A point mutation in *VIG1* boosts development and chilling tolerance in rice

Rice (Oryza sativa L.) feeds over half of the world’s population, but rising labor shortages, aging populations, and increasing costs challenge production. Paddy direct seeding reduces labor and shortens the life cycle, yet its wider adoption is limited by low seedling vigor (SV), high sensitivity to chilling (CT) at multiple stages, and reduced grain yield due to management constraints. SV—rapid early shoot and root growth—is controlled by many QTLs; identified loci such as SOR1 (modulating Aux/IAA stability) and OsSWEET3a (a GA and glucose transporter) affect root and shoot growth. Although several regulators coordinate SV and CT (e.g., OsICE1–OsMAPK3–OsTPP1, OsMADS57, OsCYP20-2–SLR1–OsFSD, OsGRF6–SLR1–OsGA20x1), impacts on grain yield are less explored. Grain yield is mainly determined by grain number per panicle (GNP), tiller number, and grain weight; major QTLs such as GnlA/OsCKX2, DEP1, and IPA1 (OsSPL14 targeted by OsmiR156) influence GNP. Notably, IPA1 integrates SV, CT, and GNP but exemplifies a trade-off: loss of IPA1 enhances SV but reduces CT and GNP. Therefore, identifying regulators that can simultaneously improve SV, CT, and GNP without detrimental trade-offs is of great breeding interest. This study identifies vig1a, a mutant with enhanced SV, CT, and GNP caused by an OsbZIP01 mutation, and reveals cooperative functions with OsbZIP18 that coordinate cell expansion/proliferation, CBF/DREB-mediated chilling responses, and GNP control.

Prior work has mapped numerous QTLs for seedling traits influencing SV, including loci affecting root and shoot growth such as SOR1 (regulating Aux/IAA protein stability) and OsSWEET3a (a dual GA and glucose transporter supporting young shoot development). Multiple genes modulate CT across development; OsICE1 phosphorylated by OsMAPK3 activates OsTPP1 to promote shoot growth and CT, OsMADS57 coordinates cold adaptation and growth, OsCYP20-2 integrates defense and elongation with effects on CT, and OsGRF6 interacts with SLR1 to tune OsGA20x1 expression in a temperature-dependent manner. Yield, particularly GNP shaped by primary and secondary branches, is governed by loci including GnlA/OsCKX2 (cytokinin degradation), DEP1 (meristem activity), and IPA1/OsSPL14 (plant architecture regulated by OsmiR156). While many regulators affect SV, CT, or GNP individually, few balance all three. IPA1 is a notable integrator but imposes a trade-off by attenuating SV while promoting CT and GNP. OsbZIP01 (also known as OsRE1/OUR1/OsbZIP1) has been implicated in heading date via repression of Ehd1, root development via auxin signaling repression, photomorphogenesis with splice-specific functions, and nutrient-use efficiency under low N/P, suggesting broader developmental roles that could intersect SV, CT, and yield.

- Mutant identification and mapping: Screened NaN3-mutagenized M2 KY131 (japonica) populations for elongated seedling traits, identifying vig1a and vig1b. Map-based cloning used a cross to KD8 and bulked segregant analysis to initially map VIG1 to chromosome 1, refined to a 90-kb interval between M5 and M6, and resequencing identified a G→A mutation (R121H) in OsbZIP01 (LOC_Os01g07880) in vig1a; vig1b carried a 14-bp deletion causing frameshift. - Genetic validation: Complementation with a 3.52-kb WT OsbZIP01 genomic fragment (promoter + CDS + downstream) rescued vig1a and vig1b phenotypes; introducing vig1a genomic fragment into vig1b yielded vig1a-like phenotypes (dominance). - Genome editing: CRISPR/Cas9 edits in OsbZIP01 generated frameshift (VIG1-NK) and C-terminal leucine zipper deletions/frameshifts (VIG1-CK) in KY131 and KD8 to test regional function; overexpression (OE) of VIG1 under ubiquitin promoter assessed dosage effects. - Homolog analysis and interaction assays: Identified OsbZIP18 and OsbZIP48 as close paralogs. Protein–protein interactions tested via yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC) in rice protoplasts, and in vitro GST pull-down; mapped interaction domain of VIG1 to aa 138–172 (leucine zipper). Assessed interactions for mutant proteins (vig1a maintains, vig1b loses interactions). - Functional genetics of paralogs: Generated OsbZIP18 and OsbZIP48 knockout lines (multiple CRISPR targets causing frameshifts/truncations) to test single-mutant phenotypes; constructed double/triple mutants (e.g., vig1b OsbZIP18-CK) by crossing. 5′ RACE verified alternative splicing (OsbZIP18-A1 isoform) and VIG1 splice variants in edited lines. - Domain-specific mutagenesis: Engineered basic-region variants of VIG1 (R121G, R128P, or basic region deletion; vig1m1/m2/m3) and transformed into vig1b background; edited the leucine zipper domain within vig1a background to disrupt VIG1–OsbZIP18 interaction. - Phenotyping: Quantified seedling shoot and root lengths (1–7 DAG), chilling tolerance (4 °C treatments with recovery; survival rates), heading date, plant height, grain number per panicle (GNP), and grain yield per plant (GYP). Cytology: SEM-based coleoptile cell measurements. - Transcriptomics and target validation: RNA-seq on 7-day-old shoots (WT, vig1a, vig1b), DEGs by DESeq (|FC| ≥ 2, p < 0.05); RT-qPCR validation of selected genes (cell expansion/division and OsDREB/CBF). Promoter analyses for A/G/C-box motifs; Dual-luciferase transient assays (LUC) in rice protoplasts for transactivation/repression and promoter binding by VIG1/OsbZIP18; yeast one-hybrid (Y1H) and EMSA for direct binding. Assessed Ehd1 and OsPID expression and genetic tests: Ehd1 knockout in vig1b, OsbZIP18 overexpression in vig1a. - Subcellular localization: EYFP-tagged proteins in rice protoplasts; OsbZIP52-mRFP as nuclear marker. - Breeding test: Developed an indica NIL (BC4F3) by backcrossing vig1a into ZF802 with background selection; evaluated SV, CT, and yield traits in field/phytotron conditions. - Growth conditions: Controlled light/temperature phytotron for seedling and CT assays; field trials in Beijing (summer) and Hainan (winter).

- Two allelic mutants of OsbZIP01 (VIG1) were identified: vig1a (G→A in exon 2 causing R121H in the basic region) and vig1b (14-bp deletion in exon 2 causing frameshift). Both enhance seedling vigor and chilling tolerance; vig1a additionally increases grain number per panicle (GNP), while vig1b decreases GNP. - vig1a seedlings showed markedly elongated shoots and roots; SEM indicated coleoptile inner cell length 1.6× WT with unchanged cell width; coleoptile length 1.8× WT. Under 4 °C for 4 days with recovery, survival was 74.2% in vig1a vs 0.83% in WT. Heading date was delayed (85 vs 71 days), and plant height and GNP increased. - Complementation with WT OsbZIP01 rescued vig1a and vig1b phenotypes; introducing vig1a allele into vig1b conferred the vig1a phenotype, showing vig1a is dominant over vig1b. - Regional function within VIG1: CRISPR edits at the C-terminal leucine zipper (VIG1-CK) produced enhanced SV and CT but reduced GNP (phenocopying vig1b), whereas frameshift near the N-terminus (VIG1-NK) had SV/CT comparable to WT but reduced GNP. Overexpression of VIG1 decreased shoot length and CT, increased root length, delayed flowering, and increased plant height and GNP. - VIG1 interacts with OsbZIP18 and OsbZIP48; interaction domain mapped to aa 138–172 (leucine zipper). vig1a protein retains interactions; vig1b loses them. - Single knockouts of OsbZIP18 or OsbZIP48 showed no obvious phenotypic alterations (SV, CT, GNP), suggesting redundancy. However, the double mutant vig1b OsbZIP18-CK phenocopied vig1a in SV, CT, and GNP (though with higher mortality and delayed flowering), indicating cooperative function between VIG1 and OsbZIP18. An alternatively spliced isoform OsbZIP18-A1 was identified and interacts with vig1a. - Basic-region mutations in VIG1 (R121G, R128P, or deletion) each conferred the vig1a phenotype when introduced into vig1b, demonstrating that altering the VIG1 basic region is sufficient to generate the vig1a-like state. - Disrupting the leucine zipper interaction domain in the vig1a background converted the phenotype to vig1b-like, reducing CT and aligning SV/GNP with vig1b, establishing that vig1a’s phenotype depends on intact VIG1–OsbZIP18 interaction. - Transcriptomics: 1268 DEGs in vig1a (892 up, 376 down) and 595 in vig1b (435 up, 160 down); 338 co-regulated. Genes linked to cell expansion/division (e.g., EXPB4, EXLA2, CYCD1-2) and cold tolerance (OsDREB1A/B) were more strongly upregulated in vig1a than vig1b, aligning with greater SV and CT. - Target regulation: VIG1 directly binds and represses EXPB4 and OsPID promoters; OsbZIP18 binds and represses EXLA2, CYCD1-2, OsDREB1A/B, and OsPID. Both proteins function as repressors in LUC assays; binding confirmed by Y1H and EMSA. - Ehd1 expression increased in VIG1-NK, vig1b, and VIG1-CK (consistent with reduced GNP) and decreased in VIG1-OE (consistent with delayed heading and increased GNP). Knocking out Ehd1 in vig1b delayed flowering by 15–16 days and increased plant height, GNP, and grain yield per plant without altering tiller number or seed setting rate. In vig1a, Ehd1 expression was unchanged, and vig1a still repressed the Ehd1 promoter in LUC assays; instead, OsPID expression was uniquely elevated in vig1a panicles, consistent with increased GNP. - Breeding relevance: An indica NIL-vig1a (in ZF802 background) exhibited significantly increased shoot/root lengths, higher CT survival after chilling, and improved plant height, GNP, and grain yield per plant, showing transferability across subspecies.

The study addresses how to simultaneously enhance seedling vigor, chilling tolerance, and grain yield in rice—key limitations for paddy direct seeding—by uncovering a cooperative module comprising VIG1 (OsbZIP01) and OsbZIP18. A specific basic-region point mutation (R121H) in VIG1 (vig1a) strengthens SV and CT and increases GNP, while a frameshift (vig1b) improves SV and CT but reduces GNP. Genetic interaction assays demonstrate that VIG1’s leucine zipper-mediated interaction with OsbZIP18 is essential for the vig1a phenotype; disrupting this interaction reverts vig1a to vig1b-like traits. Mechanistically, VIG1 and OsbZIP18 act as transcriptional repressors on distinct but overlapping downstream programs: VIG1 targets EXPB4 and OsPID, and OsbZIP18 targets EXLA2, CYCD1-2, OsDREB1A/B, and also OsPID. Their coordinated repression balances cell expansion and division with stress-responsive CBF/DREB pathways, thereby enhancing SV and CT. Yield outcomes are context-dependent: in knockout/CK lines, elevated Ehd1 correlates with reduced GNP, whereas in vig1a, unchanged Ehd1 but elevated OsPID links to increased GNP. The findings establish a strategy to decouple traditional trade-offs by precise alteration of the VIG1 basic region and maintaining interaction with OsbZIP18, offering a route to integrate vigor, stress tolerance, and yield. Transfer of vig1a into indica confirms broad breeding utility.

This work identifies a single amino-acid substitution in the basic region of VIG1 (OsbZIP01) that, in concert with OsbZIP18 via the leucine zipper interaction, simultaneously enhances seedling vigor, chilling tolerance, and grain number per panicle. The study defines regional functions within VIG1 (basic region vs leucine zipper), maps cooperative repression of downstream targets governing growth and cold response, and separates yield control via Ehd1 from increased GNP via OsPID in the vig1a context. Domain editing (basic region) can recapitulate vig1a, while disrupting the interaction domain converts vig1a to vig1b, underscoring the importance of VIG1–OsbZIP18 cooperation. Near-isogenic introduction of vig1a into an indica variety improves SV, CT, and yield, highlighting breeding potential. Future research should systematically explore additional mutations within the VIG1 basic region, delineate structural bases for altered repressive activity and partner effects, and assess field performance across diverse genetic backgrounds and environments.

- Background dependency: Phenotypic outcomes (e.g., flowering time, lethality reports for OsbZIP48 mutants) vary by genetic background; some discrepancies with prior studies likely stem from background effects. - Alternative splicing complexity: Differences between VIG1-NK and VIG1-CK suggest splice-specific functions (612-bp vs 345-bp isoforms) complicating genotype–phenotype interpretation. - Partial lethality/delays: The vig1b OsbZIP18-CK double mutant showed approximately one-third mortality and delayed flowering relative to vig1a, indicating potential fitness costs when both functions are strongly impaired. - Mechanistic gaps: While downstream targets were validated, the precise structural mechanism by which the R121H/basic-region mutations alter repression and partner interplay remains to be fully resolved; the breadth of basic-region variants capable of producing the vig1a phenotype requires further testing. - Environmental scope: CT assays were controlled; broader multi-environment field validation will strengthen generalizability for different climates and management regimes.