*2.6. Quantitative Real-Time PCR Analysis of Rice Trihelix Genes in Responses to Different Treatments under Normal and Dark Conditions*

A prediction of the *cis*-elements of the *OsMSL*s suggested that they may participate in rice dehydration stress tolerance and light-mediated signaling pathways. To verify this hypothesis, we subjected rice seedlings to ABA, hydrogen peroxide, drought, and high salt then carefully selected 12 genes expressing positively in the leaves (Figures 10 and 11). On the whole, although trihelix genes were induced by multiple treatments, their expression levels under one treatment were much higher than other treatments. For instance, six *OsMSL*s (*OsMSL25a*, *OsMSL25b*, *OsMSL28*, *OsMSL34a*, *OsMSL34b*, and *OsMSL35*) were induced by multiple treatments, but the transcript levels significantly increased under hydrogen peroxide treatment compared to other treatments. Several genes were induced after being repressed, such as *OsMSL41*, which remained downregulated up until 12 h of ABA treatment. Some trihelix genes showed high transcript levels under multiple treatments. For example, *OsMSL39* simultaneously responded to all treatments. *OsMSL28* was significantly induced by three tested treatments except ABA treatment. In conclusion, all 12 genes are induced by various abiotic stress or stress signaling molecules, but the expression levels are different and there was no significant correlation between gene expression and its classification.

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 17 of 29

**Figure 9.** Expression of the rice trihelix gene family at different developmental stages (S1: 7 days after sowing; S2: 20 days after sowing; S3: 40 days after sowing; S4: 80 days after sowing; S5: 100 days after sowing; S6: 140 days after sowing). (**A**) Numbers of expressed genes in various developmental stages. Expression data of the rice trihelix gene family genes 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 trihelix genes in various rice developmental stages. Heatmaps were created in HemI v.1.0 and based on the expression data. Expression levels are depicted by different colors on the scale. Blue and red represent low and high expression levels, respectively. **Figure 9.** Expression of the rice trihelix gene family at different developmental stages (S1: 7 days after sowing; S2: 20 days after sowing; S3: 40 days after sowing; S4: 80 days after sowing; S5: 100 days after sowing; S6: 140 days after sowing). (**A**) Numbers of expressed genes in various developmental stages. Expression data of the rice trihelix gene family genes 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 trihelix genes in various rice developmental stages. Heatmaps were created in HemI v.1.0 and based on the expression data. Expression levels are depicted by different colors on the scale. Blue and red represent low and high expression levels, respectively.

A prediction of the *cis*-elements of the *OsMSL*s suggested that they may participate in rice

*2.6. Quantitative Real-Time PCR Analysis of Rice Trihelix Genes in Responses to Different Treatments*

*under Normal and Dark Conditions*

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 19 of 29

**Figure 10.** Expression profiles of 12 selected *OsMSLs* in response to (**A**) ABA (abscisic acid), (**B**) hydrogen peroxide treatments. Data were normalized to the β-actin gene. Vertical bars indicate **Figure 10.** Expression profiles of 12 selected *OsMSLs* in response to (**A**) ABA (abscisic acid), (**B**) hydrogen peroxide treatments. Data were normalized to the β-actin gene. Vertical bars indicate standard deviations. Asterisks indicate corresponding genes significantly upregulated or downregulated compared with the control. (\* *p* < 0.05; \*\* *p* < 0.01; Student's *t*-test).

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 21 of 29

**Figure 11. Figure 11.** Expression profiles of 12 selected OsMSLs in response to ( Expression profiles of 12 selected OsMSLs in response to (**A**) drought, **A**) drought, ( (**B**) high **B**) high salt stress, salt stress, treatments. Data were normalized to the β-actin gene. Vertical bars indicate standard deviations. Asterisks indicate corresponding genes significantly upregulated or downregulated compared with the control. (\* *p* < 0.05; \*\* *p* < 0.01; Student's *t*-test).

To determine whether light influences trihelix genes expression, the expression levels of 12 *OsMSL*s from each treatment group under light and dark conditions were investigated (Figures 10 and 11). Overall, some *OsMSL*s were induced under normal conditions. For example, at 0 h, *OsMSL01*, *OsMSL25b*, *OsMSL33*, *OsMSL39*, and *OsMSL41* expression levels significantly differed between the two conditions. Several genes began to be light-regulated expressed under different treatments. For instance, after ABA exposure, the expression levels of *OsMSL16*, *OsMSL25a*, *OsMSL25b*, *OsMSL28*, *OsMSL34a*, and *OsMSL39* at different time points under normal conditions were significantly higher than those at the same intervals under dark conditions. In contrast, some genes were repressed by light at different points, such as the expression levels of *OsMSL41* at 2 h under ABA treatment, *OsMSL28* at 6 h under hydrogen peroxide treatment, *OsMSL34b* at 12 h under drought stress and *OsMSL16* at 12 h under high salty stress were higher in the dark than under normal conditions. Therefore, the relationship between trihelix genes and light is complex and trihelix gene expression is not directly regulated by light but may be controlled by multiple regulatory mechanisms.
