis.

#### The *OsMSL*s were clustered into groups I to V according to their expression characteristics in *2.5. Expression Profiles of Trihelix Genes in Rice Tissues and Developmental Stages*

different tissues (Figure 8B). *OsMSL*s in groups I and II expressed at lower levels in all tissues, but members in group I displayed relatively higher expression in root tissue. For instance, although *OsMSL19* maintained very low expression levels in most tissues, its expression displayed a distinct The expression profiles of the various rice tissues including the root, stem, leaf, and sheath of four-leaf rice seedlings were investigated (Figure 8). As shown in Figure 8A, all 43 transcripts expressed

enrichment in root. In contrast, the genes in group II, especially *OsMSL02*, *OsMSL08,* and *OsMSL12,* remained low in the various tissues. On the other hand, in *Arabidopsis*, the At5g63420 gene, the

lower levels in the roots. However, the transcription levels of *OsMSL34a* and *OsMSL34b* in group IV were high in all four tissues. Group V members also displayed tissue specific expression, but expressed at higher levels than group I and III. Besides, the genes in group V, especially *OsMSL16*, *OsMSL27*, *OsMSL28*, *OsMSL35,* and *OsMSL39*, expressed extremely highly in the leaves and sheaths, whereas their expression levels in the roots and stems were lower than those of genes in group I and

in all tissues but their expression levels varied greatly in each tissue. Particularly, there are more highly expressing trihelix genes in the leaves and sheaths than the other organs. There are almost no genes with low expression levels and most of the genes remained at high expression levels in the leaves. In contrast, comparatively fewer genes with extremely high expression exist in the stems and none of them express at an extremely high level in the roots. *Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 16 of 29

**Figure 8.** Expression of the rice trihelix gene family in various tissues (R: Root; St: Stem; L: Leaf; Sh: Sheath). (**A**) Numbers of expressed genes in each tissue. Expression data of the rice trihelix gene family were retrieved from the Expression Atlas database. Extremely high: Expression value > 6, high: 6 ≥ expression value > 4, medium: 4 ≥ expression value > 2, low: 2 ≥ expression value > 0; (**B**) Expression patterns of the trihelix genes in various rice tissues. Heatmaps were created in HemI v.1.0 and based on the expression data. Expression levels are depicted by different colors on the scale. Green and red represent low and high expression levels, respectively. **Figure 8.** Expression of the rice trihelix gene family in various tissues (R: Root; St: Stem; L: Leaf; Sh: Sheath). (**A**) Numbers of expressed genes in each tissue. Expression data of the rice trihelix gene family were retrieved from the Expression Atlas database. Extremely high: Expression value > 6, high: 6 ≥ expression value > 4, medium: 4 ≥ expression value > 2, low: 2 ≥ expression value > 0; (**B**) Expression patterns of the trihelix genes in various rice tissues. Heatmaps were created in HemI v.1.0 and based on the expression data. Expression levels are depicted by different colors on the scale. Green and red represent low and high expression levels, respectively.

The *OsMSL*s were clustered into groups I to V according to their expression characteristics in different tissues (Figure 8B). *OsMSL*s in groups I and II expressed at lower levels in all tissues, but members in group I displayed relatively higher expression in root tissue. For instance, although *OsMSL19* maintained very low expression levels in most tissues, its expression displayed a distinct enrichment in root. In contrast, the genes in group II, especially *OsMSL02*, *OsMSL08*, and *OsMSL12*, remained low in the various tissues. On the other hand, in *Arabidopsis*, the At5g63420 gene, the orthology of *OsMSL12*, is highly expressed specifically in the seeds, suggesting that also *OsMSL12* could show a dominant expression level in seeds rather than in any of these four tissues [19]. Contrary to the group I, most of the genes in group III expressed at higher levels in the stems or leaves and at lower levels in the roots. However, the transcription levels of *OsMSL34a* and *OsMSL34b* in group IV were high in all four tissues. Group V members also displayed tissue specific expression, but expressed at higher levels than group I and III. Besides, the genes in group V, especially *OsMSL16*, *OsMSL27*, *OsMSL28*, *OsMSL35*, and *OsMSL39*, expressed extremely highly in the leaves and sheaths, whereas their expression levels in the roots and stems were lower than those of genes in group I and III, respectively. In conclusion, *OsMSL*s displayed tissue expression specificity, indicating their potential roles in different mechanisms.

We also investigated the *OsMSL* expression profiles at different rice developmental stages including 7 (S1), 20 (S2), 40 (S3), 80 (S4), 100(S5), and 140(S6) days after sowing. As shown in Figure 9A, the numbers of genes with high- and extremely high expression levels are greater in S2 and S3 than in the other stages. In contrast, no genes expressed at high levels during S4, S5, or S6. The genes were subdivided into five groups according to their expression characteristics at different stages (Figure 9B). *OsMSL*s in group I, were at comparatively higher transcription levels during the S2 and S3 stages except for *OsMSL28*, which displayed high expression levels at S1 stage as well. In general, the expression levels of the genes in groups II and III were similar to those in group I, but their expression levels in S2 and S3 were lower than in group I. Notably, the group IV genes had lower expression levels than the other four clusters at almost all developmental stages. Contrary to groups II and III, high gene expression levels were observed mainly at S1 in group V. Besides, with the exception of *OsMSL35* and *OsMSL06*, all other genes in group V expressed at significantly lower levels by S2. In conclusion, *OsMSL*s play a potential role at early developmental stages.
