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

Global food security is threatened by population growth, climate change, and limited arable land. Increasing crop yields is crucial, particularly for staple crops like maize, rice, and wheat. Grain yield depends on several components, including grain size. Foxtail millet (*Setaria italica*) is a valuable model system for studying grain yield due to its similar inflorescence structure to other Poaceae crops. Grain yield is a complex trait influenced by various genetic pathways, including the ubiquitination cascade (E1, E2, and E3 enzymes). Several ubiquitin-related proteins affecting grain yield have been identified in other plants. Brassinosteroids (BRs) are phytohormones regulating plant growth and development; BRI1 is the key BR receptor. Several E3 ubiquitin ligases influence BR signaling, but the complete picture remains unclear. This research uses the *Setaria* model system to explore grain yield control, focusing on the role of E2-E3 enzyme pairs.

Previous research has highlighted the importance of protein ubiquitination in regulating various aspects of plant growth and development, including grain yield. Several ubiquitin-related proteins, such as DA1, DAR1, DA2 in *Arabidopsis* and GW2 in rice, have been implicated in this process. The ubiquitination cascade, involving E1, E2, and E3 enzymes, is crucial for controlling protein stability, degradation, and localization. Brassinosteroids (BRs) are critical plant hormones known to affect many aspects of growth and development. The receptor BRI1 plays a vital role in BR signaling, and studies have identified several E3 ubiquitin ligases influencing this pathway, including SINAT, KIB1, ELF1, TUDI, PUB12, and PUB13. However, a comprehensive understanding of how E2-E3 enzyme pairs impact grain yield remains limited. The *Setaria* model offers an accessible system to investigate this complex interaction.

The study identified two foxtail millet mutants (*sgd1-1* and *sgd1-2*) with reduced grain yield and dwarf phenotypes. Map-based cloning, MutMap analysis, CRISPR/Cas9 genome editing, and transgenic functional complementation were used to identify the causal gene, *SGD1*. Subcellular localization assays using SGD1-GFP fusion protein and ER markers (HDEL-mCherry and FM4-64) determined SGD1's location. Expression patterns were examined using RNA-seq and a SGD1-promoter-driven GUS reporter. To assess the conserved role of SGD1, homologs were identified and studied in rice, maize, and wheat using CRISPR/Cas9. Yeast two-hybrid (Y2H) screening identified SiUBC32 as a potential interacting partner of SGD1. The interaction was confirmed using Y2H assays, in vitro GST pull-down assays, split luciferase complementation assays (LCA), and in vivo Co-IP assays. Bacterial ubiquitination assays assessed the ubiquitin-conjugating activity of SiUBC32 and the ubiquitin ligase activity of SGD1. The genetic relationship between SiUBC32 and SGD1 was investigated by creating *Siubc32* single and *Siubc32/sgd1* double mutants using CRISPR/Cas9. The role of SGD1 in BR signaling was investigated by examining the response of *sgd1* mutants to exogenous BR (eBL), analyzing the expression of BR-associated genes using RNA-seq and qPCR, and assessing the interaction of SGD1 with BRII, BAK1, and BIN2 using LCA and Y2H. In vitro ubiquitination assays determined if SGD1 ubiquitinates BRII, and in vivo ubiquitination assays were used to assess the ubiquitination of BRII in WT and *sgd1* plants. The stability of BRII was also measured in the presence of cycloheximide (CHX). Finally, high-throughput resequencing data of *Setaria* germplasm was used to identify selective signatures of SGD1 during domestication and improvement. Agronomic traits and haplotype analysis were conducted to assess the association between SGD1 and grain yield. Overexpression of the elite SGD1 haplotype was performed to verify its effects on grain yield and disease resistance. RNA-sequencing was carried out to explore the broader effects of SGD1 on gene expression.

The study successfully identified *SGD1*, a RING-type E3 ubiquitin ligase crucial for grain yield regulation in foxtail millet and conserved across major Poaceae crops (rice, maize, wheat). *SiUBC32* was identified as SGD1's E2 partner, forming a functional E2-E3 ubiquitin enzyme pair. SGD1 interacts with and ubiquitinates the BR receptor SiBRI1, leading to increased SiBRI1 stability and enhanced BR signaling. The *sgd1* mutant showed decreased sensitivity to BR, reduced SiBRI1 stability, and altered expression of BR-associated genes. Genetic complementation tests and transgenic experiments confirmed the role of the SiUBC32-SGD1-SiBRI1 module in grain yield regulation. Haplotype analysis revealed that SGD1 was under selection during foxtail millet domestication and improvement. Overexpression of the elite SGD1 haplotype (*SGD1^H1*) significantly increased grain yield (12.8% per plant) and improved blast disease resistance. RNA-sequencing analysis revealed that SGD1 influences multiple biological processes, including protein processing in the ER, PS II stabilization, stress responses, and nitrogen metabolism.

This study provides strong evidence for a novel genetic module (SiUBC32-SGD1-SiBRI1) that regulates grain yield in Poaceae crops. The finding that SGD1 ubiquitination of BRI1 leads to increased stability, rather than degradation, is a significant discovery, highlighting the multifaceted roles of ubiquitination in plant growth regulation. The conservation of SGD1 function across diverse Poaceae species suggests its potential for improving grain yield in major crops. The identification of an elite SGD1 haplotype associated with increased yield and blast resistance opens up new avenues for marker-assisted selection and crop improvement. The broader impact of SGD1 on various biological processes, as revealed by RNA-sequencing, underscores its role as a central hub regulating plant growth and stress responses.

This study successfully identified and characterized the SiUBC32-SGD1-SiBRI1 genetic module in foxtail millet, demonstrating its critical role in regulating grain yield. The findings are conserved across major Poaceae crops and highlight the importance of E2-E3 pairs in plant development. The identification of an elite SGD1 haplotype with enhanced yield and disease resistance provides a valuable target for crop improvement. Future research should focus on elucidating the precise mechanisms of SGD1 action, especially its role in BR trafficking and ER quality control, and exploring the potential of SGD1 for breeding high-yield, stress-tolerant crops.

The study primarily focused on foxtail millet, and while the conserved function of SGD1 was demonstrated in other Poaceae species, further investigation is needed to confirm the precise mechanisms of action and the applicability of these findings across different genetic backgrounds and environmental conditions. While the study suggests the involvement of SGD1 in various biological processes, further research is needed to fully unravel the complex interplay of these pathways and SGD1's role in integrating them. The role of the elite haplotype needs further investigation to disentangle the specific contribution of the haplotype from the overexpression effect.