Wild rice *GL12* synergistically improves grain length and salt tolerance in cultivated rice

Cultivated rice is a staple feeding over half the world’s population, and breeding for high yield with resilience to environmental stresses is a central but difficult goal due to negative correlations between yield and stress tolerance. Grain size and shape are major determinants of yield and quality and are controlled by numerous QTLs and pathways involving phytohormones, G-proteins, proteasomal degradation, kinases, and transcription factors. Several key grain-size genes in Asian rice (e.g., GW2, GL3.1, GS5, GW8, GS2/GL2, GS3, GL7/GW7, GS9) have been identified, with natural variation at GS3 and GL3.1 contributing to subspecies differences and regulation of cell cycle and hull cell proliferation. Wild rice (Oryza rufipogon), the progenitor of cultivated rice, harbors diverse alleles valuable for yield and abiotic stress tolerance, especially salt tolerance, many of which were lost during domestication. However, resistance traits in wild rice often link with inferior yield traits, and positive yield alleles from wild rice have been scarce. Building on prior identification of qGL12 from wild rice, this study shows that the wild allele GL12W can simultaneously increase grain length and salt tolerance across indica and japonica backgrounds. A G/T promoter variation mediates regulation by OsGIF1 and OsWRKY53, providing a mechanistic basis for coordinated enhancement of yield-related traits and salt tolerance.

Prior work has mapped and cloned numerous grain-size QTLs/genes in rice. GS3 encodes a transmembrane protein whose natural variation underlies grain-length differences between indica and japonica. GL3.1/OsPPKL1, a serine/threonine phosphatase, regulates grain length by affecting Cyclin-T1;3 and grain hull cell proliferation. Transcription factor OsMADS1, phosphatase GL3.1, and regulator GS9 also influence hull cell proliferation. GS2 encodes OsGRF4, promoting cell expansion and (to a lesser extent) proliferation; OsGRF4 is negatively regulated by miR396, and mutations disrupting miR396 targeting increase OsGRF4 expression and grain size. OsGRF4 physically interacts with coactivators OsGIF1/2/3. Despite extensive knowledge of these components, the genetic and molecular integration controlling grain size and possible connections to salt tolerance remain incompletely understood. Wild rice is recognized as a reservoir of beneficial alleles for yield and abiotic stress tolerance, yet positive yield-regulating alleles from wild rice have been rarely reported, and trade-offs with yield are common in resistant wild rice genotypes.

- Genetic materials: Constructed CSSLs using indica cultivar 9311 as recurrent parent and O. rufipogon as donor. Identified qGL12 across six environments; selected CSSL41 for fine mapping. Generated F2, F3, F4 populations for mapping and recombination analysis. - Fine mapping: Bulked-segregant analysis and recombinant analysis narrowed qGL12 to ~16 kb on chromosome 12 between markers WR12.4 and RM28586 containing three ORFs (LOC_Os12g39640, LOC_Os12g39650, LOC_Os12g39660). Sequence polymorphisms in promoters and coding regions were characterized. - Candidate validation: Developed NIL carrying qGL12 using CAPS marker and MAS. Assessed expression of candidate genes in young panicles by RT-qPCR. Created independent overexpression (OE) lines in 9311 background for each candidate ORF; generated CRISPR/Cas9 knockouts (KO) of LOC_Os12g39640 in NIL background; overexpressed GL12 alleles from wild rice and 9311 in japonica backgrounds (ZH11, Nipponbare). Phenotyped grain length (GL) and 1000-grain weight (TGW). - Subcellular localization: Transient expression of GL12-GFP in tobacco epidermal cells and rice protoplasts; confocal microscopy. - Transcriptomics: RNA-seq on young panicles of NIL and NIL-KO (|log2FC|>1, FDR<0.05) with GO and KEGG enrichment; validated expression of grain size-related genes (e.g., GS2/GRFs) by RT-qPCR. - Histology and microscopy: Cross-sections and SEM of spikelet hulls to quantify cell number and length and perimeter differences among 9311, NIL, and GL12-OE. - Promoter activity and transactivation: qRT-PCR of GL12 across panicle stages; transient promoter-LUC assays in Nicotiana benthamiana; transgenic NIP lines with combinations of promoters (wild vs 9311) and coding sequences to test promoter effects. - Protein-DNA and protein-protein interactions: Yeast two-hybrid (GL12–GIF1), luciferase complementation imaging in N. benthamiana. Yeast one-hybrid with truncated promoters (Pro-1 to Pro-4) to map binding by GIF1 and WRKY53; EMSA with biotin-labeled probes centered on G/T SNP at −1117. Dual-luciferase assays in protoplasts to test WRKY53 repression of GL12 promoters. - Functional genetics of regulators: Generated GIF1 OE and KO lines (ZH11 background) and assessed GL12 expression; KO of OsGIF1 in NIL background to test grain effects. Used WRKY53 OE and KO lines (LG11 background) to test effects on GL12 expression and salt tolerance. - Salt tolerance assays: 20-day-old seedlings treated with 150 mM NaCl for 10 days followed by 7-day recovery; scored survival, fresh and dry weights; examined expression of salt-response genes (NACs, NCED3) under salt. - Population genetics: Analyzed nucleotide diversity (π) in 100-kb windows around GL12 in 56 indica, 51 japonica, 52 wild rice; mined 3K Rice Genomes and Rice Super Pan-genome datasets for promoter G/T SNP distribution; compared grain length between Hap-G and Hap-T in indica; constructed promoter haplotype networks. - General experimental details: Plant growth at Beijing and Sanya field sites; standard transformation vectors and constructs; qRT-PCR with OsACTIN control; statistical analyses as indicated in figure legends.

- qGL12 was mapped to a ~16-kb interval on Chr. 12 harboring three ORFs; LOC_Os12g39640 (a MYB transcription factor) was identified as the causal gene and named GL12. - NIL carrying the wild rice allele increased grain length (GL) and 1000-grain weight (TGW) by 8.1% and 12.9%, respectively, relative to 9311. Overexpression of LOC_Os12g39640 from wild rice in 9311 increased GL by 5.1%, while LOC_Os12g39650-OE increased GL by 2.2%. KO of LOC_Os12g39640 in the NIL reduced GL and TGW; OE of the 9311 allele did not increase GL in 9311. - In japonica backgrounds, GL12W-OE lines (ZH11, Nipponbare) significantly increased GL and TGW, whereas GL12(9311)-OE did not, indicating a stable positive effect of GL12W across genetic backgrounds. - Cellular basis: GL12 localizes to the nucleus. GL12W reprograms gene expression (8079 DEGs; 4615 up, 3464 down). Grain-size genes including GS2/GRF4 and GRF3/GRF6 were upregulated. Histology/SEM showed increased cell length and reduced longitudinal cell number in spikelet hulls of NIL and GL12-OE, indicating enhanced cell expansion drives longer grains. Cell cycle genes (CYCD4, CYCD7, CAK1, CYCA2.1, CYCB2.2) were reduced, while expansins (OsEXPB3, OsEXPB4, OsEXPA10) were elevated. - Promoter regulation: GL12W promoter is more active than GL12(9311) in transient assays; under native GL12W promoter, transgenic lines showed greater GL increases than under the 9311 promoter. A single G/T SNP at −1117 in the GL12 promoter determines binding of regulators. - GIF1 interaction: GL12 (both alleles) physically interacts with OsGIF1. GIF1 overexpression elevates GL12 expression. Yeast one-hybrid and EMSA demonstrated GIF1 binds only the GL12W promoter truncation (ProW-2) containing the G allele at −1117, with stronger binding than to the 9311 promoter (T). KO of GIF1 in NIL decreased grain length. - Salt tolerance: GL12W enhances salt tolerance. After 150 mM NaCl for 10 days plus 7-day recovery, survival was ~80% for NIL, ~60% for GL12W-OE, and ≤30% for 9311; NIL-KO was hypersensitive. In Nipponbare background, GL12W-OE also improved salt tolerance, while GL12(9311)-OE did not differ from control. Under salt, NIL showed stronger induction of salt-related genes (e.g., NACs, NCED3). GIF1-OE lines were more salt-tolerant, GIF1-KO lines were sensitive; GS2-NIL also showed increased salt tolerance. - WRKY53 repression: WRKY53 negatively regulates GL12 expression. WRKY53-KO increased GL12 expression and salt tolerance; WRKY53-OE decreased GL12. WRKY53 binds the same promoter region (Pro-2) with the G/T site; dual-luc assays showed WRKY53 represses the GL12(9311) promoter more strongly than GL12W, indicating the SNP modulates WRKY53 repression. - Evolution and domestication: Nucleotide diversity at GL12 is reduced in indica relative to wild rice, suggesting a selective sweep in indica. The promoter −1117 G/T variant is unbalanced between subspecies (indica predominantly haplotype-T; japonica predominantly haplotype-G). In indica accessions, Hap-G (n=15) exhibited significantly longer grains than Hap-T (n=894; p≈0.0104). Haplotype networks support divergence with wild rice distributed across both haplotypes and Aus tending toward indica type. - Overall model: The GL12W allele synergistically improves grain length and salt tolerance via promoter-mediated recruitment of GIF1 (activation in young panicles) and WRKY53 (repression under salt), influencing downstream grain size genes (e.g., GS2) and salt-response genes (e.g., NACs, NCED3).

The study addresses the long-standing challenge of improving yield-related traits alongside abiotic stress tolerance by uncovering a wild rice allele, GL12W, that enhances grain length and salt tolerance concurrently. Mechanistically, a single promoter G/T SNP at −1117 enables differential transcriptional regulation: GIF1 binds and activates GL12W in young panicles, promoting expression of grain size regulators such as GS2/GRFs and expansins to enhance cell expansion and grain length; under salt stress, WRKY53 preferentially binds and represses GL12 (more strongly for the 9311 promoter), modulating salt-responsive pathways including NACs and NCED3. This dual regulation provides a dynamic balance that supports both improved grain traits and enhanced salt tolerance. Population genetic analyses indicate that selection at GL12 occurred primarily in indica, with the T haplotype prevalent and associated with shorter grains compared to the G haplotype, contributing to subspecies divergence. While GL12W improves grain length and salt tolerance, its impact on overall yield is tempered by reduced grain number per panicle, underscoring the complexity of yield architecture and trade-offs. Nevertheless, integrating GL12W with complementary alleles could mitigate trade-offs and inform breeding strategies that decouple negative correlations between yield and stress tolerance.

This work identifies GL12 (LOC_Os12g39640) from wild rice as a MYB transcription factor whose wild allele (GL12W) simultaneously increases grain length and salt tolerance across indica and japonica backgrounds. A key promoter G/T SNP at −1117 governs binding by GIF1 and WRKY53, enabling context-dependent activation in panicles and repression under salt stress. GL12W reprograms gene expression to favor cell expansion (upregulating expansins, GS2/GRFs) while modulating salt-response genes (NACs, NCED3). Population analyses reveal subspecies divergence at GL12, with evidence of selection in indica and haplotype-specific grain-length differences. Although GL12W does not by itself increase overall yield due to reduced grain number per panicle, it provides a valuable genetic resource and mechanistic insight for designing breeding strategies that combine yield and stress resilience. Future research should dissect the detailed biochemical and regulatory networks of the GIF1–GL12–GS2 module, explore stacking with alleles that enhance grain number, and evaluate field performance across diverse environments.

- Despite improved grain length and salt tolerance, GL12W/NIL lines exhibited decreased grain number per panicle and did not significantly increase overall yield under normal or salt stress conditions. - The precise molecular mechanism by which GIF1 engages the GL12 promoter (including potential DNA-binding facilitation) and the full regulatory network connecting GL12 to GS2/GRFs and salt-response pathways require further elucidation. - Salt tolerance assays were conducted under controlled conditions (150 mM NaCl, seedling stage); broader multi-environment, multi-developmental stage field validations are needed to confirm agronomic utility. - The haplotype–phenotype association for the promoter G/T SNP in indica relies on available datasets with an imbalanced number of Hap-G vs. Hap-T accessions, which may affect statistical power.