SiMYBS3, Encoding a Setaria italica Heterosis-Related MYB Transcription Factor, Confers Drought Tolerance in Arabidopsis

**Background:** Drought is a major constraint on global grain production, and developing drought-tolerant crop varieties is essential for food security. Heterosis, where F1 hybrids outperform their parents, has been successfully used in agriculture, but its molecular basis—particularly for drought tolerance—remains poorly understood. Foxtail millet (Setaria italica) is a drought-tolerant C4 model plant with a small genome, and the hybrid Zhangza 19 is known for superior drought resistance. MYB transcription factors are the largest TF family in plants and are implicated in both heterosis and abiotic stress responses. This study aimed to identify drought-resistant heterosis-related genes from Zhangza 19 via transcriptome analysis and functionally validate a candidate MYB gene, SiMYBS3, in Arabidopsis.

**Methods:** Three-week-old seedlings of Zhangza 19 and its female parent A2 and male parent DH2 were subjected to natural drought stress (soil moisture 20%). Leaf samples were collected on day 9 for RNA extraction. RNA-seq was performed on the BGISEQ-500 platform, yielding 821,298,598 clean reads from 855,140,406 raw reads. DEGs were identified using DESeq2 (|log2FC| > 1, p < 0.05). WGCNA was used to construct co-expression networks and identify drought-associated modules. Heterosis-related genes were defined as those with FPKM > 1 in both parents without differential expression but FPKM > 50 in the F1, or those with 1 < log2FC < 2 in both parents and log2FC > 3 in the F1. Cis-acting elements in the 2 kb upstream promoter regions were analyzed using PlantCARE. SiMYBS3 was cloned and subcellular localization was assessed via GFP fusion in tobacco leaves. Transactivation activity was tested in yeast (pGBKT7 system). Overexpression lines (OX-1, -2, -3) in Arabidopsis Col-0 and the mybs3 T-DNA insertion mutant (SAIL_205_B08C1) were phenotyped under 200 mM mannitol, 1 µM ABA, and natural drought stress.

**Key Results:** A total of 5597 DEGs were identified across all groups: 2283 in the F1 (1212 up, 1071 down), 3323 in the male parent (1392 up, 1931 down), and 1808 in the female parent (1005 up, 803 down). WGCNA identified 19 modules; three (green, Grey60, yellow) were significantly associated with drought resistance, yielding 607 drought-related genes (module membership > 0.7, significance > 0.6). A total of 286 heterosis-related genes were identified (279 M-F-F1 type and 7 with differential multiples). Eighteen genes overlapped between the drought and heterosis sets. Among these, Seita.9G321800 (SiMYBS3) was highly expressed in Zhangza 19 after drought, contained ABRE and MBS drought-responsive elements, and showed high homology with MYBS3 in maize, rice, and sorghum. SiMYBS3 expression increased progressively under drought stress. The SiMYBS3 protein localized to the nucleus and cytoplasm and exhibited transcriptional activation activity in yeast. Under 200 mM mannitol and 1 µM ABA, germination rates on day 7 were significantly higher in overexpression lines than in WT, and lowest in the mybs3 mutant. Under natural drought stress, survival rates were 81.7–94.5% in overexpression lines, 33.6% in WT, and 11.9% in the mybs3 mutant. Overexpression lines also flowered earlier than WT and the mutant.

**Clinical Implications:** Although this is a plant biology study without direct clinical application, the findings have significant agricultural implications. SiMYBS3 is a drought-responsive MYB transcription factor that enhances drought tolerance, reduces ABA sensitivity, and promotes early flowering when overexpressed in Arabidopsis. The gene was identified from a drought-resistant foxtail millet hybrid and appears to act through an ABA-dependent signaling pathway (ABRE element in its promoter). SiMYBS3 represents a promising candidate for molecular breeding to improve drought resistance in foxtail millet, wheat, and other crops, potentially contributing to food security in arid regions.