Drought significantly hinders crop production, exacerbated by climate change and increasing agricultural water demands. Rice, a staple food for over half the world's population, consumes 70% of China's agricultural water, highlighting the urgent need for drought-resistant cultivars. Upland rice, domesticated in rain-fed regions, grows in aerobic soils unlike lowland rice cultivated in flooded paddy fields. Genetic differences between upland and lowland rice exist regarding drought resistance and productivity, yet the underlying genetic mechanisms remain unclear. While linkage analyses and GWAS have been employed to study drought resistance, cloning genes involved in this complex trait regulated by multiple loci with minor effects is challenging. Cell walls, the plant's first line of defense, play crucial roles in abiotic stress resistance. Mutations affecting cell wall structure and composition can alter drought resistance. COBRA proteins regulate cellulose microfibril organization crucial for cell expansion, and rice contains 11 COBRA family members, some linked to cell wall formation. ERF transcription factors, acting as activators or repressors, are involved in drought response; ERF3 represses, while ERF71 activates, drought resistance. This study uses GWAS on upland and lowland rice to identify and characterize *DROT1*, a drought resistance gene encoding a COBRA family protein, and investigates its regulatory network involving ERF3 and ERF71. The study also explores the natural variation in the *DROT1* promoter and its potential use in rice breeding.

Previous research has explored drought resistance in rice using linkage analyses and GWAS, identifying several QTLs and genes associated with drought tolerance. However, cloning genes contributing to this complex trait, which involves many loci with small effects, has proven challenging. Studies have highlighted the importance of cell walls in plant stress responses, with mutations affecting cell wall composition and structure impacting drought resistance. The COBRA family of proteins, crucial for cellulose microfibril organization and cell expansion, has been implicated in cell wall development. ERF transcription factors have emerged as key regulators in the drought response, with some ERF genes acting as positive regulators and others as negative regulators of drought tolerance. Despite these findings, the specific interactions and regulatory pathways involved in drought resistance in rice, especially the interplay between cell wall structure and transcriptional regulation, require further investigation. This study bridges this gap by integrating GWAS, introgression line analyses, and transcriptomic profiling to identify a novel drought resistance gene and elucidate its regulatory mechanisms.

The study used an integrative approach involving GWAS, introgression line analysis, and transcriptomic profiling to identify and characterize the drought resistance gene *DROT1*. **GWAS:** A panel of 271 japonica rice accessions (59 upland, 212 lowland) was phenotyped under field drought conditions, evaluating Leaf Rolling Index (LRI) and Leaf Color Index (LCI) to calculate a Drought Resistance Index (DRI). GWAS using 2,070,333 SNPs identified seven loci associated with DRI and LRI. **Introgression Lines:** 88 introgression lines (ILs) with IRAT109 (upland) segments in the Yuefu (lowland) background were used to validate the GWAS findings. ILs showing increased relative shoot length under PEG treatment confirmed the role of candidate loci. **Candidate Gene Identification:** Local LD analysis and sequence comparisons narrowed down the *qDR10b* region to 25 candidate genes. Transcriptomic data highlighted three genes with higher expression in upland rice, with *Os10g0497700* (*DROT1*) showing strong dehydration-induced expression. Gene-based association analysis confirmed *DROT1* as a candidate gene. **Functional Validation:** *DROT1* knockout mutants (*drot1-1*, *drot1-zh11*) and overexpression lines (OEI, OEY) were generated using CRISPR/Cas9. These lines were phenotyped under drought conditions (pot, field, PEG treatment), evaluating survival rates, plant height, biomass, and grain yield to assess drought resistance. RNAi lines were also generated to further confirm the role of *DROT1* expression. **Expression Analysis:** Tissue-specific expression of *DROT1* was determined using GUS staining and laser capture microdissection (LCM). The effect of PEG stress and drought stress on *DROT1* expression was also examined. Promoter activity was analyzed using GUS reporter assays. Subcellular localization was determined by fusing *DROT1* with GFP and mCherry tags. **Cell Wall Analysis:** Cellulose, hemicellulose, and lignin contents were measured in leaf tissues using chemical methods and FTIR micro-spectroscopy. Cellulose crystallinity was analyzed by X-ray diffraction (XRD). TEM and SEM were used to examine the cell wall structure of sclerenchyma cells. **Transcriptional Regulation:** The interaction of *DROT1* with ERF3 and ERF71 was examined through gene expression analysis in transgenic lines, dual-luciferase reporter assays, Y1H assays, ChIP assays, and EMSA. Genetic interactions between *DROT1*, *ERF3*, and *ERF71* were also explored. **Haplotype Analysis:** Haplotype analysis of *DROT1*, *ERF3*, and *ERF71* in 743 rice accessions was performed to explore the natural variation and potential elite alleles of *DROT1*. Phylogenetic analysis and introgression analysis were conducted to investigate the origin and spread of *DROT1* haplotypes.

This study successfully identified and functionally characterized *DROT1*, a novel drought resistance gene in rice. Key findings include: 1. **Identification of *DROT1*:** GWAS and subsequent analyses identified *DROT1* (Os10g0497700), encoding a COBRA-like protein, as a major contributor to drought resistance in upland rice. 2. **Tissue-Specific Expression:** *DROT1* is specifically expressed in vascular bundles, suggesting a crucial role in water transport and retention under drought stress. 3. **Functional Role of *DROT1*:** Knockout mutants of *DROT1* showed significantly reduced drought resistance, while overexpression lines exhibited enhanced drought tolerance across various drought stress assays (pot, field, PEG treatment), improving survival rate, plant height, biomass and grain yield. RNAi lines further confirmed the direct relationship between *DROT1* expression and drought resistance. 4. **Mechanism of Drought Resistance:** *DROT1* improves drought resistance by regulating cell wall properties, specifically increasing cellulose content and maintaining cellulose crystallinity, particularly in vascular bundles, enhancing water transport and retention. This is supported by the observed increase in cellulose content and crystallinity index in overexpression lines under drought, and the opposite in knockout lines. Microscopic analysis confirmed structural differences in cell walls of overexpression lines compared to controls. 5. **Regulatory Network:** *DROT1* is directly regulated by the drought-responsive transcription factors ERF3 and ERF71. ERF3 acts as a repressor, while ERF71 functions as an activator of *DROT1* expression. This regulatory mechanism fine-tunes *DROT1* expression in response to drought stress, balancing growth and survival. Genetic interaction experiments supported the hypothesis that ERF3 and ERF71 function in the same pathway as *DROT1*. 6. **Natural Variation and Haplotype Analysis:** Haplotype analysis revealed that an elite haplotype (Hap3) of *DROT1*, with a C-to-T SNP in its promoter, is strongly associated with increased *DROT1* expression and drought resistance in upland rice. This haplotype appears to have originated in wild rice (*O. rufipogon*) and introgressed into cultivated rice. Combined haplotype analysis of *DROT1*, *ERF3*, and *ERF71* further indicated that specific combinations are enriched in upland rice and are associated with increased drought resistance.

This study provides substantial evidence that *DROT1*, a COBRA-like protein, plays a critical role in enhancing drought resistance in upland rice. The findings address the research question by identifying a key gene and elucidating its mechanisms of action, including its role in cell wall modification and its interaction with drought-responsive transcription factors. The identification of a functional SNP in the *DROT1* promoter, associated with enhanced drought resistance, has significant implications for rice breeding. The results highlight the importance of coordinated regulation between growth and stress response and suggest potential targets for improving drought tolerance in rice. This study successfully links genomic variation, gene expression regulation, and physiological traits, providing a comprehensive understanding of drought adaptation in upland rice. The identification of *DROT1* and its regulatory network provides valuable resources for breeding drought-resistant rice cultivars. The potential of exploiting the elite haplotype (Hap3) of *DROT1* is significant for breeding improved upland rice varieties.

This research identified *DROT1*, a gene encoding a COBRA-like protein, as a key determinant of drought resistance in upland rice. *DROT1* enhances drought resistance by modulating cell wall structure and is regulated by the transcription factors ERF3 and ERF71. A specific haplotype of *DROT1*, Hap3, is strongly associated with increased drought resistance in upland rice, possibly originating from wild rice. These findings provide valuable insights into drought adaptation mechanisms and offer potential targets for breeding drought-tolerant and high-yielding rice varieties. Future research could focus on further exploring the regulatory network of *DROT1*, investigating the epistatic interactions with other drought-related genes, and developing marker-assisted selection strategies for deploying Hap3 in breeding programs.

While the study provides strong evidence for the role of *DROT1* in drought resistance, several limitations should be noted. First, the study primarily focused on japonica rice, and further research is needed to determine whether *DROT1* plays a similar role in other rice subspecies. Second, while the study explored the regulatory network of *DROT1*, further research is needed to fully understand the complex interactions among *DROT1*, ERF3, and ERF71 and other potential regulatory elements. Finally, the study mainly employed controlled experiments; further research under diverse field conditions is needed to fully assess the generalizability of the findings.