Genome-Wide Identification of Ovarian Tumor Proteases Gene Family and Knockout of TaOTU6 Increases Grain Width and Weight in Wheat

TL;DR

This study identifies a gene family in wheat linked to grain size and shows that knocking out one gene increases grain width and weight.

Contribution

The novel contribution is the discovery that TaOTU6 negatively regulates wheat grain size through interaction with TaUBC13.

Findings

Knockout of TaOTU6-7B increases grain width and weight in wheat.

TaOTU6-7B interacts with TaUBC13, which may positively regulate grain size.

49 OTU genes were identified and classified into four subfamilies in wheat.

Abstract

Deubiquitinating enzymes (DUBs) play essential roles in diverse plant biological processes, yet the ovarian tumor proteases (OTUs), a major DUB subfamily, have not been systematically characterized in wheat, and their functions in grain development remain unclear. Here, we identified 49 OTU genes (TaOTUs) in the hexaploid wheat genome and classified them into four subfamilies based on phylogenetic relationships, with nomenclature assigned according to homology. TaOTU6-7B was highly expressed during early and mid-grain development and was responsive to gibberellin and jasmonic acid. Its expression differed significantly between large-grain wheat Pindong34 (PD34) and small-grain wheat MY11847 at 7 and 11 days after flowering. To elucidate its function, we used CRISPR/Cas9 to generate loss-of-function mutants by knocking out the three homoeologs (TaOTU6-7A, -7B, and -7D). These mutants exhibited significantly increased grain width and weight relative to wild type. Moreover, TaOTU6-7B directly interacted with TaUBC13, whose expression was markedly elevated in the TaOTU6 knockout background, suggesting that TaUBC13 may positively regulate wheat grain size. Collectively, this study establishes the TaOTU gene family in wheat, reveals TaOTU6 as a negative regulator of grain width and weight, and provides valuable genetic resources and a theoretical foundation for high-yield wheat breeding.

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1. Introduction

Common wheat (Triticum aestivum L.) is one of the world’s three major cereal crops, providing a substantial proportion of daily calories and protein for approximately 60% of the global population and playing a central role in global food security [1]. Although wheat yields have risen markedly since the Green Revolution, food security remains threatened by sustained population growth and the escalating impacts of climate change [2]. Consequently, increasing wheat yield will remain a core breeding objective in the foreseeable future [3]. Wheat yield is a complex quantitative trait governed by multiple genes and shaped by genetic, environmental, and genotype-by-environment interactions [4,5]. Agronomically, yield is determined by three principal components: spike number per unit area, kernel number per spike, and thousand kernel weight (TKW) [6]. Among these, grain size (GS) shows a strong positive correlation with grain weight [7]. Identifying GS-associated genes and elucidating their regulatory networks are therefore essential for developing high-yield, high-quality wheat cultivars [8].

Numerous quantitative trait loci (QTLs) influencing wheat GS have been identified and fine-mapped, and several key genes controlling GS have been cloned and functionally characterized [9,10,11,12]. These genes predominantly act through pathways including the ubiquitin–proteasome system, G-protein signaling, mitogen-activated protein kinase (MAPK) cascades, phytohormone perception and homeostasis, and transcriptional regulation [13,14]. Ubiquitination is a fundamental regulatory mechanism in eukaryotes, controlling protein stability and function, and plays a critical role in seed development [15,16]. In wheat, grain width2 (GW2) encodes a RING-finger E3 ubiquitin ligase; loss-of-function of GW2 increases grain width and weight by promoting cell proliferation in spikelet hulls, thereby enhancing yield [17,18]. The E3 ligase Decreased Grain Size 1 (DGS1) interacts with the GS regulator SMALL GRAIN 3 (SMG3); disruption of either gene produces smaller grains, whereas their overexpression leads to larger grains [19]. CHANG LI GENG 1 (CLG1), another E3 ligase, interacts specifically with GW6a and stabilizes it via targeted ubiquitination, thereby increasing GS [20].

In parallel with ubiquitination, deubiquitination is a tightly regulated process that modulates protein stability by removing single ubiquitin molecules or polyubiquitin chains from substrates. The dynamic balance between ubiquitination and deubiquitination is crucial for the precise control of signaling pathways [21]. Deubiquitination is also involved in various biological functions, including the plant immune response [22], regulating flowering [23], root development [24], and plant growth [21]. Deubiquitinating enzymes (DUBs) are broadly classified into metalloproteases containing a JAMM domain or cysteine proteases. The ovarian tumor protease (OTU) family, which comprises highly specific ubiquitin isopeptidases that remove ubiquitin modifications from substrate proteins, exhibits strong evolutionary conservation from plants to humans [25,26]. OTU proteins typically harbor conserved domains, including the OTU catalytic domain with an active cysteine protease triad, a ubiquitin-interacting motif (UIM)-like motif (phi-xx-A-xxxs-xxx-Ac), a nuclear localization signal, and a ubiquitin-associated (UBA) domain [15]. Functional studies in plants have shown that AtOTU6 deubiquitinates monoubiquitinated histone H2B and represses target genes involved in growth and development [27]. In rice, downregulation of OsOTU1 enhances meristematic activity, leading to reduced tiller number, increased grain number, higher grain weight, and ultimately increased yield [28]. Despite these advances, functional analyses of OTU proteins in wheat remain limited, particularly regarding their roles and mechanisms in regulating GS and yield.

Gene-editing technologies, especially the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system, have revolutionized crop improvement by enabling precise and efficient genome modification, thereby accelerating functional genomics and breeding [29,30,31]. Continuous optimization of CRISPR/Cas9 system has greatly expanded its application in wheat research [32]. For yield improvement, targeted editing of the three homoeologs of TaGW2 significantly increased grain width and yield [33]. Similarly, knockout of negative yield regulators such as TaGW7 and TaGASR7 effectively increased grain weight [34]. For quality traits, CRISPR/Cas9-mediated mutation of TaSBEIIa produced high-amylose wheat with elevated resistant starch content [35]. In disease resistance, disruption of the susceptibility gene MLO conferred durable resistance to powdery mildew, and inactivation of TaPsIPK1, a gene associated with susceptibility to stripe rust caused by Puccinia striiformis f. sp. tritici (Pst), enhanced broad-spectrum resistance without affecting key agronomic traits [36,37]. Collectively, these examples demonstrate that genome editing enables rapid functional validation of genes, efficient generation of elite germplasm, and substantial acceleration of crop improvement [38].

In this study, we identified 49 OTU family members in the wheat genome. Sequence and expression analyses highlighted TaOTU6-7B as a strong candidate regulator of GS. Using genome-editing technology, we knocked out TaOTU6-7B and its homoeologs, demonstrating that TaOTU6 acts as a negative regulator of wheat grain width and weight. We further confirmed the physical interaction between TaOTU6-7B and TaUBC13 and analyzed the expression pattern of TaUBC13, suggesting that it may also function as a GS regulator in wheat. Together, these findings establish TaOTU6 as a key negative regulator of grain development and provide a promising target for yield improvement through targeted gene editing.

2. Results

2.1. Identification and Structural Analysis of the OTU Gene Family in Wheat

A total of 49 OTU genes were identified in the wheat genome and designated TaOTU1 to TaOTU49 based on chromosomal location (Figure 1A). These genes were distributed across 18 wheat chromosomes, with the highest density on chromosomes 2B, 5B, 5D, and 7B, each harboring four TaOTUs. Among homoeologous groups, group 5 contained the largest number of TaOTUs (11 members), whereas no TaOTU genes were detected in homoeologous group 1. Overall, 15, 18, and 16 TaOTUs were identified in the A, B, and D subgenomes, respectively, indicating no major subgenome-level bias in gene abundance. Sequence analysis showed that the coding sequences (CDS) ranged from 477 to 1632 bp, corresponding to proteins of 18.02 to 59.38 kDa. The predicted isoelectric points varied from 4.5 to 9.24, and all TaOTU proteins exhibited negative grand average of hydropathicity (GRAVY) values, indicating hydrophilic properties (Table S2).

Gene structure analysis revealed substantial diversity among TaOTUs (Figure 1B). Exon numbers ranged from 2 to 13, with TaOTU28 containing the highest number of exons, while TaOTU22 and TaOTU25 each contained only two. Analysis of untranslated regions showed that TaOTU4, TaOTU6, TaOTU11, TaOTU14, TaOTU17, TaOTU19, TaOTU21, and TaOTU23 lacked both 5′ and 3′ untranslated regions (UTRs), whereas TaOTU12, TaOTU15, and TaOTU45 possessed only a 3′ UTR. These structural differences indicate considerable diversification of gene architecture during the evolution of the OTU family in wheat.

2.2. Phylogenetic Relationship, Gene Duplication, and Synteny Analysis of TaOTUs

To clarify evolutionary relationships, TaOTUs were renamed according to homology (Table S3). A phylogenetic tree was constructed using full-length protein sequences from 49 TaOTUs, 12 AtOTUs, and 20 OsOTUs (Figure 2A, Table S4). All OTU genes clustered into four subfamilies (Groups I–IV). Group II consisted exclusively of monocot OTU genes from wheat and rice and lacked dicot genes from Arabidopsis, indicating that this group is monocot-specific. Broader phylogenetic analysis across wheat, Hordeum vulgare L., Zea mays L., Setaria italica, Sorghum bicolor L., Oryza sativa L., and Arabidopsis further demonstrated that the evolutionary relationships among OTU genes within monocots are closer than those between monocots and dicots. (Figure S1).

The allohexaploid nature of wheat, comprising A, B, and D subgenomes, results in many genes being retained as three homoeologous copies. Differences in expression and even function exist among these homoeologs [36]. In total, 39 duplication events were detected within the wheat OTU gene family, all of which were segmental duplications, with no tandem duplications observed (Figure 2B, Table S5). This pattern indicates that segmental duplication was the primary driver of OTU family expansion in wheat. To assess cross-species conservation, collinearity analyses were conducted among wheat, Arabidopsis, rice, and barley. Six, 31, and 46 collinear gene pairs were identified between wheat and Arabidopsis, wheat and rice, and wheat and barley, respectively (Figure 2C, Table S6). The highest number of collinear genes was identified between wheat and barley, suggesting derivation from a common ancestral lineage and potentially conserved functions. Non-synonymous substitution rates/Synonymous substitution rates (Ka/Ks) ratios were calculated to evaluate selection pressures and divergence times of duplicated and orthologous OTU gene pairs. The average Ka/Ks ratio for duplicated TaOTUs within wheat was 0.203, while average ratios for orthologous pairs between wheat and Arabidopsis, rice, and barley were 0.1483, 0.1644, and 0.1846, respectively. All ratios were <1, indicating that these genes have undergone purifying selection during evolution.

2.3. Cis-Acting Elements and Conserved Motifs Analysis of TaOTUs

Promoter regions (2000 bp upstream of the ATG start codon) were analyzed to identify cis-regulatory elements and infer transcriptional regulation and potential functions of TaOTUs. 20 cis-acting elements were identified and classified into four functional categories: growth and development, hormone responsiveness, light responsiveness, and stress responsiveness (Figure 3A, Figure S2). Growth and development-related elements included the O2-site, CAT-box, RY-element, circadian element, and GCN4_motif. The O2-site is associated with grain protein metabolism, the RY-element mediates seed-specific expression, and the GCN4_motif regulates endosperm-related gene expression, collectively suggesting roles for TaOTUs in grain development. Hormone-responsive elements included CGTCA-motif and TGACG-motif (jasmonic acid, JA), TGA-element and AuxRR-core (auxin, AUX), ABRE (abscisic acid, ABA), TCA-element (salicylic acid, SA), and P-box and GARE (gibberellin, GA), indicating extensive hormonal regulation of TaOTUs. Light-responsive elements included G-box, Sp1, and ACE, while stress-responsive elements comprised LTR, TC-rich repeats, ARE, MBS, and others.

Conserved motifs within TaOTU proteins were identified using MEME v5.2.0 software (Figure 3B). Three motifs were detected, of which motif 2 was present in all TaOTUs, representing the most conserved feature of the family. Motif 1 was found in all but one member (TaOTU12-2B), suggesting that this gene is the most divergent within the family. Motif 3 was unique to Group IV members. Overall, proteins within the same subfamily exhibited similar motif architectures, supporting their phylogenetic clustering and evolutionary relationships.

2.4. Gene Expression Profiles of TaOTUs

Spatiotemporal expression patterns of TaOTUs were examined using RNA-seq datasets from diverse tissues and hormone treatments (Figure 4A,B). Nine TaOTUs showed relatively high expression across multiple tissues. Notably, TaOTU9-3B and TaOTU9-3D exhibited strong spike-specific expression during flowering. In addition, TaOTU6-7A, TaOTU6-7B, and TaOTU14-6D were highly expressed during early and mid-stages of grain development. Hormone treatment analyses revealed that TaOTU2-6A and TaOTU2-6D were significantly induced three hours after 6-Benzylaminopurine (6BA) treatment, whereas TaOTU9-3A and TaOTU9-3D were markedly repressed. GA treatment significantly reduces the expression of 12 TaOTUs, including TaOTU15-2A, within one hour. JA treatment led to significant induction of eight TaOTUs, such as TaOTU3-2B, and repression of seven TaOTUs, including TaOTU15-2A, after one hour (Figure 4C).

Expression patterns were further analyzed using grain transcriptome data from large-grain wheat PD34 and small-grain wheat MY11847 at three developmental stages (3, 7, and 11 days after flowering, DAF) (Figure 4D). TaOTU14-6A, TaOTU14-6B, TaOTU14-6D, TaOTU6-7A, TaOTU6-7B, and TaOTU6-7D exhibited relatively high expression in developing grains (Figure 4E). Among these, TaOTU14-6B (11 DAF), TaOTU14-6D (3 and 7 DAF), TaOTU6-7A (7 DAF), and TaOTU6-7B (7 and 11 DAF) were expressed at higher levels in MY11847 than in PD34, suggesting that these genes may act as negative regulators of grain development. Notably, TaOTU6-7B was predominantly expressed during early and mid-grain development, was responsive to both GA and JA treatments, and exhibited significantly higher expression in MY11847 grains at 7 and 11 DAF compared with PD34. These findings strongly implicate TaOTU6-7B as a key regulator of wheat grain development.

2.5. Expression Characteristics of TaOTU6-7B

Based on expression patterns, TaOTU6-7B was selected as a candidate gene for further functional analysis. The gene was cloned from Chinese Spring (CS), revealing a 960 bp coding sequence encoding a 319 amino-acid protein (Figure S3). Subcellular localization assays demonstrated that the protein localizes to both the nucleus and cytoplasm (Figure S4A). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis across tissues showed that TaOTU6-7B expression was highest at 3 and 7 days after flowering, indicating a likely role in early grain development and regulation of grain size (Figure S4B).

2.6. Knockout of the TaOTU6-7B and Its Homeologs Increases Wheat Grain Width and Weight

To investigate gene function, two guide RNAs were designed to simultaneously target TaOTU6-7B and its homoeologs (TaOTU6-7A and TaOTU6-7D) in the A and D subgenomes (Figure 5A). Using gene gun-mediated CRISPR/Cas9 transformation in the BobWhite-Cas9 background, 17 T_0_ mutant plants were obtained (Table S10). In the T_1_ generation, homozygous mutants with distinct genotypes (aaBBDD, AAbbDD, AABBdd, aabbDD, and aabbdd) were identified (Figure 5A). Grain traits of mutant and wild-type (WT) plants were evaluated in the T_2_ generation (Figure S6). The aaBBDD genotype showed no significant change in TKW, whereas AAbbDD, AABBdd, aabbDD, and aabbdd exhibited significant increases. Grain length (GL) was significantly reduced in AAbbDD and aabbdd, while aaBBDD, AABBdd, and aabbDD showed no significant change relative to WT. Grain width (GW) increased significantly in all genotypes except aaBBDD. Notably, the aabbdd genotype showed the greatest increases, with GW rising by 13.19% and TKW by 30.52%, despite a reduction in GL.

The T_3_ mutant seeds were grown under field conditions, and their phenotypes were comprehensively evaluated (Figure 5B,C). TKW was significantly higher in AAbbDD, AABBdd, aabbDD, and aabbdd relative to WT, with the aabbdd genotype showing the largest increase (13.35%), whereas aaBBDD showed no significant change. GL did not differ significantly among genotypes. For GW, aaBBDD did not differ from WT, and AAbbDD, AABBdd, and aabbDD showed slight but non-significant increases, whereas aabbdd exhibited a significant increase (10.05%). No significant differences were observed between mutants and WT in plant height, spike length, or spikelet number. Grain quality analysis revealed that crude protein content was significantly increased and starch content significantly decreased in AAbbDD, AABBdd, aabbDD, and aabbdd genotypes (Figure 5C). Transverse sections of grains at 15 days after flowering showed that pericarp cell length did not differ between aabbdd and WT, whereas cell width was significantly increased (Figure 5D–G). The enlarged pericarp cell area likely accounts for the increased GW and TKW in mutant grains. Moreover, mutant grain sections exhibited a larger circumference than WT, further confirming wider and larger grains (Figure 5H).

2.7. TaOTU6-7B Interacts with TaUBC13

The ubiquitin–proteasome pathway plays a central role in GS regulation, and OTU proteins function as deubiquitinating enzymes that remove ubiquitin from substrate proteins. To elucidate how ubiquitination and deubiquitination coordinate to regulate wheat grain size, we examined the interaction between TaOTU6-7B and the wheat homolog of OsUBC13, an E2 ubiquitin-conjugating enzyme. TaUBC13 was cloned, and its interaction with TaOTU6-7B was confirmed using yeast two-hybrid (Y2H) assays (Figure 6A), dual luciferase (Dual-LUC) assays (Figure 6B), and bimolecular fluorescence complementation (BiFC) assays (Figure 6C). qRT-PCR analysis revealed that TaUBC13 expression was highest in grains at 11 and 19 days after flowering (Figure 6D). Furthermore, TaUBC13 expression was significantly upregulated in the TaOTU6-7B triple mutant background (Figure 6E). Together, these results support a cooperative regulatory mechanism between TaUBC13 and TaOTU6-7B in controlling wheat grain size and yield-related traits.

3. Discussion

3.1. Identification and Analysis of the TaOTU Gene Family Reveal a Potential Role for TaOTU6-7B in Regulating Wheat Grain Development

Advances in genome annotation and comparative genomics have enabled comprehensive phylogenetic analyses that clarify evolutionary relationships and functional diversification within gene families, providing an effective framework for inferring gene function [39]. The OTU family belongs to the DUBs that remove ubiquitin from substrate proteins, and it is evolutionarily conserved from plants to humans. Although OTU genes have been identified in model plants such as Arabidopsis and rice, their biological functions remain largely unexplored [15,40]. To determine whether OTU genes in wheat contribute to GS regulation, we systematically identified and characterized the OTU gene family using integrated bioinformatics, comparative genomics, evolutionary analysis, and genetic approaches. The 49 wheat OTU genes identified here were analyzed for homology, chromosomal distribution, gene structure, conserved domains, and promoter cis-acting elements. Phylogenetic analysis classified these genes into four subfamilies, with members within each subfamily sharing similar gene structures and conserved motifs. Extensive variation in exon-intron organization among TaOTUs suggests substantial structural diversification during evolution, which may underlie functional divergence within the family. Comprehensive expression profiling further enabled the identification of candidate genes potentially involved in grain development, which were subsequently functionally validated using CRISPR/Cas9-mediated genome editing and molecular analyses.

Cis-acting elements are non-coding DNA sequences in promoter regions that regulate gene expression by binding specific transcription factors, thereby shaping tissue specificity, developmental timing, and stress responsiveness [41]. Analysis of TaOTU promoters revealed abundant cis-elements associated with grain development, including the O2-site (grain protein metabolism), RY-element (seed-specific regulation), and GCN4_motif (endosperm-related gene expression), indicating that OTU genes may be broadly involved in wheat grain development. In addition, numerous hormone-responsive elements were identified. Plant hormone perception and homeostasis are central regulators of GS [42], and signaling pathways mediated by JA [43], AUX [44], and GA [45] have all been implicated in controlling grain size and yield. Among the 49 TaOTUs, 44 contained a total of 280 JA-responsive elements, 29 contained 41 AUX-responsive elements, and 31 contained 45 GA-responsive elements, further supporting the hypothesis that TaOTUs participate in hormone-mediated regulation of grain development.

Spatiotemporal expression patterns of genes provide critical insights into stage- and tissue-specific gene function [46]. Expression analyses across developmental stages and tissues enable the identification of candidate functional genes for subsequent validation. For example, the wheat NF-Y complex component TaNF-YA3 exhibits expression patterns similar to starch biosynthesis genes, suggesting a role in embryo starch accumulation [47,48]. Expression profiling of TaWOX genes during callus proliferation identified TaWOX9 and TaWOX5 as potential enhancers of wheat transformation efficiency, which was later confirmed experimentally [49,50]. In the present study, RNA-seq analyses across tissues, hormone treatments, and contrasting large- and small-grain wheat varieties revealed that TaOTU6-7B is highly expressed during early and mid-grain development and is responsive to GA and JA. Moreover, its expression differed significantly between large-grain wheat PD34 and small-grain wheat MY11847 at 7 and 11 days after flowering. Collectively, these findings strongly suggest that TaOTU6-7B functions as a negative regulator of GS.

3.2. Targeted Knockout of the Negative Regulator TaOTU6 via Genome Editing Simultaneously Improves Wheat Grain Width and Weight

Genome editing enables precise modification of endogenous genes to enhance desirable traits or eliminate unfavorable ones, thereby accelerating crop improvement [51,52]. Unlike transgenic approaches, genome editing introduces targeted mutations without foreign DNA insertion, and many regulatory frameworks now distinguish genome-edited crops from genetically modified organisms, gradually easing regulatory constraints while maintaining biosafety standards [53,54]. As a result, genome editing technologies, particularly CRISPR/Cas9, have become a powerful tool for both functional genomics and practical crop breeding.

Grain size is a key agronomic trait defined by length, width, and thickness, with GW being a major determinant of grain weight and yield [55,56]. Ubiquitination and deubiquitination are central to GS regulation. For example, GW2, a RING-type E3 ubiquitin ligase, negatively regulates GW by ubiquitinating and promoting the degradation of WG1, thereby affecting yield [57]. OsUBP15, encoding a ubiquitin-specific protease with in vitro deubiquitination activity, acts as a positive regulator of GS when overexpressed [58]. OTU family members are also DUBs implicated in seed size regulation in other species [59]. AtOTU1 functions as a histone deubiquitinase that represses DA1 and DA2, key regulators of organ and seed size [60]. In rice, WTG1, encoding an Otubain-like protease homologous to human OTUB1, plays a critical role in GS control, with mutations leading to wider grains and leaves [61]. In the previous analysis, the TaOTU6-7B appeared to be a negative regulator gene for wheat GS.

Using genome editing, we knocked out TaOTU6-7B and its homoeologs, generating mutants with distinct genotypes. Phenotypic analyses showed that GW and TKW were significantly increased in AAbbDD, AABBdd, aabbDD, and aabbdd mutants relative to the WT. Similar results have been reported for wheat TaOTUB1 homoeolog knockouts, which also exhibit significantly increased grain size and yield [62]. Interestingly, while previous studies reported no significant differences in grain size and yield between AABBdd and WT, our study observed no significant differences between aaBBDD and WT, likely reflecting differences in genetic background among germplasms. Moreover, compared with AAbbDD and AABBdd, the aabbdd mutant exhibited a substantially greater increase in GW and TKW, indicating functional divergence among homoeologous gene copies across the A, B, and D subgenomes and a cumulative effect of B- and D-subgenome copies on GW and TKW regulation (Figure S6). The phenotypes of the mutants in the field were further measured. The GW and TKW of the mutant with genotype aabbdd increased significantly, which was consistent with the traits observed in the greenhouse. But the magnitude of mutant effects on grain traits differed between field and greenhouse conditions (Figure 5C and Figure S6). This underscores the necessity of field trials for translating functional gene discoveries into practical agricultural applications, as field performance ultimately determines socio-economic impact. In addition, crude protein content was significantly higher in the mutants than in the WT, indicating that TaOTU6 knockout not only enhances grain width and weight but also improves grain quality. Together, these findings identify TaOTU6 as a valuable genetic target for simultaneously improving wheat yield and quality.

3.3. Interaction Between TaOTU6-7B and TaUBC13 and Their Antagonistic Expression Patterns During Wheat Grain Development

Recent studies indicate that TaOTUB1 regulates GS by delaying flowering and shortening the grain-filling period, and physically interacts with TaRCN2, the wheat homolog of the rice flowering regulator OsRCN2 [62]. OsUBC13 functions as an E2 ubiquitin-conjugating enzyme and has been shown to interact with OsPUB9, suppressing its degradation and thereby modulating leaf angle and GS [63]. We cloned the wheat homolog TaUBC13 and experimentally confirmed its physical interaction with TaOTU6-7B (Figure 6A–C). Expression profiling revealed that TaUBC13 is highly expressed in grains at 11 and 19 days after flowering, whereas TaOTU6-7B shows higher expression at 3, 7, and 15 days after flowering and lower expression at 11 and 19 days. Thus, TaUBC13 and TaOTU6-7B exhibit reciprocal expression dynamics during grain development. Notably, TaUBC13 expression is significantly elevated in homozygous TaOTU6 mutants (Figure 6D,E and Figure S4), indicating that TaUBC13 may also function in regulating grain size and weight and acts antagonistically to TaOTU6-7B. This indicates that TaUBC13 may also be a potential functional gene regulating grain size and weight and has opposite functions to TaOTU6-7B. Together, these findings highlight a coordinated regulatory network between ubiquitination and deubiquitination in controlling GS and GW, providing ideas for further understanding the molecular mechanism by which ubiquitination and deubiquitination regulate wheat grain development.

4. Materials and Methods

4.1. Plant Materials and Phenotypic Assessment

Hexaploid wheat cultivar CS was grown in the experimental field of Northwest A&F University (34°16′ N, 108°4′ E), Yangling, Shaanxi Province. Gene editing receptor material BobWhite-Cas9 and derived mutants were cultivated in a controlled greenhouse under a 16-h light/8-h dark cycle at 23 °C/18 °C and 10,000 lx. T_3_ homozygous mutant lines and BobWhite-Cas9 control plants were planted in the transgenic experimental field of Northwest A&F University. GL, GW, and TKW were measured using the Wan Shen image analysis system. Nicotiana benthamiana plants were used for subcellular localization, Dual-LUC assays, and BiFC assays, grown under a 16-h light/8-h dark cycle at 25 °C/20 °C and 10,000 lx.

4.2. Genome-Wide Identification of OTU Genes in Wheat

Wheat genome sequences and GFF3 annotation files (IWGSC v1.1) were retrieved from Ensembl Plants “https://plants.ensembl.org/index.html (accessed on 10 November 2024)”. The OTU domain (PF02338) from PFAM “http://pfam-legacy.xfam.org/ (accessed on 12 November 2024)” was used to query the wheat proteome via HMMER 3.0 [64]. For each gene, the longest transcript was retained, and incomplete sequences lacking start or stop codons were discarded. Candidate sequences were filtered with E-value < 1 × 10^−10^ and validated using NCBI-CDD “https://www.ncbi.nlm.nih.gov/cdd/ (accessed on 12 November 2024)”, identifying 49 wheat OTU proteins. Chromosomal positions were mapped using TBtools (v0.664432), and genes were numbered according to chromosome location. Using similar approaches, 12 Arabidopsis OTUs and 20 rice OTUs were identified for comparative phylogenetic analysis. Theoretical isoelectric points (pI), molecular weights (MW), and GRAVY values were calculated using ExPASy “https://www.expasy.org/ (accessed on 22 November 2024)”, and domain positions were determined via NCBI-CDD.

4.3. Phylogenetic Analysis, Gene Structure, and Conserved Motif Identification

Multiple sequence alignment of wheat OTUs, along with Arabidopsis and rice OTUs, was performed using Clustal W v2.0 [65]. Phylogenetic trees were constructed via the Neighbor-Joining (NJ) method in MEGA11.0 [66]. Exon-intron structures were retrieved from genome annotation files. Conserved motifs in OTU proteins were identified using MEME v5.2.0 “https://meme-suite.org/meme/ (accessed on 18 November 2024)” and visualized in TBtools.

4.4. Cis-Acting Element, Gene Duplication, and Synteny Analysis

Promoter sequences (2000 bp upstream of ATG) were extracted using TBtools and analyzed in PlantCARE “http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 10 December 2024)” to predict cis-regulatory elements [67]. OTU gene duplication and syntenic relationships were assessed using MCScanX (v1.0.0) and visualized with Circos (v0.69-9). Collinearity among wheat, Arabidopsis thaliana, rice, and barley OTUs was analyzed using Advanced Circos in TBtools. Synonymous (Ks) and nonsynonymous (Ka) substitution rates were calculated with KaKs_Calculator v2.0. Divergence times of duplicated gene pairs were estimated using L T = (Ks/2λ) × 10^−6^ Mya, with λ = 6.5 × 10^−9^ [68].

4.5. Gene Expression Profiles Analysis

Spatial-temporal expression patterns of TaOTUs were analyzed using RNA-seq datasets from five tissues (root, stem, leaf, spike, and grain) under different developmental stages and hormone treatments, retrieved from URGI Wheatomics “http://wheatomics.sdau.edu.cn/ (accessed on 21 December 2024)”. Expression in large-grain PD34 and small-grain MY11847 at 3, 7, and 11 DAF was also analyzed. FPKM values were normalized using Z-score, and heatmaps were generated in TBtools [69].

4.6. Cloning and Expression Analysis of TaOTU6-7B

Total RNA was extracted from CS leaves using TRNzol Universal Reagent (TIANGEN, Beijing, China) and reverse transcribed into cDNA with a FastKing cDNA First Strand Synthesis Kit (TIANGEN, Beijing, China). Gene-specific primers were used to amplify the TaOTU6-7B CDS (Table S1). The ORF was cloned into pYJ-GFP to generate 35S::TaOTU6-7B-GFP, which, along with 35S::GFP control and nuclear marker AtHY5-RFP, was transformed into Agrobacterium tumefaciens GV3101. Leaves of 3–4-week-old N. benthamiana were infiltrated and imaged using an Olympus laser confocal scanning microscope (Olympus, Tokyo, Japan) 48 h post-infiltration.

RNA extraction from various tissues of BobWhite-Cas9 and the acquisition of cDNA followed previously described protocols. The obtained cDNA was used as a template for the polymerase chain reaction. qRT-PCR was performed using cDNA from BobWhite-Cas9 tissues with LightCycler^®^ 96 (Roche, Basel, Switzerland), using TaActin2 as a reference. Three biological replicates with three technical replicates each were included, and relative expression levels were calculated via the 2^−ΔΔCt^ method [70].

4.7. Generation of Transgenic Plants

Two sgRNAs were designed to simultaneously target the exons of TaOTU6 (-7A, -7B, and -7D). The pCBC-MT1T2 vector was used as the intermediate vector, and the double sgRNA was driven and expressed by the TaU6 and OsU3 promoters, respectively. The GRF-GIF fusion gene driven by the maize ubiquitin promoter was used to enhance the regenerative efficiency of wheat transformation [71]. Particle bombardment of sgRNA expression vectors was performed on BobWhite-Cas9 embryos 20 days after flowering. Positive seedlings were detected by Sanger sequencing at the seedling stage.

4.8. Verified Interaction

Y2H assays. TaOTU6-7B CDS was cloned into pGBKT7 (bait), and TaUBC13 CDS into pGADT7 (prey). Vectors were co-transformed into Y2H Gold yeast, with BK-P53 + AD-T and BK-Lam + AD-T as positive and negative controls.

BiFC assay. TaOTU6-7B and TaUBC13 CDSs were fused to nEYFP and cEYFP, co-expressed with p19 in N. benthamiana through Agrobacterium tumefaciens infiltration, and YFP fluorescence was observed 48 h post-infiltration.

Dual-LUC assay. TaOTU6-7B and TaUBC13 were cloned into 35S::nLUC and 35S::cLUC vectors, co-infiltrated with p19 into N. benthamiana leaves via Agrobacterium tumefaciens infiltration, and LUC activity was measured at 48 h. All assays were repeated ≥3 times.

4.9. Statistical Analysis

Data were analyzed using SPSS (v22.0). One-way ANOVA with Duncan’s test was applied at p < 0.01 or p < 0.05. Analyses were based on three independent replicates. Figures were prepared using Origin 2018 and Photoshop CS6.

5. Conclusions

In this study, 49 wheat OTU genes were identified and classified into four subfamilies. Sequence, expression, and functional analyses implicate TaOTU6-7B as a negative regulator of grain size. CRISPR/Cas9-mediated knockout of TaOTU6-7B and its homoeologs significantly increased grain width and weight, demonstrating its potential as a target for crop improvement. Interaction with TaUBC13 highlights a coordinated regulatory network between ubiquitination and deubiquitination, providing mechanistic insights into wheat grain development.