Rice yield is significantly determined by grain size, a quantitative trait influenced by various genetic factors and environmental conditions. While numerous QTLs regulating grain size have been identified, their roles in abiotic stress tolerance remain largely unknown. Abiotic stresses such as drought, salinity, and extreme temperatures severely impact crop yields. Plants combat these stresses through various mechanisms, including the accumulation of antioxidants like flavonoids, which are glycosylated by UDP-glucosyltransferases (UGTs). This study focuses on understanding the genetic regulation of grain size and abiotic stress tolerance, specifically investigating the role of a QTL designated GSA1.

Previous research has extensively explored QTLs influencing grain size in rice, revealing the involvement of signaling pathways mediated by phytohormones, G-proteins, proteasomal degradation, protein kinases, and transcription factors. Studies have also identified QTLs associated with abiotic stress tolerance in rice, such as *SKC1*, *COLD1*, and *TT1*. However, the molecular mechanisms underlying the synergistic regulation of grain size and abiotic stress tolerance remain largely unexplored, particularly regarding the role of flavonoid glycosylation.

The researchers employed a combination of techniques to identify and characterize GSA1. They began by constructing a set of chromosome segment substitution lines (CSSLs) using an African rice variety (CG14) and an Asian rice variety (Wuyunjing, WYJ). QTL mapping identified GSA1 on chromosome 3. Near isogenic lines (NILs) were developed to investigate the effects of GSA1 on grain size and other agronomic traits. High-resolution mapping narrowed down the GSA1 locus, leading to the identification of a candidate gene encoding a UDP-glucosyltransferase (UGT83A1). The functionality of GSA1 was investigated through overexpression in WYJ and CRISPR/Cas9 knockout lines. Tissue-specific expression analysis, cell number and size measurements, and auxin level measurements were conducted to understand GSA1's role in grain development. A widely targeted metabolomics approach was used to analyze the effects of GSA1 on phenylpropanoid metabolism, specifically flavonoid and lignin biosynthesis. The glucosyltransferase activity of GSA1 was investigated by heterologous expression in *Escherichia coli*. Finally, abiotic stress tolerance assays (salt, heat, drought) were performed on transgenic lines to determine GSA1's role in stress response. Statistical analyses, such as Student's t-tests and Duncan's multiple range test, were employed to compare the results.

GSA1, a QTL on chromosome 3, was identified as a positive regulator of grain size and abiotic stress tolerance. The GSA1 gene encodes a UDP-glucosyltransferase (UGT83A1) with activity toward both flavonoids and monolignols. Natural variations in GSA1 led to smaller grains due to altered flavonoid-mediated auxin levels and related gene expression (decreased auxin levels, PIN1 protein levels and decreased auxin-related genes expression). GSA1 regulates cell proliferation and expansion during spikelet development. Under abiotic stress, GSA1 is crucial for redirecting metabolic flux from lignin biosynthesis to flavonoid biosynthesis. This redirection results in increased flavonoid glycoside accumulation, enhancing abiotic stress tolerance (increased tolerance under salt, heat and drought stress). Overexpression of GSA1 resulted in larger grains and improved tolerance to salt, heat, and drought stress. Conversely, knockout of GSA1 resulted in smaller grains and reduced abiotic stress tolerance. Specifically, the A349T substitution within the conserved PSPG box domain of GSA1 is critical for its glucosyltransferase activity.

This research provides a detailed mechanism explaining how GSA1, a UDP-glucosyltransferase, regulates both grain size and abiotic stress tolerance in rice. The findings emphasize the importance of metabolic flux redirection in plant development and stress response. The interplay between flavonoid glycosylation, auxin signaling, and lignin biosynthesis is crucial for grain size determination. The ability of GSA1 to modulate this metabolic flux under abiotic stress conditions offers a potential target for crop improvement. The results suggest that enhancing flavonoid glycoside accumulation via GSA1 manipulation could lead to crops with both increased yield and enhanced stress resistance.

This study successfully identified and characterized GSA1, a key QTL regulating grain size and abiotic stress tolerance in rice. GSA1 encodes a UDP-glucosyltransferase with broad substrate specificity. The study's findings highlight the importance of flavonoid glycosylation and metabolic flux redirection in achieving high yields and stress resilience. Future research could focus on identifying and characterizing additional genes involved in this regulatory pathway. Further investigation is warranted to leverage GSA1 for molecular breeding strategies aimed at improving rice production under various environmental conditions.

The study primarily focused on salt stress, although other abiotic stress conditions were also examined. Further research is needed to comprehensively examine GSA1's function under other environmental stresses, particularly in field settings. The study primarily used a limited number of rice varieties, and future investigations should explore the role of GSA1 in a broader range of genetic backgrounds.