| Drought is a significant environmental constraint on grain production and crop yield,making droughttolerant crop varieties essential for food security.Hybrid breeding can help facilitate crop production by exploiting heterosis.Foxtail millet(Setaria italica L.),which is the main multigrain crop in the Shanxi Province,is also a model plant for stress tolerance studies on C4 crop species,and it is both barren-tolerant and drought-tolerant,has a small genome and short growth cycle.Zhangza19 is a hybrid variety with a high yield under drought stress and strong adaptability,making it widely planted in China.This variety is an important drought-tolerant foxtail millet germplasm resource that provides crucial information and genetic resources for improving drought tolerance.Exploring the drought resistance genes in Zhangza19 is of great significance for studying drought response mechanisms and heterosis.Therefore,it is essential to investigate drought-resistant heterosis genes in Zhangza19 for drought response mechanisms and heterosis research.In this study,we subjected the drought-resistant hybrid Zhangza19 and its maternal A2 and paternal DH2 to drought stress.Foxtail millet seedlings were used for physiological and biochemical measurements under drought stress conditions for 0,3,6,9,and 12 days.To identify drought-related heterotic genes in Zhangza19,we performed WGCNA and differential gene expression analyses based on the transcriptome data.Si MYBS3 was selected as a candidate gene for subsequent functional verification.The main findings are summarized as follows:1.The photosynthetic rate decreased with increasing drought stress in Zhangza19 and its parent plants.Stomatal closure was the primary limitation in photosynthesis under mild drought conditions.As the drought intensified,non-stomatal factors were the main limitations to photosynthesis in A2,and the observed decline in the photosynthetic rate may have been caused by both stomatal and non-stomatal factors in Zhangza19 and DH2.Furthermore,absorbed light energy was more efficiently directed toward photochemistry in Zhangza19 and DH2 by the increased fraction of open PSII reaction centers,and the electron transfer intensity was maintained at a higher level under drought stress.These results suggest that Zhangza19 and DH2 have better drought resistance than A2.In addition,PSI photoinhibition was not observed in Zhangza19 under drought stress,indicating that Zhangza19 exhibited stronger drought resistance than its parent DH2.2.Drought stress caused a decrease in relative water content and an increase in cellular O2˙-and H2O2 levels and MDA content increased under drought stress in Zhangza19 and its parents.As drought stress treatment duration increased,the activities of SOD,POD,and CAT were different among A2,DH2 and Zhangza19,which increased first and then decreased in A2,while increased and remained at a high level in DH2,and still showed upward trends in Zhangza19.This suggests that Zhangza19 and its parents have a high antioxidant capacity for scavenging free radicals.However,prolonged drought could disturb the balance between the scavenging and the production of ROS in foxtail millet,leading to ROS accumulation and membrane lipid peroxidation.Zhangza19 exhibited a stronger antioxidant activity than A2 and DH2.3.With the extension of the drought,the proline content in the leaves of both parents and Zhangza19 increased gradually,particularly in Zhangza19 and DH2,which had higher proline levels than A2.The soluble protein content in DH2 and Zhangza19 also increased gradually with the extension of the drought.In contrast,the soluble protein content in A2 initially increased and then decreased.DH2 and Zhangza19 exhibited stronger osmotic regulation under drought stress conditions.4.A total of 5597 differentially expressed genes(DEGs)were identified in Zhangza19 and its parents under drought stress conditions.Nineteen gene modules were identified using WGCNA,and three modules(Green,Grey60,and Yellow)were found to be closely related to drought resistance in the parents and the F1 generation.The 286 M-F-F1-type DEGs in Zhangza19,A2,and DH2 were related to heterosis.Among these,18 genes were upregulated only in the F1 generation following drought stress,.which were related to heterosis and involved in response to drought stress.5.One gene encoding MYBS3 transcription factor Seita.9G321800 and showing upregulated expression after drought stress was highly homologous with MYBS3 in maize,rice,and sorghum.The gene was named Si MYBS3.The transactivation assay showed that Si MYBS3 had transcriptional activation activity in yeast cells and subcellular localization demonstrated that it was localized in the nucleus and cytoplasm.Overexpression of Si MYBS3 in Arabidopsis thaliana resulted in increased drought tolerance,insensitivity to ABA,and earlier flowering.In conclusion,Zhangza19 and DH2 were shown to be drought-tolerant varieties,whereas A2 was sensitive to drought stress.Zhangza19 exhibited a stronger ability to resist drought stress than DH2.Si MYBS3 was identified as a drought-related heterotic gene through the transcriptome expression data analysis of Zhangza19 and its parents.Overexpression of Si MYBS3 in Arabidopsis resulted in improved drought tolerance and avoidance.These findings suggest that Si MYBS3 could be an important candidate gene for enhancing crop drought resistance.These results support the exploration of heterosis genes associated with drought resistance in different varieties. |
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