An E2-E3 pair contributes to seed size control in grain crops

Global food security is threatened by population growth, climate change, and shrinking arable land, making yield improvement a central goal in crop research. In Poaceae crops (maize, rice, wheat, and foxtail millet), grain yield is determined by panicle/cob number, grains per panicle, and thousand-grain weight; grain size is a key component. Setaria italica (foxtail millet) is an emerging model for Poaceae due to shared inflorescence architecture and genetic tractability, enabling insights transferable to major cereals. Protein ubiquitination (E1-E2-E3 cascade) regulates plant growth and yield traits, yet the specific roles of E2-E3 enzyme pairs in grain yield remain insufficiently understood. Brassinosteroid (BR) signaling, mediated by the receptor BRI1, broadly controls growth and stress responses; known E3 ligases (e.g., PUB12/13) ubiquitinate BRI1 to regulate its internalization, but BR signaling is only modestly affected in pub12/13, implying additional E3s. This study asks whether a specific E2-E3 pair regulates grain size and yield in Poaceae, identifies the underlying genes in Setaria, elucidates their mechanism (including potential modulation of BR signaling), and assesses conservation and breeding utility across cereals.

Prior studies identified ubiquitin-related regulators of seed size, including DA1, DAR1/DAR2 in Arabidopsis and GW2 in rice, highlighting ubiquitination’s role in organ size control. BR signaling components are modulated by several E3 ligases (SINAT, KIB1, ELF1, TUD1, PUB12/13), with PUB12/13 promoting BRI1 endocytosis and degradation; however, residual BRI1 internalization and near-normal BR sensitivity in pub12/13 indicate additional E3s act on BRI1. In foxtail millet, the LRR-RLK DPY1 orchestrates early BR signaling and affects leaf architecture. The rice ortholog of SGD1, TT3.1, confers thermotolerance by protecting chloroplasts under heat stress, suggesting potential conserved roles in yield under stress. Collectively, the literature supports ubiquitination as a nexus for growth and stress signaling, with unresolved questions about specific E2-E3-substrate modules regulating BR receptor dynamics and cereal yield.

- Forward genetics and cloning: Two EMS-induced foxtail millet mutants (sgd1-1, sgd1-2) with small grains and semi-dwarfism were phenotyped (plant height, panicle traits, grain metrics). Positional cloning, map-based cloning, and MutMap identified causal mutations in Seita.9G123200 (SGD1). Sanger sequencing validated mutations. - Genome editing and complementation: CRISPR/Cas9 knockout of SGD1 in foxtail millet (CR-sgd1 lines) phenocopied mutants; complementation with native promoter-driven SGD1 (pSGD1::SGD1-GFP) restored WT phenotypes. Cross-species validation included CRISPR knockouts of OsSGD1 in rice (Kitaake) and TaSGD1A/B/D in wheat (Fielder), and transgenic rescue of foxtail millet sgd1 with maize ZmSGD1. - Cytology and subcellular localization: Resin sections and SEM assessed cell size vs number contributions to grain size. SGD1-GFP colocalization with FM4-64 and HDEL-mCherry determined plasma membrane and ER localization. - Protein interaction assays: Split-ubiquitin membrane Y2H screens identified SiUBC32 as an SGD1 interactor. Interactions were validated by directed Y2H, in vitro GST pull-down, split luciferase complementation in Nicotiana benthamiana, and co-immunoprecipitation in Setaria protoplasts. - Enzymatic activity and ubiquitination: A bacterial reconstituted ubiquitination system (E1 UBA1, E2s, ubiquitin-FLAG) tested SiUBC32 E2 activity and SGD1 E3 ligase activity (truncated SGD1c). RING domain mutants (C426A, H443A; mSGD1c) assessed catalytic dependence. In vitro ubiquitination assays tested SGD1c-mediated ubiquitination of SiBRI1 kinase domain (SiBRI1c). In vivo ubiquitination of SiBRI1 was examined in protoplasts co-expressing SiBRI1-HA and FLAG-Ub in WT vs sgd1. - Genetic interactions: CRISPR/Cas9 created Siubc32 single mutants and sgd1/Siubc32 double mutants to analyze additive/overlapping roles in growth and yield traits. - BR signaling assays: eBL treatments tested leaf angle, leaf droopiness, and primary root growth responses in WT vs sgd1. RNA-seq and qPCR profiled BR-associated gene expression, including BZR1 targets and feedback on BR biosynthetic genes (SiD2, SiCYP51G3) and BR-responsive genes (SiGLR2.7, SiCBF2, SiBRH1). - Protein stability: Immunoblotting with anti-SiBRI1 measured BRI1 abundance in WT, sgd1, and SGD1 overexpressors (OE-SGD1). Cycloheximide (CHX) chase assays assessed BRI1 stability over time across genotypes. BZR1 phosphorylation status was evaluated as BR output marker. - Genetic complementation: Overexpression of SiBRI1-GFP in sgd1 background assessed rescue of growth and grain traits. - Population genomics and haplotypes: Whole-genome resequencing of 1681 Setaria accessions (530 wild, 694 landraces, 457 cultivars). Selective sweep analyses used FST and nucleotide diversity (π) ratios. Haplotype analysis of SGD1 across germplasm associated with 13 agronomic traits measured in field trials (n=960 accessions). - Breeding and field evaluation: Overexpression of elite SGD1 haplotype (Ubi::SGD1H1) in foxtail millet (Ci846 background) generated independent lines evaluated for grain area, 1000-grain weight, panicle length, and grain yield per plant; disease resistance (blast) assessed after inoculation. Statistical analyses included Student’s t-tests and ANOVA with Tukey’s multiple comparisons, as noted in figure legends.

- Identification and function of SGD1: Map-based cloning and MutMap revealed SGD1 (Seita.9G123200) encodes a RING-type E3 ubiquitin ligase with a Fragile-X-F 7-TM domain and C3HC4 RING. sgd1 mutants and CR-sgd1 lines showed reduced plant height, shorter panicles, fewer grains, smaller grains due to decreased cell expansion (not cell number), causing severe yield loss. - Subcellular localization: SGD1 localizes to the plasma membrane and endoplasmic reticulum (ER), suggesting roles in membrane signaling and ER-associated processes. - Conservation across Poaceae: CRISPR knockout of OsSGD1 in rice reduced panicle size, grain number, and grain size; CRISPR Ta-sgd1A/B/D wheat triple mutants showed decreased panicle length, grain number per panicle, grain size, and 100-grain weight; maize ZmSGD1 transgene rescued Setaria sgd1 phenotypes. This demonstrates conserved yield regulation across cereals. - E2-E3 pairing with SiUBC32: SGD1 directly interacts with the E2 enzyme SiUBC32 (ER-localized) in Y2H, split-luciferase, pull-down, and Co-IP assays. In a bacterial system, SiUBC32 showed E2 activity and, with SGD1c, catalyzed ubiquitination (lost with RING mutations C426A/H443A). Siubc32 mutants phenocopied sgd1 for growth and grain size; sgd1/Siubc32 double mutants showed additive reductions in plant height, flag leaf and panicle length, and slightly greater reductions in grain size traits, indicating partially overlapping functions. - BR signaling involvement and hyposensitivity of sgd1: sgd1 exhibited BR-defective phenotypes (short bristles, compact leaves) and hyposensitivity to eBL. Leaf angle increased from 32.1° to 46.5° in WT with 5 µM eBL for 3 days; sgd1 angle was 34.65 ± 1.77° under the same treatment. High eBL (0.01–0.1 µM) inhibited WT root growth but not sgd1. Among putative BZR1-target genes, 53.61% were DEGs in WT+eBL vs mock, 71.90% in BR hypersensitive dpy1, and only 18.25% in sgd1. BR biosynthetic genes (SiD2, SiCYP51G3) were upregulated in sgd1, consistent with feedback from reduced BR signaling. - SGD1 targets and stabilizes BRI1: SGD1 interacts with the SiBRI1 C-terminal kinase domain (Y2H, pull-down, Co-IP) and ubiquitinates SiBRI1 in vitro; in vivo SiBRI1 ubiquitination was reduced in sgd1. Catalytically active SGD1 increased BRI1 levels in protoplasts; mSGD1 did not. In plants, BRI1 and dephosphorylated BZR1 increased in OE-SGD1 and decreased in sgd1. CHX-chase showed faster BRI1 decay in sgd1 and stabilized BRI1 in OE-SGD1. Overexpressing BRI1 in sgd1 partially rescued growth and grain traits. Together, SGD1-mediated ubiquitination enhances BRI1 stability and BR signaling rather than promoting degradation. - Yield improvement via elite haplotype and selection signals: Selective sweep analyses (top 1% FST and high π ratio) indicate SGD1 underwent selection during domestication and improvement, with selection focused in promoter/intron regions and conserved exons/RING domain. Haplotype H1 was strongly enriched in cultivars (70.5%) and associated with higher panicle and grain weights and more seeds per panicle; H2 predominated in wild (98.7%) with lowest yield. Overexpressing SGD1H1 increased grain area, 1000-grain weight, and panicle length, delivering ~12.82% higher grain yield per plant. OE lines also showed enhanced blast disease resistance. - Broader pathways: RNA-seq of sgd1 and OE-SGD1 revealed enrichment of DEGs in protein processing in ER, PSII stabilization, responses to heat and other stresses, defense responses, chlorophyll biosynthesis, nitrogen metabolism, and organ growth, suggesting multi-pathway contributions to yield.

This study addresses the gap in understanding how specific E2-E3 ubiquitination pairs regulate grain yield in cereals. By identifying SGD1 (a RING E3) and its E2 partner SiUBC32, and showing their conserved roles in Setaria, rice, maize, and wheat, the work establishes an E2-E3 module critical for panicle and seed development. Mechanistically, SGD1 directly ubiquitinates the BR receptor BRI1, leading to its stabilization and enhanced BR signaling, which promotes cell expansion and yield traits. The BR hyposensitivity of sgd1, reduced BRI1 abundance/stability, and partial rescue by BRI1 overexpression collectively link SGD1 activity to BR pathway output. The findings suggest non-degradative roles of ubiquitination on BRI1 (e.g., promoting chaperone function or trafficking), contrasting with PUB12/13-mediated ubiquitination that favors BRI1 endocytosis and degradation. Two non-exclusive mechanisms are proposed: (1) SGD1 may regulate BRI1 trafficking among ER, Golgi, TGN/EE, and PM, potentially cooperating with SNAREs, thus counterbalancing PUB12/13 and optimizing receptor residence; (2) SGD1, with UBC32, may participate in ER-associated quality control/ERAD, ensuring proper folding/maturation of BRI1 and limiting misfolded receptor turnover, as supported by ER stress/ERAD gene upregulation in sgd1. Population genetic evidence shows strong selection at SGD1 during domestication and improvement, with the elite haplotype H1 associated with superior yield. Overexpression of SGD1H1 conferred significant yield gains and disease resistance in field settings, underscoring breeding potential. More broadly, results validate Setaria as an efficient model to dissect ubiquitination-regulated traits translatable to major cereals, accelerating gene discovery for yield improvement.

- SGD1 encodes a RING-type E3 ligase that, together with E2 partner SiUBC32, is a key regulator of grain size and yield in Setaria and functions conservatively in rice, maize, and wheat. - SGD1 directly interacts with and ubiquitinates BRI1, stabilizing the receptor and enhancing BR signaling, thereby promoting cell expansion and yield components. - Natural variation at SGD1 shows strong domestication/improvement selection; the elite haplotype H1 is associated with higher yield, and its overexpression increases grain yield per plant by ~12.8% and improves disease resistance. - SGD1 appears to integrate growth and stress pathways, influencing ER protein processing, photosystem stability, and stress responses, offering a multifaceted route to yield enhancement. Future directions include clarifying how SGD1-mediated ubiquitination stabilizes BRI1 (trafficking vs ERAD quality control), mapping ubiquitination sites and linkage to PUB12/13 actions, testing different SGD1 haplotypes in uniform genetic backgrounds (including NILs), and deploying the SiUBC32–SGD1–BRI1 module in breeding programs for high-yield, stress-resilient cereals.

- The precise mechanism by which SGD1-mediated ubiquitination stabilizes BRI1 is unresolved (e.g., effects on trafficking vs ER quality control; interplay with PUB12/13 and ubiquitination site specificity). - Functional overlap between SGD1 and SiUBC32 is partial, and additional E2/E3 partners or substrates likely contribute to phenotypes. - Yield gains in overexpression lines may reflect both expression level and haplotype effects; controlled comparisons of different SGD1 haplotypes in the same background and nearly isogenic lines are needed. - Many mechanistic assays were performed in protoplasts or controlled environments; broader multi-location field trials across environments and species will refine translational potential.