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

Rice, a staple food for over half the world's population, faces challenges due to an aging population, labor shortages, and increasing production costs. Paddy direct-seeding offers a solution by reducing costs and labor, but its success hinges on seedling vigor (SV), chilling tolerance (CT), and high productivity. Modern rice varieties, originating in tropical and subtropical regions, often exhibit poor SV and high cold sensitivity, leading to reduced grain yield in direct-seeding systems. While QTLs associated with shoot and root length have been identified (e.g., *SOR1*, *OsSWEET3a*), understanding the coordination of growth and stress response remains limited. Similarly, while genes affecting CT have been studied (e.g., *OsICE1*, *OsMADS57*, *OsCYP20-2*, *OsGRF6*), the simultaneous control of SV, CT, and grain yield remains poorly understood. Grain yield is determined by grain number per panicle (GNP), tiller number, and grain weight, with GNP being particularly important. Numerous QTLs affecting GNP have been identified (e.g., *Gnla/OsCKX2*, *DEPI*, *IPAI*), but integrating the control of SV, CT, and GNP remains a challenge. This study aims to identify and characterize regulators that can simultaneously enhance these three vital traits, improving rice production, particularly in direct-seeding systems. The focus will be on identifying the molecular mechanisms involved in the interaction of these genes and providing strategies for improving rice yield in direct-seeding systems.

Extensive research has explored the genetic basis of seedling vigor, chilling tolerance, and grain yield in rice individually. Several studies have identified quantitative trait loci (QTLs) associated with shoot and root length, providing insights into genes controlling early growth. For example, *SOR1* regulates root length by modulating Aux/IAA protein stability, while *OsSWEET3a* impacts shoot development through its dual function as a gibberellin and glucose transporter. Research on chilling tolerance has identified genes involved in cold acclimation and stress response, including *OsICE1*, *OsMADS57*, *OsCYP20-2*, and *OsGRF6*. These genes often interact with other pathways to modulate plant growth and cold tolerance. The regulation of grain yield has also been extensively investigated, with studies focusing on genes affecting grain number per panicle (GNP), such as *Gnla/OsCKX2*, *DEPI*, and *IPAI*. However, limited research has focused on the integrated regulation of SV, CT, and GNP. While *IPAI* has been shown to influence all three, it demonstrates a trade-off effect, promoting SV while negatively impacting CT and GNP. This highlights the need for further research into genes capable of simultaneously enhancing all three traits.

This study employed a multi-faceted approach combining mutant screening, map-based cloning, gene editing, transgenic complementation, yeast two-hybrid assays, bimolecular fluorescence complementation (BiFC), in vitro pull-down assays, RNA sequencing, real-time PCR, luciferase reporter assays, yeast one-hybrid assays, electrophoretic mobility shift assays (EMSA), and phylogenetic analysis. Initially, the *vigla* and *viglb* mutants were identified from a NaN3-mutagenized M2 population of KY131 (japonica). Map-based cloning localized the causal mutation to *OsbZIP01*. CRISPR/Cas9 gene editing was used to generate *VIGI-NK* and *VIGI-CK* lines to further investigate the function of *OsbZIP01*. Homologous genes, *OsbZIP18* and *OsbZIP48*, were also investigated using CRISPR/Cas9 knockout. Yeast two-hybrid and BiFC assays examined protein-protein interactions between *VIGI*, *OsbZIP18*, and *OsbZIP48*. RNA sequencing compared the transcriptomes of WT, *vigla*, and *viglb* to identify differentially expressed genes. Real-time PCR validated the expression levels of selected genes. Luciferase assays assessed the direct binding of *VIGI* and *OsbZIP18* to the promoters of target genes. Yeast one-hybrid and EMSA experiments further confirmed these interactions. Finally, near-isogenic lines (NILs) of *vigla* were developed in the indica rice variety ZF802 to evaluate the potential for broader application.

The study identified two allelic mutants, *vigla* and *viglb*, with enhanced seedling vigor and chilling tolerance. Map-based cloning and subsequent analysis confirmed that *OsbZIP01* (renamed *VIGI*) was the causal gene. The *vigla* mutant harbored a G-to-A point mutation leading to an R121H substitution in the basic region of VIGI. *viglb* had a 14-bp deletion causing a frameshift mutation. *vigla* was found to be dominant over *viglb*. CRISPR/Cas9 knockout lines (*VIGI-NK* and *VIGI-CK*) showed that different regions of *VIGI* had distinct functions. Knocking out *OsbZIP18* or *OsbZIP48* resulted in no significant phenotypic changes. However, simultaneous mutation of *VIGI* and *OsbZIP18* phenocopied *vigla*. Yeast two-hybrid and BiFC assays demonstrated interactions between VIGI, OsbZIP18, and OsbZIP48. RNA sequencing revealed differentially expressed genes in *vigla* and *viglb*, including those related to cell expansion, cell division, and cold tolerance. Further analyses, including luciferase assays, Y1H, and EMSA, indicated that *VIGI* and *OsbZIP18* cooperatively regulated downstream target genes involved in these processes. Finally, NIL-*vigla* in indica rice showed improved seedling vigor, chilling tolerance, and grain yield, highlighting its potential for broader application.

This study's findings demonstrate that *VIGI*, in conjunction with *OsbZIP18*, plays a crucial role in regulating seedling vigor, chilling tolerance, and grain yield in rice. The synergistic action of these two bZIP transcription factors provides a mechanism for balancing these often-conflicting traits. The specific point mutation in *vigla* leads to a disruption in the function of both *VIGI* and *OsbZIP18*, resulting in the observed enhanced phenotypes. The results highlight the complexity of gene regulation in plants, where single point mutations can lead to significant changes in multiple traits due to protein-protein interactions and downstream effects on gene expression. The fact that the *vigla* phenotype could be phenocopied by manipulating the basic region of *VIGI* points to the importance of this region in protein-protein interactions and subsequent gene regulation. The successful development of NIL-*vigla* in indica rice further broadens the potential application of this mutation for improving rice production.

This research identifies *VIGI* and its interaction with *OsbZIP18* as a key mechanism for regulating seedling vigor, chilling tolerance, and grain yield in rice. The *vigla* mutant, with its superior performance in both japonica and indica rice, presents a promising target for improving paddy direct-seeding systems. Future research could explore the detailed mechanisms of the *VIGI*-*OsbZIP18* interaction and the full extent of its downstream effects on gene expression. Furthermore, investigating the potential for stacking this mutation with other beneficial alleles could further enhance rice productivity.

The study primarily focused on two specific rice varieties (KY131 and ZF802), limiting the generalizability of the findings to other rice genotypes. Further studies are needed to investigate the performance of *vigla* across a wider range of genetic backgrounds and environmental conditions. While the study identifies key downstream targets of *VIGI* and *OsbZIP18*, a more comprehensive understanding of the entire regulatory network would benefit from further investigation. The relatively small sample sizes used in some experiments could also affect the statistical power of certain findings.