Natural variation of *DROT1* confers drought adaptation in upland rice

Drought increasingly limits rice production as water resources tighten, highlighting the need for cultivars that require less water and tolerate drought. Upland rice, adapted to aerobic conditions, shows superior drought resistance to lowland rice, yet the genetic basis of this adaptation remains unclear. Prior QTL mapping and GWAS efforts indicate drought tolerance is a complex trait controlled by many small-effect loci. Cell wall composition, particularly cellulose deposition regulated by COBRA-like proteins, and ERF-family transcription factors have been implicated in abiotic stress responses, but how these pathways integrate to control drought resistance and growth trade-offs is unresolved. This study aims to identify genetic loci underlying drought resistance differences between upland and lowland japonica rice and to functionally characterize the causal gene(s), their regulatory mechanisms, and natural variation contributing to adaptation.

The authors review evidence that cell walls are central to abiotic stress responses; mutations affecting cell wall composition can alter drought tolerance in cereals. COBRA family proteins guide cellulose microfibril organization; rice BC1 loss-of-function causes brittle culms, underscoring conserved COBRA roles in cell wall formation. Several ERF transcription factors modulate drought responses: ERF3 acts as a repressor reducing drought tolerance, while others such as OsLG3 (ERF62) and ERF71 act positively, with ERF71 linked to cell wall lignification. However, the role of COBRA proteins in drought response and their transcriptional regulation by ERFs in balancing growth and stress resistance had not been established.

Study design and populations: A GWAS panel of 271 japonica accessions (59 upland, 212 lowland) with 2,070,333 SNPs (MAF ≥ 0.05) was phenotyped under field drought in a rain-proof shed. Drought phenotypes were quantified using Leaf Rolling Index (LRI) and Leaf Color Index (LCI), combined into a Drought Resistance Index (DRI) via BLUP, PCA and membership function weighting. Population structure was assessed by PCA. GWAS was performed using a Compressed Mixed Linear Model (GAPIT v2), with significance threshold P < 1e-4 (permutation-based), defining loci with ≥2 consecutive significant SNPs; local LD decay delineated candidate intervals. Introgression lines and PEG assays: 270 IRAT109 (upland) × Yuefu (lowland) ILs were developed; 88 ILs with ≤3 donor segments were screened under 15% PEG6000 for 10 days to assess relative shoot length; key ILs (e.g., IL349 carrying qDR10b; IL10 carrying qDR4a/qDR4b) were prioritized. Gene identification and expression: Within qDR10b, sequence comparison (IL349 vs Yuefu) identified SNPs; transcriptomics (two upland, two lowland) highlighted differentially expressed candidates. qRT-PCR validated dehydration induction. Gene-based association pinpointed significant SNPs in Os10g0497700 (DROT1), including promoter s18975900 and an exon SNP. Transgenics: CRISPR/Cas9 knockouts: drot1-1 in IL349 (143-bp deletion), drot1-zh11, drot1-n (Nipponbare), and erf71 mutants; erf71/drot1-n double mutant via crossing. Overexpression lines OEI (IRAT109 CDS) and OEY (Yuefu CDS) in Nipponbare; DROT1 RNAi lines in Nipponbare. ERF3 and ERF71 overexpression lines used for regulatory tests and genetic interaction (OE-ERF3/OEI). Drought evaluations: Pot drought (15 days without water, then rewatering) and survival scoring; field trials in paddy vs severe drought (no irrigation; ~90 days) to measure plant height, aboveground biomass, and relative values (drought/paddy). Moderate drought yield trials used rain-proof shed with controlled irrigation (eight waterings), random plots (1.9 m squares), plot biomass and yield per hectare, and per-plant yield from >30 plants. Expression pattern and localization: DROT1 promoter::GUS (IRAT109) for tissue specificity; laser capture microdissection of vascular bundles vs parenchyma to validate by qRT-PCR (CesA4 marker). Promoter activity assays using GUS reporters driven by ProY (Yuefu), ProI (IRAT109), and ProT (Yuefu with s18975900 C→T) under dehydration. Subcellular localization: proDROT1::DROT1-GFP/mCherry in rice roots; plasmolysis with 1 M sorbitol; confocal imaging. Cell wall analyses: Chemical quantification of cellulose (glucose), hemicellulose (xylose+arabinose), and lignin (acetyl bromide method) from leaves of OE and knockout lines under paddy and drought fields. FTIR micro-spectroscopic imaging with fast NNLS fitting for in situ semi-quantification in lateral veins and segmented vascular bundles. Cell wall ultrastructure by TEM/SEM to measure sclerenchyma wall thickness. X-ray diffraction to compute cellulose relative crystallinity index (RCI) under paddy vs drought. Regulatory assays: Dual-luciferase transient expression in rice protoplasts to test ERF3/ERF71 effects on DROT1 promoters (IRAT109 and Yuefu). Yeast one-hybrid binding to promoters. ChIP-qPCR using 35S:ERF3-Flag and 35S:ERF71-Flag to enrich promoter fragments containing GCC boxes (F1–F4). EMSA with MBP-ERF3 and MBP-ERF71(1–200 aa) on P1–P3 GCC-box probes and mutants to confirm direct binding and competition. Natural variation and evolution: Haplotype analysis of DROT1 across 743 accessions (489 cultivated, 254 wild) using 23 SNPs spanning ~3.6 kb; maximum-likelihood phylogeny and minimum-spanning network; geographic distribution mapping for Hap3/Hap8; combined haplotype analysis of DROT1–ERF3–ERF71 in 402 cultivars (12 major combined haplotypes). Introgression analysis of the DROT1 Hap3 region (18.1–20.5 Mb on chr10) between indica and tropical japonica using subgroup-specific SNP frequencies.

• GWAS in 271 japonica accessions identified seven loci for DRI and seven for LRI; qDR10b overlapped both traits. IL validations under PEG supported qDR10b (IL349) and qDR4a/qDR4b (IL10) effects. • Within qDR10b, Os10g0497700 (renamed DROT1) emerged as the top candidate: higher expression in upland vs lowland, strong dehydration induction, and two significant SNPs associated with DRI including promoter s18975900. • Promoter variation: s18975900 (C→T) in the DROT1 promoter enhances transcription; GUS assays showed ProI > ProY, and ProT (Yuefu promoter with T) > ProY under dehydration, linking the T allele to higher expression. • Function: DROT1 positively regulates drought resistance. CRISPR knockout drot1-1 (IL349 background) showed reduced survival and growth under drought; OEI/OEY overexpression increased survival and growth. Under severe drought field (~90 days), drot1-1 had significantly reduced relative plant height and biomass vs IL349; OE lines had increased relative height and biomass vs NT. Under moderate drought, drot1-1 reduced plot biomass and yield per hectare; OE lines maintained or improved biomass and, for OEI, increased per-plant yield. • Physiology: OE lines exhibited lower water loss rates; knockouts lost water faster. • Tissue expression and localization: DROT1 is specifically expressed in vascular bundles and localized predominantly to the cell wall. • Cell wall impacts under drought: Overexpression increased cellulose content (no change under paddy), with hemi-cellulose increased in OE and knockout under drought; lignin increased in OEY only. FTIR imaging and NNLS fitting showed increased cellulose (and lignin in OE) in vascular bundles of OE lines; drot1-1 had reduced cellulose in vascular bundles under drought. Sclerenchyma walls were thicker in OE leaves and stems. XRD revealed higher cellulose crystallinity (RCI) in OE lines under both paddy and drought; drot1-1 had reduced RCI vs IL349 under drought. • Regulation by ERFs: ERF3 overexpression reduced DROT1 expression and drought survival; ERF71 overexpression increased DROT1 expression. Both ERF3 and ERF71 are more expressed in vascular bundles. Dual-luciferase assays showed ERF3 represses, ERF71 activates DROT1 promoters (stronger activation on IRAT109 promoter). Y1H, ChIP-qPCR, and EMSA confirmed direct binding of both ERFs to GCC boxes (P1–P3) in the DROT1 promoter, including the region harboring s18975900. • Genetic interactions: Overexpressing DROT1 suppressed the drought-sensitive phenotype of ERF3 overexpression (OE-ERF3/OEI ≈ OEI survival). The erf71/drot1-n double mutant resembled drot1-n in drought sensitivity, indicating DROT1 acts downstream in the same pathway. • Natural variation: Eight DROT1 haplotypes were identified; Hap3 uniquely carries the T at s18975900, is enriched in upland rice, and associates with higher DROT1 expression and survival under drought relative to other haplotypes when ERF3/ERF71 backgrounds are controlled. Combined haplotypes (DROT1–ERF3–ERF71) showed upland-specific combinations (e.g., DEE1-1-6, DEE3-1-4). Evolutionary and geographic analyses suggest Hap3 originated from wild rice in Southeast Asia, entered indica, and later introgressed into tropical japonica, contributing to upland adaptation.

This study links a cell-wall structural gene, DROT1 (a COBRA-like protein), to field-relevant drought resistance in rice, explaining part of the phenotypic divergence between upland and lowland ecotypes. DROT1 is induced by drought and specifically expressed in vascular bundles, where it enhances cellulose deposition and maintains cellulose crystallinity, reinforcing vascular and sclerenchyma walls to sustain water transport and prevent tissue collapse under water deficit. The promoter SNP s18975900 (C→T) increases DROT1 expression and underlies superior drought performance of upland accessions. Mechanistically, DROT1 integrates into an ERF-mediated regulatory module: ERF3 represses and ERF71 activates DROT1 by binding GCC motifs in its promoter. The balance between ERF3 and ERF71 expression across drought phases likely fine-tunes DROT1 levels to negotiate growth–stress trade-offs. Genetic interaction tests place DROT1 downstream of ERF71 and epistatic to ERF3 overexpression effects. Population analyses reveal an elite DROT1 Hap3 that is enriched in upland rice and likely introgressed from indica-type wild rice into indica and subsequently into tropical japonica, contributing to aerobic drought adaptation. These findings provide causal molecular variation and a regulatory framework for breeding drought-resilient upland-type traits into elite cultivars.

An integrative GWAS–genetics–omics approach identified DROT1 (Os10g0497700), a vascular bundle-expressed COBRA-like protein, as a positive regulator of drought resistance in rice. DROT1 enhances cellulose content and preserves cellulose crystallinity under drought, strengthening cell walls and improving growth and survival under water deficit. DROT1 expression is directly repressed by ERF3 and activated by ERF71, enabling dynamic regulation during drought responses. A promoter C→T SNP (s18975900) defines Hap3, an elite haplotype enriched in upland rice, associated with higher DROT1 expression and drought tolerance, and likely originating from wild rice before introgression into tropical japonica. These insights provide targets and haplotypes for marker-assisted selection and transgenic strategies to improve drought adaptation in rice. Future work should quantify how DROT1 expression levels scale with comprehensive drought resistance metrics across diverse germplasm, dissect interactions with broader cell wall biosynthesis networks, and resolve the detailed evolutionary trajectory and agronomic impacts of Hap3 during domestication and breeding.

The study acknowledges challenges in fully quantifying drought resistance across germplasm: survival rate under severe stress captures only part of drought performance, while traits like relative biomass and plant height under moderate drought also matter. Knowledge gaps remain in the detailed evolutionary history of upland rice and the precise origin and spread of DROT1 Hap3. As drought resistance is highly influenced by background genetics and environment, minor-effect loci and genotype-by-environment interactions may modulate DROT1’s impact.