UDP-glucosyltransferase regulates grain size and abiotic stress tolerance associated with metabolic flux redirection in rice

Rice yield is determined by quantitative traits including grain size. Many QTLs and pathways (phytohormones, G-proteins, protein kinases, transcription factors) regulating grain size have been identified, yet how grain yield traits are coordinated with abiotic stress tolerance is unclear. Abiotic stresses (salinity, drought, heat/cold) elevate ROS, which can be scavenged by antioxidants including flavonoids. Glycosylation modulates flavonoid stability and activity, but QTLs linking flavonoid glycosylation to abiotic stress tolerance in rice are largely unknown. The study aims to identify and functionally characterize a QTL that simultaneously modulates grain size and abiotic stress tolerance and to uncover the mechanistic basis involving phenylpropanoid metabolic flux and flavonoid glycosylation.

Prior work has mapped numerous rice grain size QTLs and genes influencing seed/grain traits through ubiquitin-proteasome, G-protein, MAPK, and hormone signaling. Several major stress-related loci (e.g., SKC1 for salt tolerance, COLD1 for chilling tolerance, TT1 for thermotolerance) have been cloned in rice. Flavonoids, which accumulate under stress, act as antioxidants; their glycosylation by plant UGTs affects solubility, stability, and bioactivity, including anthocyanin regulation. Flavonoids can modulate auxin transport; Arabidopsis tt mutants with altered flavonoid biosynthesis show reduced auxin transport and smaller seeds. Despite these insights, a direct QTL controlling rice grain size that is mechanistically tied to flavonoid glycosylation and abiotic stress tolerance had not been elucidated.

- Genetic materials: Developed chromosome segment substitution lines (CSSLs) using African rice CG14 (donor) and Asian rice Wuyunjing (WYJ, japonica; recurrent). Identified a grain-size QTL on chromosome 3 (GSA1). Constructed NIL-GSA1CG14 and NIL-GSA1WYJ. - Fine mapping: Used BC4F2 populations (n≈5260 screened) and newly designed markers to delimit GSA1 to a 29.47-kb region containing five genes (three UGTs, a UDP-glucose-6-dehydrogenase, and a small peptide gene). Sequenced candidates and promoters; identified nonsynonymous variants A349T (PSPG box) and A246V in LOC_Os03g55040. - Population genetics: Assessed nucleotide diversity and selection signatures across ~3.3 kb (promoter + ORF) using Rice3K, O. rufipogon, O. glaberrima, and O. barthii datasets; sliding-window π analyses. - Transgenics: Overexpressed GSA1WYJ (CaMV 35S promoter) in WYJ; overexpressed GSA1CG14; generated CRISPR/Cas9 knockouts of GSA1 and nearby UGTs (LOC_Os03g55030, LOC_Os03g55050); performed genetic complementation by introducing genomic GSA1WYJ into NIL-GSA1CG14. Agrobacterium-mediated transformation; selection with hygromycin; validation by PCR/sequencing. - Phenotyping: Measured 1000-grain weight, grain length and width (multiple plants/replicates); grain filling/milk stage dynamics (fresh/dry weight, size, water content). Microscopy: scanning electron microscopy of spikelet hull epidermal cells; histological cross-sections at booting stage to quantify cell size and number. - Gene expression: qRT-PCR for tissue-specific GSA1 and pathway genes (phenylpropanoid, lignin, flavonoid/anthocyanin, auxin biosynthesis/transport/response); RNA-seq of young panicles; western blot for OsPIN1 protein levels. - Hormone assays: LC-ESI-MS/MS quantification of endogenous IAA and ICA in young caryopses and panicles. - Metabolomics: Widely targeted LC-ESI-MS/MS profiling in young panicles, spikelet hulls, caryopses, and seedlings under normal vs salt stress; analysis of flavonoid aglycones/glycosides and monolignols; Z-score heatmaps. - Enzyme assays: Heterologous expression of GSA1WYJ, GSA1CG14, and point mutants (A246V, A349T) in E. coli; in vitro UGT assays using UDP-glucose donor and acceptors (kaempferol, quercetin, naringenin; p-coumaryl, coniferyl, sinapyl alcohols); HPLC and LC-MS identification; kinetic measurements (Km for kaempferol and UDP-glucose). - Lignin measurement: Thioglycolic acid method to quantify lignin in spikelet hulls and mature caryopses. - Abiotic stress assays: Seedling survival after 120 mM NaCl (7 d), heat 42 °C (~26 h), and 16% PEG8000 (14 d) followed by recovery; compared WYJ, overexpression, knockout, NILs, and complementation lines; qRT-PCR of OsC4H, OsCAD7, OsC1, OsANS before/after NaCl. - Statistical analyses: Student's t-tests, Duncan's multiple range tests as appropriate; replicates detailed per figure; source data provided.

- Cloning and identity of GSA1: Fine mapping and sequencing identified LOC_Os03g55040 (UGT83A1) as the causal gene at GSA1. Natural variants include A349T in the conserved PSPG box and A246V in GSA1CG14. - Effect on grain size: NIL-GSA1CG14 showed reduced 1000-grain weight (-9.29%), grain length (-3.78%), and width (-4.8%) vs NIL-GSA1WYJ. Overexpressing GSA1WYJ increased 1000-grain weight (+12.06%), length (+4.41%), and width (+5.27%). GSA1 knockout or overexpressing GSA1CG14 decreased grain size; complementation restored NIL-GSA1WYJ phenotype. - Cellular basis: NIL-GSA1CG14 spikelet hulls had reduced outer epidermal cell length/width and fewer cells in length/width directions; reduced parenchyma cell number in cross-sections, indicating diminished cell proliferation and expansion. - Auxin linkage: NIL-GSA1CG14 had significantly lower IAA in young caryopses and panicles; down-regulated auxin biosynthesis (e.g., TSG1, OsTAR1/TARL1/TARL2), transport (OsPIN1 family), and response genes; OsPIN1 protein level reduced. - Phenylpropanoid homeostasis: Metabolomics showed higher aglycone flavonoids (kaempferol, quercetin, naringenin) and monolignols (in young caryopses) but lower flavonoid glycosides (e.g., naringenin-7-O- and quercetin-7-O-glucosides) in NIL-GSA1CG14; lignin content was reduced in spikelet hulls and mature caryopses. Key pathway genes (PAL4, COMT, CCR1, CAD7, CHS, CHI, F3H, ANS, OsC1, OsP1) were downregulated in NIL-GSA1CG14 young panicles. - Enzymatic function: GSA1 catalyzed 7-O-glucosylation of kaempferol and quercetin (and naringenin) and glucosylation of monolignols (not at 4-OH). GSA1CG14 had weaker activity than GSA1WYJ. Mutational analysis showed A349T in the PSPG box is critical for activity. Kinetics: GSA1WYJ displayed Km ≈ 25.54 ± 5.43 μM for kaempferol (vs 42.78 ± 23.26 μM for GSA1CG14) and higher affinity for UDP-glucose (Km 51.06 ± 7.81 μM vs 134.6 ± 21.63 μM). - Abiotic stress tolerance: Overexpression lines had significantly higher survival under salt (120 mM NaCl), heat (42 °C), and PEG-induced osmotic stress; GSA1CG14 overexpression and GSA1 knockout lines were more sensitive; complementation restored tolerance. - Stress-induced metabolic reprogramming: Under salt stress, OsC4H was induced (less so in NIL-GSA1CG14 and not in KO), OsCAD7 was downregulated in WYJ and OE but not in KO or NIL-GSA1CG14, while flavonoid/anthocyanin regulators OsC1 and OsANS were strongly upregulated (more in OE and NIL-GSA1WYJ than NIL-GSA1CG14/KO). Metabolomics indicated metabolic flux redirected from lignin (reduced monolignols) toward flavonoid glycosides and anthocyanins (elevated apigenin-7-O-glucoside, chrysoeriol glucosides, anthocyanins) in GSA1WYJ backgrounds; this redirection was impaired in NIL-GSA1CG14. - Selection signals: Nucleotide diversity analyses suggest directional selection of GSA1 in O. glaberrima and O. sativa ssp. japonica during domestication.

The study demonstrates that GSA1, a UDP-glucosyltransferase, provides a mechanistic link between grain size regulation and abiotic stress tolerance by coordinating phenylpropanoid metabolism. By glucosylating flavonoids and monolignols, GSA1 influences both lignin biosynthesis (supporting cell proliferation/expansion and structural development) and flavonoid glycoside accumulation (supporting ROS scavenging under stress). The weak GSA1CG14 allele results in aglycone flavonoid accumulation, suppressed auxin biosynthesis/transport gene expression, reduced PIN1 protein, and impaired cell proliferation/expansion, collectively yielding smaller grains. Under abiotic stress, GSA1 is necessary for induction of the central phenylpropanoid pathway and for redirecting metabolic flux away from lignin (downregulating CAD7, lowering monolignols) toward flavonoid glycosides and anthocyanins (upregulating OsC1 and OsANS), enhancing stress tolerance. These findings position GSA1 as a central node integrating growth and stress via metabolic flux control and auxin-related processes, offering a strategy to improve crop yield and resilience.

GSA1 (LOC_Os03g55040/UGT83A1) is a cloned rice QTL that positively regulates grain size and abiotic stress tolerance by glucosylating flavonoids and monolignols and orchestrating phenylpropanoid metabolic flux. Natural variation (A349T in the PSPG box) diminishes enzyme activity, disrupts flavonoid glycosylation and lignin biosynthesis, lowers auxin levels/signaling, and reduces grain size and stress tolerance. Overexpression of GSA1 enhances grain size and tolerance to salt, heat, and osmotic stress by promoting stress-induced flux toward flavonoid glycosides and anthocyanins. GSA1 is a promising genetic resource for molecular breeding aimed at simultaneously improving yield components and abiotic stress resilience. Future work could define in vivo glycosylation sites and partners, elucidate transcriptional regulation of GSA1 under stress, and evaluate performance across environments and genetic backgrounds.

The study primarily uses controlled-environment seedling stress assays; field-level performance and multi-environment stability were not reported. Enzymatic activities and glycosylation positions for monolignols were determined in vitro, with in vivo glycosylation sites unresolved. While correlations between flavonoid profiles and auxin transport/signaling are strong, direct causal genetic or biochemical links between specific glycosides and auxin transport machinery remain to be demonstrated. Regulatory factors upstream of GSA1 induction under stress were not identified.