Pangenome-Wide Identification, Evolutionary Characterisation and Stress- Responsive Dynamics of SIZ1-Type SUMO E3 Ligase Gene Family in Bread Wheat (Triticum aestivum L.)

Pangenome-Wide Identification, Evolutionary Characterisation and Stress- Responsive Dynamics of SIZ1-Type SUMO E3 Ligase Gene Family in Bread Wheat (Triticum aestivum L.) | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Pangenome-Wide Identification, Evolutionary Characterisation and Stress- Responsive Dynamics of SIZ1-Type SUMO E3 Ligase Gene Family in Bread Wheat (Triticum aestivum L.) Hemant Sharma, Mallana Gowdra Mallikarjuna, Garudapalya Muniswamy Keerthi, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9114016/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Background: SUMOylation, a key post-translational modification in plants, modulates diverse stress responses through SUMO E3 ligases (SIZ1). The SIZ1 gene family remains uncharacterized in bread wheat ( Triticum aestivum L.), despite its proven functional relevance in model systems. Results: The pangenome survey of bread wheat identified 15 TaSIZ1 genes, unevenly distributed across the genomes. The reference genome Chinese Spring harboured 14 TaSIZ1 genes distributed across homoeologous groups 1, 3, 4, 5, and 7. These intron-rich TaSIZ1 genes with 14–17 exons encode nuclear-localised, hydrophilic SIZ proteins (pI 4.85–6.60) that carry a conserved zf-MIZ1 domain. The TaSIZ1 family expansion occurred predominantly through whole-genome and segmental duplications under purifying selection (Ka/Ks < 1). The synteny and orthology among wheat, barley, rice, maize, and sorghum SIZ1 genes further confirmed evolutionary conservation, with strong purifying selection (Ka/Ks: 0.21–0.46). Promoter analysis revealed abundant stress- and hormone-responsive cis-elements (ABRE, MBS, ARE, GT1), alongside major transcription factor binding sites for ERF, WRKY, MIKC_MADS, and MYB families. Expression profiling showed higher basal activity of TaSIZ1-1A, 1B, 1D, 3B, and 3D in vegetative tissues and induction by heat, drought, powdery mildew, and leaf rust. Conclusions: Our study showed that TaSIZ1 genes are structurally conserved but show functional divergence across homoeologs and genotypes. The strong, specific induction of selected copies underscores their utility in enhancing stress resilience. This study provides the first comprehensive framework for understanding TaSIZ1 regulation in wheat and nominates candidate genes for multi-stress engineering and wheat breeding. Post-translational modification SUMOylation SUMO E3 ligase Evolution Expression dynamics Stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Background Post-translational modifications (PTMs) regulate the function, stability, and interaction networks of proteins, thereby influencing numerous biological processes. SUMOylation is one of the crucial PTMs, where a Small Ubiquitin-like Modifier (SUMO) protein is covalently attached to target proteins, modulating their activity, localisation, or stability. SUMOylation modifications are conserved among eukaryotes and are involved in critical cellular processes, including transcription, DNA repair, response to abiotic and biotic stresses, and cell cycle regulation [ 1 , 2 ]. SUMOylation involves a cascade of enzymatic reactions, including the activation (E1), conjugation (E2), and ligation (E3) of SUMO to target proteins. The E3 SUMO ligases facilitate substrate specificity and enhance the efficiency of SUMO transfer. Among known E3 ligases, the SAP and Miz1 (SIZ1)-type SUMO E3 ligase is particularly well-studied. SIZ1 plays a pivotal role in plant stress and developmental processes [ 3 ]. In Arabidopsis thaliana , the SIZ1 mediate the SUMOylation of key proteins of pathways controlling photomorphogenesis, flowering, phosphate starvation, and freezing tolerance, showing responses to cold, drought, and heat stress conditions, highlighting its significance in stress adaptation [ 4 – 7 ]. Functional studies across species demonstrate that overexpression of SIZ1 homologs, such as OsSIZ1 in rice and cotton, significantly enhances tolerance to abiotic stresses, including drought, heat, and salt, while also improving photosynthesis, biomass accumulation, and yield under adverse conditions [ 8 , 9 ]. Recently, alternative splicing of SIZ1 has been shown to generate distinct isoforms with stress-specific localisation and activity, underscoring the dynamic regulation of SUMOylation in response to environmental fluctuations [ 10 ]. Furthermore, SIZ1 plays a key role in enhancing salicylic acid (SA) signalling, which is essential for systemic acquired resistance (SAR) against biotrophic pathogens. It facilitates the SUMOylation of key defence regulators, such as NPR1 (Non-Expressor of Pathogenesis-Related Genes 1), thereby stabilising NPR1 and promoting the expression of defence-related genes [ 2 , 3 , 11 ]. Additionally, SIZ1 helps balance growth and immune responses, preventing the detrimental effects of constitutive defence activation. In Arabidopsis thaliana , the siz1 mutants exhibit enhanced resistance to pathogens, such as Pseudomonas syringae , but suffer from growth retardation due to excessive salicylic acid (SA) signalling [ 12 ]. SIZ1 also interacts with the jasmonic acid (JA) pathway, which fine-tunes the crosstalk between SA- and JA-mediated defence to optimise plant immunity [ 12 – 14 ]. Similarly, SIZ1 provides temperature-sensitive immunity, where the SIZ1 function is essential for maintaining disease resistance across different environments [ 14 ]. These evidences underline SIZ1 as a key regulator of plant immunity, making it a valuable target for improving crop disease resistance. Bread wheat ( Triticum aestivum L.) is a major staple crop of global importance for food and nutrition with a large and complex genome [ 15 ]. The identification and characterisation of SIZ1-like genes help in understanding the SUMOylation pathway, which is crucial for improving stress resilience and yield stability. Previous studies have identified components of the SUMOylation machinery in model plants, such as Arabidopsis and rice ( Oryza sativa ), but the comprehensive identification, evolutionary and functional characterization of SUMO E3 ligases in wheat remains limited [ 8 , 9 , 16 – 20 ]. Recent advancements in bioinformatics tools and the availability of high-quality wheat genome assemblies (IWGSC RefSeq v2.1) have facilitated the identification of gene families across the wheat genome [ 15 ]. Utilising wheat genomic resources, a genome-wide identification and analysis of SIZ1-type SUMO E3 ligases in wheat can provide insights into their gene structure, evolutionary and regulatory relationships and expression responses. This information can serve as a foundation for functional studies and the development of wheat varieties with enhanced stress tolerance through molecular breeding or genetic engineering. Thus, the present investigation aimed to identify and characterise SIZ1-like SUMO E3 ligase genes in the wheat genome, determine the evolutionary relationships between wheat SIZ1 homologs and those from other cultivars, and decipher the regulatory and functional response of the identified SIZ1 genes to various stresses. Materials and Methods Sequence Retrieval and Identification of SIZ1 Genes and Proteins The whole-genome sequences and annotation files of common wheat ( Triticum aestivum ) and the sequences of SIZ1 from Arabidopsis thaliana (AtSIZ1, AT5G60410) and rice (OsSIZ1, Os05t0125000) were downloaded from the EnsemblPlants database ( https://plants.ensembl.org/ ). The wheat pangenomes were retrieved from the wheat pangenomes database ( https://wheatgenomes.com/ ). The protein sequences (CDSs) of AtSIZ1 and OsSIZ1 were used to perform BLASTp search (E-Value ≤ 1e-5) against the wheat proteome assembly (IWGSC RefSeq v2.1). HMMSearch (E-value ≤ 1e-5) was performed across the whole wheat proteome for pfam profile PF02891. The non-redundant hits from both the BLASTp and HMMSearch were again subjected to HMMScan to confirm the presence of the PF02891 domain and finalise the number of SIZ1 genes in wheat. Further, to mine SIZ1 in related species for evolutionary analysis, the identified wheat SIZ1 (TaSIZ1) proteins were used as query sequences to perform BLASTp searches (E-Value ≤ 1e-5) against the rice, sorghum, maize and barley proteomes downloaded from EnsemblPlants Database ( https://plants.ensembl.org/ ), and the HMMSearch and HMMScan were performed as explained above. Analysis of Chromosome Location, Gene Structures, and Motifs The physical locations and gene structures of the identified SIZ1 genes were retrieved from the GFF file downloaded from the EnsemblPlants database and mapped using TBtools ( https://github.com/CJ-Chen/TBtools ) [ 21 ]. Conserved motifs within SIZ1 proteins were identified using the MEME Suite ( http://meme-suite.org/tools/meme ), and motif annotation was verified using the HMMER tool ( https://www.ebi.ac.uk/Tools/hmmer/search/phmmer ). Physico-chemical properties and Gene Ontology (GO) Terms Analysis Physicochemical properties, including molecular weight, isoelectric point (pI), and amino acid composition, were calculated using the ProtParam tool ( http://web.expasy.org/protparam ) [ 22 ]. Subcellular localization and Gene Ontology (GO) annotations of SIZ1 proteins were predicted using the WoLFPSORT ( https://wolfpsort.hgc.jp ) server and the ShinyGO tool ( http://bioinformatics.sdstate.edu/go ), respectively. Evolutionary Analyses of SIZ1 Family in Wheat and Related Species Multiple Sequence Alignment and Phylogenetic Analysis The SIZ1 proteins from wheat, rice, maize, barley and sorghum were aligned with MUSCLE [ 23 ]. The multiple alignment was trimmed with the trimAL v1.2 tool ( http://trimal.cgenomics.org ) [ 24 ]. The phylogenetic tree was constructed using the maximum likelihood method. The best model identified using Bayesian information criterion and phylogenetic tree was constructed using 5000 ultrabootstraps using IQ-TREE version 2 [ 25 ] software. The tree was realised in iTOL [ 26 ]. Analysis of SIZ1 family expansion and selection pressure Identification of TaSIZ1 paralogs was performed through self-BLASTp search (e-value < 1e-5) within the wheat proteome using BLAST + 2.10.1 ( https://www.ncbi.nlm.nih.gov/ ). Subsequently, DupGen_finder software was used to harvest the various duplications in the wheat genome [ 27 ]. For identification of orthologs across the target species, viz., wheat, rice, maize, barley, and sorghum, the SIZ1 sequences were subjected to OrthoFinder software ( https://github.com/davidemms/OrthoFinder ) [ 28 ]. The nonsynonymous rate (Ka), synonymous rate (Ks) and evolutionary constraint (Ka/Ks) were calculated for each of the paralog and ortholog pairs of SIZ1 using KaKs calculator v.3.0 ( https://ngdc.cncb.ac.cn/tools/kaks ) [ 29 ] Synteny and Collinearity Analysis The whole proteomes of related cereals, viz ., rice, maize, barley and sorghum, were aligned with the wheat proteome using the BLASTp with an e-value < 1e-10 and the best five hits per query. The MCScanX software was used to identify syntenic and collinear blocks [ 30 ] and visualised with TBtools v2.052 ( https://bio.tools/tbtools ) [ 21 ]. Regulatory analysis SIZ1 Genes Prediction of Cis-acting Elements in SIZ1 Promoters Sequences of 1.5 kb upstream regions of SIZ1 genes were retrieved and scanned for cis-acting elements using the PlantCARE server ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ). Only response elements with a matrix value > 5 on the sense strand were fetched for subsequent analysis and interpretation [ 31 ]. SIZ1-Regulatory Network Analysis Putative microRNA (miRNA) targets in SIZ1 genes were predicted using the psRNATarget server ( https://plantgrn.noble.org/psRNATarget/ ) with default parameters. The Transcription factor (TF) was predicted using PlantRegMap ( https://plantregmap.gao-lab.org/binding_site_prediction.php ). Further, the regulatory network of TaSIZ1 s was visualised with Cytoscape [ 32 ]. Gene Expression Analysis In-silico Expression Analysis Transcriptome data from the Wheat Expression Database ( http://www.wheat-expression.com/ ) were retrieved to analyse the expression of SIZ1 genes across different tissues and stress conditions. The heatmaps were generated using TBtools. Quantitative real-time PCR analysis of TaSIZ1 genes Plant material and treatments The leaf rust–susceptible and drought-tolerant cultivar C306 and the leaf rust–resistant but comparatively drought-sensitive cultivar HD2888 were used to study the TaSIZ1s expression (Supplementary Table 1). Seeds were surface-sterilised with 0.1% HgCl₂ for 2 minutes, rinsed thoroughly with sterile distilled water, and treated with 0.5% Bavistin before sowing. Plants were grown in a controlled growth chamber under greenhouse conditions in soil-filled pots. For the leaf rust expression analysis, 14-day-old seedlings were inoculated with the 77 − 5 strain of Puccinia triticina . Leaf samples were collected at 0, 24, 48, and 96 hours post-inoculation. For drought stress treatment, 14-day-old seedlings of both genotypes were transferred to a nutrient solution supplemented with 10% polyethene glycol (PEG-6000) to simulate osmotic stress. Leaf tissues were harvested after 7 days of treatment. All collected samples were immediately flash-frozen in liquid nitrogen, stored at − 80°C, and later used for total RNA isolation. RNA extraction and quantitative real-time PCR Six highly expressed TaSIZ1 gene copies (based on in-silico expression analysis) were selected for qRT-PCR validation. The primers used for RT-qPCR of TaSIZ1 genes were synthesised using Primer Express Software v3.0.1 (Thermo Fisher Scientific; Supplementary Table 2). Total RNA was isolated using the TRIzol reagent (Ambion, Naugatuck) as per the manufacturer’s specifications and treated with RNase-free DNase I (Invitrogen, California, United States) for 15 minutes to degrade any residual genomic DNA. First-strand cDNA was synthesised from DNase I-treated total RNA using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, Massachusetts, United States) according to the manufacturer’s instructions. RT-qPCR was performed in optical 96-well plates using a CFX96 (Bio-Rad) and SYBR Green (Bio-Rad). The Wheat β-actin gene (accession number AB181991) was used as an endogenous control. The CT values were analysed through the 2 −ΔΔCT method [ 33 ]. Results Mining and Copy Number of SIZ1 genes in Wheat Pangenome Cultivars and Related Species A pangenome-wide identification of the SIZ1 gene family, using the hexaploid wheat reference genome (cv. Chinese Spring) along with 12 high-quality cultivar assemblies from the pangenome project, revealed 15 SIZ1 genes (Table 1 ; Supplementary Table 3; Fig. 1 ). Genes were systematically renamed based on their chromosomal positions in Chinese Spring as TaSIZ1-1A to TaSIZ1-7D , with corresponding copies in each cultivar assigned the same names. In Chinese Spring, 14 TaSIZ1 genes were identified with three homoeologs each on chromosomes 1, 4, 5, and 7 of the A, B, and D genomes, and two on chromosome 3 ( TaSIZ1-3B and TaSIZ1-3D ). Similarly, the genotypes Lancer, Landmark, and Mattis showed 14 TaSIZ1 genes on chromosomes 1, 4, 5, and 7 of the A, B, and D genomes, and two on chromosome 3 ( TaSIZ1-3A and TaSIZ1-3B ). The lowest number of TaSIZ1 genes (12) is reported in the Alchemy and Kariega genotypes, with non-significant hits on 1B, 4D, 7D, and 7A, 7B, 7D chromosomes, respectively (Fig. 1 ). Further, mining SIZ1s in related species, viz ., barley, maize, rice, and sorghum, yielded 5, 4, 3, and 4 SIZ1s, respectively (Supplementary Table 4). All the predicted SIZ1 proteins had the characteristic zinc-finger MIZ (zf-MIZ; PF02891) domain, with Zn²⁺-coordinating Cys/His residues located in the central region of the protein (Fig. 1 A), which confirms that the identified sequences represent SIZ1-type SUMO E3 ligases. Additionally, the target species, including wheat, showed non-uniform distributions of SIZ1 genes across their genomes (Supplementary Tables 3–4; Fig. 1 ). Table 1 The distribution and range of various physicochemical properties of TaSIZ1 sequences mined from thirteen pangenome species. Gene No. of genomes* Gene Length (bp) cDNA Length (bp) CDS Length (bp) Gene % GC content Protein Length (aa) GRAVY Aliphatic Index Instability Index Molecular Weight (kDa) Aromaticity Isoelectric Point (pl) TaSIZ1-1A 13/13 14874–16412 3105–4826 2619–2625 41.12–42.15 872–874 -0.61–0.60 70.76–71.22 45.69–46.19 96.91–97.02 0.07 4.97 TaSIZ1-1B 12/13 14559–16368 3001–4042 2622–2625 40.57–41.29 873–874 -0.59–0.59 72.12–72.35 44.97–45.84 96.99–97.11 0.07 4.90 TaSIZ1-1D 13/13 15318–21517 3070–5037 2622–4512 40.82-47.00 873–930 -0.6–0.58 69.55–73.11 45.00-48.70 96.91-103.32 0.07 4.92–5.06 TaSIZ1-3A 6/13 7520–7878 3057–3572 2787–2805 42.48–43.06 928–934 -0.54–0.48 79.08–81.73 46.39–51.31 102.44-103.32 0.05 4.85–4.99 TaSIZ1-3B 12/13 6580–8015 2775–3705 2775–2793 42.13–42.98 924–930 -0.53–0.52 78.47–79.01 45.93–46.46 102.26-103.04 0.05 4.87–4.93 TaSIZ1-3D 4/13 6490–7634 2787–3363 2787–2901 42.06–42.57 928–966 -0.5–0.49 79.39–80.61 45.68–47.93 102.64-106.72 0.05 4.92–4.94 TaSIZ1-4A 13/13 8317–9057 2334–3074 2334–2334 40.54–41.55 777–777 -0.55–0.55 73.87-74.00 38.93–39.17 85.16–85.20 0.06 5.16 TaSIZ1-4B 13/13 8764–12143 2334–4667 2334–3972 40.67–41.52 777–837 -0.57–0.56 72.79–74.13 38.25–43.56 85.12–91.88 0.06 5.02–5.78 TaSIZ1-4D 12/13 8358–9442 2334–3021 2334–2517 40.63–41.50 777–838 -0.58–0.56 73.28–73.87 38.57–43.42 85.26–91.99 0.06 5.20–5.84 TaSIZ1-5A 13/13 8258–9238 2379–3359 2379–2583 40.99–41.98 792–860 -0.61–0.59 79.03–80.97 40.71–48.5 87.73–94.79 0.05 5.27–5.56 TaSIZ1-5B 13/13 8939–9504 2986–3154 2385–2661 40.59–41.96 794–886 -0.63–0.46 74.28–80.54 41.88–62.01 87.97–97.16 0.05 5.15–6.60 TaSIZ1-5D 13/13 8852–10028 2904–3613 2355–2622 41.50-42.26 784–873 -0.62–0.59 78.29–79.45 40.74–50.43 86.54–96.34 0.05 5.20–5.69 TaSIZ1-7A 12/13 8688–10327 2788–3319 2352–2739 38.65–40.71 783–912 -0.47–0.44 72.45–74.06 62.29–64.13 85.01–98.89 0.05 6.36–7.02 TaSIZ1-7B 11/13 9003–9723 2941–3373 2385–2661 40.18–41.86 794–886 -0.6–0.46 74.28–80.54 41.88–62.01 87.97–96.28 0.05 5.15–6.60 TaSIZ1-7D 12/13 8075–8782 2825–3222 2316- 2652 38.57–40.61 771–883 -0.45–0.40 73.44–74.98 60.3-61.88 83.9-95.97 0.05 6.11–6.45 Physico-Chemical Properties of SIZ1 Genes and Protein Sequences Genomic lengths range from about 6.6 kb ( TaSIZ1-3B in Kariega) to over 21 kb ( TaSIZ1-1D in Chinese Spring) owing to differences in intron number and size rather than changes in the core CDS. In contrast, cDNA lengths are generally 3.0-3.6 kb, with a few longer transcripts up to 5,037 bp (Alchemy: TaSIZ1-1D ), suggesting alternative splicing or longer UTRs in certain genotypes. CDS lengths are mostly 2,316–2,811 bp, with some longer variants up to 4,512 bp, resulting in protein lengths of 771–966 amino acids. TaSIZ1s from chromosome 4 (TaSIZ1-4A/4B/4D) consistently showed shorter proteins of about 777 amino acids (aa), while TaSIZ1s from chromosomes 3 and 5 code longer proteins (930–966 aa), likely due to domain extensions or low-complexity region insertions. The GC content of TaSIZ1 genes is moderate and highly conserved, typically ranging from 38.5% to 47.0%. Chromosome-1D TaSIZ1s have the highest GC content (46–47%) compared to 7A and 7D TaSIZ1s (38–39% GC) (Supplementary Table 3). Physicochemical analysis showed that TaSIZ1 proteins are highly similar across all cultivars, reinforcing the strong functional conservation within the SIZ1 SUMO E3 ligases across the target pangenome species. Predicted molecular weights range from approximately 83.90 kDa to 106.7 kDa, consistent with CDS length variation. Aromaticity values for TaSIZ1 were low (about 0.05–0.07), typical of regulatory proteins rich in charged and polar residues. All TaSIZ1 proteins have strongly negative GRAVY scores (− 0.63 to − 0.40), reflecting a hydrophilic nature, as observed in nuclear-localised proteins, and allowing extensive protein–protein interactions. Despite this, they display relatively high aliphatic indices (69–82), suggesting reasonable thermostability. Most proteins have instability indices above 40 and are predicted to be unstable, a common property of signalling and scaffold proteins. However, chromosome-4 homoeologs (TaSIZ1-4A, TaSIZ1-4B, and TaSIZ1-4D) have instability indices below 40 and are classified as stable, indicating this clade may form more rigid or long-lived structural components within the SIZ1 network. Predicted isoelectric points (pI) are mostly in the acidic to near-neutral range (pI 4.85–7.02), so nearly all TaSIZ1 proteins carry a negative net charge at pH 7, with only a few group-7A/7D members approaching neutrality or slight basicity (Supplementary Table 3–4). Gene structure and motif analysis of TaSIZ1 Genes Structural analysis of the TaSIZ1 genes showed that all members have multiple exons and introns, with a highly conserved exon-intron organisation across homoeologs. The genes are intron-rich, with exon counts ranging from 14 to 17 and intron counts from 13 to 16. TaSIZ1-1A and TaSIZ1-1B are the most complex, each with 17 exons and 16 introns, while TaSIZ1-1D has 16 exons and 15 introns. Most other genes ( TaSIZ1-3B , TaSIZ1-4A , TaSIZ1- 4B, TaSIZ1- 4D, TaSIZ1-5A , TaSIZ1-5B , TaSIZ1-5D , TaSIZ1-7B ) share 16 exons and 15 introns. TaSIZ1-3D and TaSIZ1-7A have 15 exons and 14 introns, and TaSIZ1-7D has 14 exons and 13 introns. Phase 0 introns are most common, accounting for about 43.48% of the total, followed by phase 2 with 32.61% and phase 1 with 23.91% (Fig. 2 A, B). Motif analysis showed that TaSIZ1 proteins contain multiple conserved motifs, with each gene having 4 to 10 motifs (Fig. 2 ). The majority of TaSIZ1 members possess nearly all motifs (9–10), while TaSIZ1-3D, TaSIZ1-7A, and TaSIZ1-7D have fewer, indicating structurally simplified variants. Motif 1, along with Motifs 4, 6, and 9, was found across the TaSIZ1 genes and was the most conserved, whereas Motifs 8 and 10 were found less commonly in the TaSIZ1 family (Fig. 2 A, C). Gene Ontology (GO) Terms and Sub-Cellular Localisation Analysis GO annotation indicates that all 14 TaSIZ1 proteins from cv. Chinese spring was significantly enriched for the molecular function GO term zinc ion binding (GO:0008270). Further, the 12 of 14 TaSIZ1s (excluding TaSIZ1-3D; TaSIZ1-7A) were significantly enriched for Sumo ligase activity (GO:0061665), Sumo transferase activity (GO:0019789), Aminoacyltransferase activity (GO:0016755), Ubiquitin-like protein transferase activity (GO:0019787), Transition metal ion binding (GO:0046914) and Acyltransferase activity (GO:0016746). Similarly, for biological function category the these 12 TaSIZ1s were showed significant enrichments for Protein sumoylation (GO:0016925), Peptidyl-lysine modification (GO:0018205), Peptidyl-amino acid modification (GO:0018193), Protein modification by small protein conjugation (GO:0032446), Post-translational protein modification (GO:0043687), Protein modification by small protein conjugation or removal (GO:0070647) under the biological function category (Supplementary Table 5). Further, the subcellular localisation predictions indicate that TaSIZ1 proteins are predominantly nuclear localised, with confidence scores of 0.89–0.96. (Supplementary Table 6). Evolutionary Analyses of the SIZ1 Family in Wheat and Related Species Multiple Sequence Alignment and Phylogenetic Analysis The multiple sequence alignment of SIZ1 proteins using MUSCLE produced an intact alignment of the PFAM domain PF02891 from 345 to 395 amino acids, indicating sequence conservation and suitability for phylogenetic analysis (Fig. 3 A). The IQ-Tree model find option identified JTT+G4 based on the Bayesian Information Criterion (BIC) as best model for phylogenetic tree construction. The phylogenetic analysis grouped SIZ1 proteins from wheat, barley, rice, maize, and sorghum into three major clades based on percent identity (> 50%) among the protein sequences (Supplementary Table 7), corresponding to wheat homoeologous chromosome groups 1/3, 4/5, and 7 (Fig. 3 B). Clade I contains wheat TaSIZ1 proteins from chromosomes 4A, 4B, 4D, 5A, 5B, and 5D, barley HvSIZ1-4H/5H, OsSIZ1b, ZmSIZ1b, SbSIZ1a, and SbSIZ1d from barley, rice, maize, and sorghum, respectively. Wheat chromosome 5 homoeologs show high per cent identity (95.42–95.71%), and HvSIZ1-5H shares 92.29–93.19% identity with them. Wheat chromosome-4 proteins are nearly identical (91.98–99.05% identity), with HvSIZ1-4H showing 89.12–93.55% identity. OsSIZ1b is moderately similar (67–72%) to Triticeae SIZ1 proteins in the clade. The Panicoid pair ZmSIZ1b and SbSIZ1d is closely related (85.54%) but only 62.61–63.38% identical to TaSIZ1-5A, TaSIZ1-5B and TaSIZ1-5D, indicating an earlier divergence between Triticeae and Panicoideae. SbSIZ1a has emerged as the most distant and outlier member of this phylogenetic tree. The clade II (yellow) includes wheat TaSIZ1 proteins from chromosomes 1A, 1B, 1D, 3B, and 3D, as well as HvSIZ1-1H/3H from barley and OsSIZ1a, ZmSIZ1a, ZmSIZ1c, and SbSIZ1c from rice, maize, and sorghum. Within this clade, Triticeae proteins are highly conserved: HvSIZ1-1H shares 97.03–98.05% identity with TaSIZ1-1A/1B/1D, and HvSIZ1-3H shares 91.41–91.53% identity with TaSIZ1-3B/3D. Maize and sorghum sequences shared 73–77% identity with wheat and barley but remained highly similar to each other (> 90.00%). Clade III (green) includes TaSIZ1-7A, 7B and 7D proteins from chromosome 7, HvSIZ1-7H from barley, OsSIZ1c and OsSIZ1d from rice, ZmSIZ1d from maize, and SbSIZ1b from sorghum. The three wheat homoeologs are again highly conserved (96.12–97.40%) and share high identity (90.41–92.45%) with HvSIZ1-7H (Supplementary Table 7, Fig. 3 B). Duplication and selection pressure analysis of TaSIZ1 Gene duplications drive the expansion of gene families by generating new members that contribute to functional diversity and enable the evolution of new functions. We analysed the types of duplications and the selection pressures that influenced the expansion of TaSIZ1 genes in the wheat genome. The results indicate that single-gene duplications play a significant role in the expansion of SIZ1 (Supplementary Table 8 and Fig. 4 a). In total, 24 paralog pairs were found among the 14 TaSIZ1s of the Chinese Spring cultivar, encompassing > 50% of whole-genome duplications (13), followed by segmental (9) and dispersed duplications (2) (Fig. 4 b). Selection pressure was assessed by analysing the ratio of non-synonymous (Ka) to synonymous (Ks) substitutions, which indicates the direction and strength of natural selection on protein-coding genes. Ka/Ks analysis of 24 wheat paralogous pairs showed Ka/Ks < 1, indicating strong purifying selection and functional conservation during the expansion of TaSIZ1 sequences in the wheat genome (Supplementary Table 8; Fig. 4 c). Pairs with very low Ks (< 0.10) among the 14 duplication pairs observed within chromosomal group but between the homoeologous genomes A, B, D, except the duplication pair TaSIZ1-3D-TaSIZ1-7D (Supplementary Table 8), suggesting their recent origin. On the other hand, the paralogs between the chromosomal groups showed higher synonymous mutation rates (Ks > 0.45), suggesting that these pairs arose from ancient polyploidy events (Supplementary Table 8; Fig. 4 c). Ortholog analysis of SIZ1 in wheat and related species Ortholog analysis of TaSIZ1 genes and their counterparts in barley, rice, maize, and sorghum identified many-to-one relationships except two pairs of one-to-one orthologs between wheat and barley (TaSIZ1-3D–HvSIZ1-3H; TaSIZ1-4A–HvSIZ1-4H) and many-to-many ortholog clade group between wheat and maize (TaSIZ1-1A/1B/1D/3D–ZmSIZ1a/c). Among the orthologs between wheat and barley, three many-to-one (TaSIZ1-1A/1B/1D–HvSIZ1-1H; TaSIZ1-7A/7B/7D–HvSIZ1-7H; TaSIZ1-5A/5B/5D–HvSIZ1-5H) orthologs with mean Ks values of 0.11, suggesting their recent divergence as compared to the rest of the species (Supplementary Table 9; Fig. 5 a). The many-to-one orthologs observed between wheat-rice orthologs (TaSIZ1-1A/1B/1D/3D–OsSIZ1a; TaSIZ1-7A/7B/7D–OsSIZ1c; TaSIZ1-4A/5A/5B/5D–OsSIZ1b) and the mean Ks value of 0.61 was found among these orthologs (Supplementary Table 9; Fig. 5 b). Interestingly, wheat-maize orthologs showed a mean Ks value of 0.74 for many-to-many and many-to-one (TaSIZ1-4A/5A/5B/5D–ZmSIZ1b; TaSIZ1-7A/7B/7D–ZmSIZ1d) orthologs (Supplementary Table 9; Fig. 5 c). The highest range (0.60–4.30) and mean (2.56) of Ks values among all the many-to-one ortholog pairs (TaSIZ1-1A/1B/1D/3D–SbSIZ1c; TaSIZ1-7A/7B/7D–SbSIZ1b; TaSIZ1-4A/5A/5B/5D–SbSIZ1d) of wheat-sorghum suggesting varying divergence times owing to various evolutionary events like episodic bursts of gene duplication or biased gene retention etc (Supplementary Table 9; Fig. 5 d). All these wheat TaSIZ1s diverged under strong purifying selection (Ka/Ks < 1) for all the orthologs from rice (0.31), barley (0.27), maize (0.29), and sorghum (0.30). Synteny and Collinearity Analysis To further understand deep evolutionary insights, the syntenic and collinearity was worked out between the wheat SIZ1 genes with barley, rice, maize and sorghum (Supplementary Table 10; Fig. 6 ). Synteny analysis of the SIZ1 gene families in wheat (Ta), barley (Hv), rice (Os), and maize (Zm) showed a highly conserved collinear relationship (Supplementary Table 10; Fig. 6 ). Nineteen SIZ1-containing syntenic blocks harboured 19 pairs of collinear SIZ1s between wheat and barley, indicating the strongest syntenic signals owing to their evolutionary lineage. Following barley, maize, sorghum, and rice, wheat also showed synteny with SIZ1 genes, with wheat spanning 13, 7, and 6 synteny blocks, each containing a respective number of SIZ1 gene pairs (Supplementary Table 10; Fig. 6 ). Regulatory analysis SIZ1 Genes Cis-regulatory elements analysis Scanning of 1.5 kb upstream of the TaSIZ1 promoter identified numerous cis-acting regulatory elements, suggesting diverse regulatory potential (Fig. 7 ; Supplementary Tables S11–S13). A total of 45 distinct cis-element types were detected across all TaSIZ1 promoters. Each promoter harboured multiple elements, including abundant core promoter motifs such as the TATA and CAAT boxes. Light-responsive elements such as G-box, Box 4 , and I-box occurred in many promoters. Among the hormone-responsive elements, the ABA-responsive ABRE motif was observed most frequently (71) across the sub-genomes. The MeJA-responsive CGTCA-motif (37) and TGACG-motif (13) were also found in the promoter sequences of TaSIZ1s across the sub-genomes. Gibberellin-responsive elements ( P-box, GARE-motif ) and auxin-responsive elements ( AuxRR-core, TGA-element ) were detected at lower frequencies. Multiple stress-related cis-elements were present as well, including ARE ( anaerobic response element ), LTR ( low-temperature response element ), MBS ( MYB-binding site ), WUN-motif (wound-response element), and STRE (stress response element) (Fig. 7 ; Supplementary Tables S11–S13). Interestingly, some cis elements are confined to specific homoeologs or sub-genomes. The auxin-responsive TGA-element was found only in B-genome promoters, and a circadian rhythm-related element appeared in just two promoters ( TaSIZ1-1B; TaSIZ1-7D ). Genome-specific differences in cis-element abundance were also observed: D-genome promoters harboured roughly twice as many cis-elements (392 total) as A-genome (176) or B-genome (177) promoters (Fig. 7 ; Supplementary Tables S11–S13). TaSIZ1-7D exhibited the highest motif density, containing 23 ABREs and 21 G-box elements, 9 AREs and 7 LTRs in addition to TATA (25) and CAAT (28) boxes, indicating complex regulation in response to stresses. Genes such as TaSIZ1-1B and TaSIZ1-1D also showed rich promoter compositions. TaSIZ1-1B had 12 ABREs and 12 G-boxes, while TaSIZ1-1D harboured nine CGTCA-motifs (MeJA-responsive) and six LTRs (low-temperature responsive). Notably, TaSIZ1-5A featured a rare 3-AF3 binding site and a GCN4 Motif, suggesting a specialised regulatory pattern. Rare or unique cis-elements were gene-specific. Auxin-responsive motifs ( TGA-element, AuxRR-core ) appeared exclusively in TaSIZ1-4B , while salicylic acid-related TCA-elements and the CAG-motif were confined to TaSIZ1-4D . The AE-box was observed only in TaSIZ1-5D . Moreover, motifs related to circadian regulation (circadian), the LAMP-element , and Gap-boxes were limited to TaSIZ1-1B and TaSIZ1-7D . The WUN-motif (wound response) appeared only in TaSIZ1-3B and TaSIZ1-3D . This cis-element landscape revealed that, while specific motifs, such as ABRE , were universally distributed, many genes exhibited distinct combinations of hormone-, stress-, or development-related elements. These patterns suggest functional divergence in transcriptional regulation among TaSIZ1 genes (Fig. 7 ; Supplementary Tables S11–S13). Regulation of TaSIZ1s through MicroRNAs and Transcription Factors A total of 63 unique miRNAs were predicted to regulate 14 TaSIZ1 genes in the Chinese Spring cultivar. After removing duplicates, the number of unique miRNA targets per TaSIZ1 gene ranges from 7 to 24. TaSIZ1-1B and TaSIZ1-1D have the highest number of unique miRNAs (24), while TaSIZ1-3D has the fewest (7). The most common miRNAs across the family are tae-miR1120, tae-miR1122, tae-miR1127, tae-miR1130, tae-miR1137, tae-miR1139, tae-miR9655, tae-miR9773, tae-miR9780, and tae-miR6197, each targeting multiple TaSIZ1 copies in groups 1, 3, 4, 5, and 7. In contrast, low-frequency miRNAs include rare or single targets such as tae-miR9664 (TaSIZ1-1D), tae-miR9667 (TaSIZ1-1A), tae-miR1136 and tae-miR5175 (TaSIZ1-7A), and tae-miR1119 (TaSIZ1-1D). Other sparsely distributed miRNAs, such as tae-miR6201 and tae-miR7757, are limited to group 1 copies (Supplementary Table 14; Fig. 8 a,b,f). The prediction of transcription factor binding sites for TaSIZ1 genes revealed broad transcriptional regulation by 37 transcription factors at 3684 binding sites (Supplementary Table 15). Among the predicted TFs, ERF (1327), BBR-BPC (388), MIKC_MADS (222), C2H2 (203), MYB (187), and bZIP (119) were found to regulate all 14 TaSIZ1 genes in the Chinese spring cultivar (Supplementary Table 15; Fig. 8 c,f). Interestingly, the TF E2F/DP were predicted only in the homoeologs of chromosome 5 ( TaSIZ1-5A; TaSIZ1-5B; TaSIZ1-5D ), where the TF AAR-B was found in the homoeologs of chromosomes 3 ( TaSIZ1-53; TaSIZ1-3D ) and five only (Supplementary Table 15; Fig. 8 c). Further, among the genes, TaSIZ1-1A and TaSIZ1-1D showed the highest number of transcription factors (491), followed by TaSIZ1-3B (465) and TaSIZ1-1B (330). In contrast, TaSIZ1-3D (102) showed a lower number of TFs (Fig. 8 d). The TaSIZ1 genes of all the sub-genomes showed a majority of the TFs (34) identified in one or other gene (Fig. 8 e). Interestingly, TFs SRS and RAV were limited to A and B sub-genomes ( TaSIZ1-4A,4B ), and TALE to sub-genome B ( TaSIZ1-4B ) (Fig. 8 c,e). The regulatory network was constructed using unique TFSs and miRNAs to capture a regulatory snapshot of TaSIZ1 genes in the Chinese Spring cultivar (Fig. 8 f). The TaSIZ1 network comprised 114 nodes, 596 unique edges, and an average of 10.46 neighbours, with a density of 0.046. The network confirmed that TFs play a major regulatory role in the expression and regulation of TaSIZ1, compared with miRNAs, by targeting multiple TaSIZ1 genes, although there are more unique regulatory miRNAs than unique TFs (Supplementary Table 15; Fig. 8 f). Gene Expression Analysis In-silico Expression Analysis Tissue-specific expression of TaSIZ1 genes Tissue-specific expression analysis of TaSIZ1 genes revealed apparent variation among homoeologs. The root (The TaSIZ1-1A, TaSIZ1-1B , and TaSIZ1-1D copies displayed the highest expression levels, especially in roots (2.84–4.05 FPKM), stems (1.91–3.24 FPKM), grains (2.46–3.66), followed by spike (2.15–2.96). Similarly, TaSIZ1-3B (root: 3.64; stem: 2.90; grain: 2.92; spike, 2.18)and TaSIZ1-3D (root: 3.35; stem: 2.57; grain: 2.92; spike, 2.07) showed a similar trend of expression across all target tissues, except leaves. Furthermore, the remaining TaSIZ1s also showed moderate expression. However, leaf tissue showed lower TaSIZ1 expression levels (0.34–1.35) (Fig. 9 a). The expression analysis of TaSIZ1 genes under Powdery Mildew and stripe rust infection Expression analysis of TaSIZ1 genes based on FPKM values revealed apparent differences among homoeologs in response to pathogen infection. TaSIZ1-3B showed high expression among all genes under non-inoculated conditions (3.12); higher expression was observed in response to powdery mildew infection at 48 h (4.13) and 72 h (4.17). However, under leaf rust inoculation, TaSIZ1-3B expression did not increase with post-inoculation hours (Fig. 9 b,c). A similar expression pattern was observed for TaSIZ1-3D in response to powdery mildew and leaf rust infections (Fig. 9 b,c). Similarly, the homoeologs of chromosome 1 showed enhanced expression in response to powdery mildew infection over post-inoculation hours (Fig. 9 b), whereas no elevated or differential expression patterns were found for leaf rust infection (Fig. 9 c). The expression patterns suggest that TaSIZ1 genes are found to be more responsive to powdery mildew infection rather than leaf rust. The expression analysis of TaSIZ1 genes under Heat and Drought Expression profiling of TaSIZ1 genes under drought, heat, and combined stress conditions revealed strong, differential regulation among homoeologs. Under control conditions, TaSIZ1-1A, TaSIZ1-1B, TaSIZ1-1D , and TaSIZ1-3B exhibited moderate expression levels (2.0–2.5 FPKM), whereas most other members showed low expression (< 1.0 FPKM). During drought stress, expression of these chromosome 1 and 3 copies increased slightly at one hour (2.5–3.0 FPKM) and remained steady at six hours. Under heat stress, a sharp induction was observed, particularly in TaSIZ1-3B reaching approximately 4.5 FPKM at six hours, while the chromosome 1 copies also showed moderate increases (2.5–3.0 FPKM). Under combined drought and heat stress, expression patterns resembled those seen under heat alone, with TaSIZ1-3B being the most highly expressed gene (4.0 FPKM), followed by moderate expression of chromosome 1 homoeologs (2.5 FPKM) (Fig. 9 d). Under control conditions, TaSIZ1-1B (2.77), TaSIZ1-1D (1.94), TaSIZ1-3B (2.90), and TaSIZ1-3D (2.30) showed relatively high expression (2.0–2.9 FPKM) as compared to the rest of the TaSIZ1 genes (Fig. 9 d). However, these genes showed elevated expression in response to prolonged heat (2.63–4.30), and combined heat and drought (2.22–3.68) stresses of 6h (Fig. 9 d), suggesting that these TaSIZ1 genes are responsive to prolonged heat stress conditions in a given set of genotypes in the target experiment. Interestingly, TaSIZ1-1A showed the least expression across the stress conditions and control. The remaining TaSIZ1 expression showed only minor changes, largely independent of stress treatments (Fig. 9 d). qRT-PCR Expression Analysis of TaSIZ1 genes in wheat The genotypes C-306 and HD2888 showed contrasting responses to leaf rust resistance and drought tolerance (Fig. 10 a-c), indicating the underlying changes in the expression patterns at the genome level. Expression profiling of six TaSIZ1 genes across three leaf rust post-inoculation time points (24 h, 48 h, and 96 h) revealed transcriptional differences between the resistant genotype HD2888 and the susceptible genotype C306. For TaSIZ1-1A , C306 showed slight upregulation at 24 h (1.1-fold) and 96 h (1.35-fold), but downregulation at 48 h (–1.95-fold). In contrast, HD2888 showed strong, consistent upregulation, reaching a 4.1-fold increase at 96 hours. For TaSIZ1-1B , C306 maintained low expression, with a downregulation at 24 h (–1.11-fold) and a marginal increase at later stages, whereas HD2888 showed pronounced upregulation at 48 h (3.6-fold). Similarly, TaSIZ1-1D showed low expression in C306 (1.05–1.2-fold), whereas HD2888 showed marked upregulation at 24 h (5.2-fold) and 96 h (6.0-fold). For TaSIZ1-3B , C306 showed downregulation at both 24 h (–0.25-fold) and 96 h (–1.85-fold), while HD2888 consistently exhibited upregulation (2.0–3.1-fold). Finally, TaSIZ1-3D was weakly expressed in C306 (1.0–1.45-fold), whereas HD2888 was strongly upregulated at all stages, with the highest fold change at 24 h (4.6-fold) (Fig. 10 d). Expression analysis of TaSIZ1 genes in the drought-stressed plants for seven days revealed distinct differences between the drought-tolerant genotype C306 and the comparatively drought-sensitive genotype HD2888. Drought stress resulted in upregulation of all target TASIZ1 genes in both tolerant and sensitive genotypes (Fig. 10 e). However, the tolerant genotype C306 showed significantly higher fold changes for TaSIZ1-1A (C306: 3.24; HD2888: 2.12), TaSIZ1-1B (C306: 2.50; HD2888: 1.80), TaSIZ1-3B (C306: 4.21; HD2888: 3.09), and TaSIZ1-3D (C306: 4.60; HD2888: 2.45); whereas TaSIZ1-1D showed the fold changes in C306 as compared to HD2888 (C306: 2.91; HD2888: 3.70 ) expression (Fig. 10 e). Discussion Expansion and Evolution of TaSIZ1 Gene Family The TaSIZ1 gene family in wheat has expanded significantly due to ancient genome duplications and polyploidy. We identified multiple TaSIZ1 loci distributed across five homoeologous chromosome groups (1, 3, 4, 5, and 7) in bread wheat. This distribution mirrors the presence of SIZ1 genes on five chromosomes in diploid barley (1H, 3H, 4H, 5H, 7H), suggesting that the ancestral grass genome already harboured several SIZ1 paralogs. Certainly, cereals have experienced at least two rounds of whole-genome duplication [ 34 , 35 ], which likely generated multiple SIZ1 loci that were retained over evolution. Subsequent allopolyploidisation in bread wheat (AABBDD) tripled these loci, resulting in a complement of homoeologous TaSIZ1 copies from each progenitor genome. Most of these homoeologs have been maintained, indicating purifying selection and an essential conserved function. Supporting this, the duplicated TaSIZ1 pairs showed Ka/Ks < 1.0, suggesting strong selective constraint on TaSIZ1 coding sequences; none of the TaSIZ1 duplicates showed evidence of positive selection. Hence, the retention of TaSIZ1 copies is supported by the theory of polyploid redundancy, which buffers the loss of critical stress-responsive genes in wheat [ 36 , 37 ]. The presence of TaSIZ1 genes across the pangenome of species further indicates that they are an essential part of the wheat core gene set. However, we did observe subtle presence/absence and copy number variations. Notably, a SIZ1 homoeolog on chromosome 3A is missing or pseudogenized in the Chinese Spring reference, whereas it is present in other cultivars, reflecting intraspecific variation in gene content. Such presence/absence variation (PAV) is not uncommon in wheat and can arise from structural rearrangements during breeding programmes [ 35 ]. For example, we detected a cultivar-specific duplication on 4A in one genotype, suggesting rare tandem duplication or gene copy fragmentation events. Nevertheless, the fact that all three sub-genomes contribute at least four TaSIZ1 genes emphasises the expansion of this family relative to diploid models. In a dicot model, Arabidopsis thaliana , only a single SIZ1 gene exists ( AtSIZ1 ) [ 9 ]. Diploid cereals also generally carry more than one SIZ1-type gene. The rice genome encodes two to three SIZ1 homologs [ 9 ] (Supplementary Table S4) and sorghum and maize (paleotetraploids) possess ~ 4 SIZ1 each (Supplementary Table S4). These findings indicate that SIZ1-type SUMO E3 ligases form a small multigene family in plants, with lineage-specific expansion. Phylogenetic analysis clustered wheat SIZ1 proteins with their cereal orthologs, which is consistent with speciation. The wheat SIZ1 proteins share ~ 59–81% identity with rice (Supplementary Table S7) and > 30% identity with Arabidopsis SIZ1, demonstrating that SIZ1 is highly conserved yet has diversified within the grass lineage. Interestingly, OsSIZ1 and OsSIZ2 can functionally complement an Arabidopsis siz1 mutant, indicating that the core SUMO ligase activity of SIZ1 has been conserved from 140–150 million years of monocot-dicot divergence [ 19 , 38 ]. The expansion of TaSIZ1 copies in wheat, therefore, likely provides additional regulatory scope or specialisation rather than entirely novel functions. The presence of intact coding sequences across the TaSIZ1 paralogs, with no premature stop codons or frame-shifts, and their promoter regions showing no evidence of transposon-driven degeneration, supports the notion of conserved constraint with potential sub-functionalization. Further, maintenance of multiple TaSIZ1 genes despite potential redundancy implies that each copy may contribute in distinct contexts, viz ., developmental stage, tissue, or stress type (Fig. 9 a), buffering the plant against losing this critical SUMO E3 ligase function and offering an additional level of resilience in wheat [ 35 ]. Structural Features and Regulatory Divergence of SIZ1 Sequences Despite their expansion, the TaSIZ1 proteins are structurally conserved, characteristic of the Siz/PIAS family of SUMO E3 ligases. All identified TaSIZ1 proteins contain the canonical domains known from Arabidopsis and yeast SIZ1, an N-terminal SAP domain (a DNA-binding module), the PINIT domain, a centrally located Siz/PIAS-specific RING (SP-RING) domain, and a C-terminal region harbouring SUMO-interaction motifs (SIMs) and nuclear localisation signals (NLS) [ 39 , 40 ]. We confirmed the presence of the Cys_3His-Cys_4 SP-RING motif in every TaSIZ1 through assuring the whole domain of SIZ1, which is essential for recruiting the SUMO-conjugating enzyme and catalysing SUMO transfer [ 40 ]. Thus, each TaSIZ1 protein is biochemically capable of catalysing SUMOylation, much like OsSIZ1 or AtSIZ1 genes. Most TaSIZ1 genes span large genomic regions, ranging from 7.62 to 21.51 kb, and contain 14–17 exons per gene. The exon/intron structure is conserved mainly among homoeologs. The conserved gene structure further supports a common origin. Nonetheless, some divergence exists in non-coding regions and untranslated regions, which could influence gene regulation. We identified numerous cis -regulatory elements within the 1.5 kb promoters of TaSIZ1 genes, pointing to divergent regulatory controls. All TaSIZ1 promoters are enriched in stress-and hormone-responsive motifs, but the composition and abundance of these elements vary by gene (Supplementary Table S11). For example, many TaSIZ1 promoters contain several ABRE motifs, MBS elements, AREs, and low-temperature-responsive elements. Elements associated with defence and pathogen response, such as the WUN motif, TGACG motifs (elicitor-responsive), and TC-rich repeats, are also present, indicating potential regulation by biotic stress signals. Such differential cis-element profiles imply that although TaSIZ1 genes may be functionally redundant at the protein level, they could be differentially regulated at the transcriptional level in response to specific cues. This kind of sub-functionalization is common in duplicated stress-responsive genes, allowing finer control over when and where each copy is expressed [ 35 , 41 ]. Beyond promoter motifs, we investigated post-transcriptional and transcriptional regulators of TaSIZ1 by predicting several microRNA (miRNA) binding sites in TaSIZ1 mRNAs and Transcription factors (Supplementary Table S14-15). Notably, some homoeologs have unique miRNA target sites that others lack, suggesting homoeolog-specific miRNA-mediated regulation. For example, a conserved miR9664 target site is present in TaSIZ1-D copy but not in its A or B counterparts, hinting that miR9664 might selectively attenuate that homoeolog’s expression under stress [ 41 , 42 ]. Similarly, 37 distinct TF families were predicted for 14 TaSIZ1 promoters (Supplementary Table S15). Among these, ERF/AP2, BBR-BPC, MIKC-type MADS, C2H2 zinc-finger, MYB, and bZIP transcription factors appear to bind all TaSIZ1 promoters, as anticipated, since these TF families include many master regulators of stress and development. For example, bZIP and MYB factors mediate ABA and drought responses, and AP2/ERF factors mediate ethylene/jasmonate and abiotic stress signals [ 6 , 7 ]. Differences in TF binding motifs also mirror the subgenome bias. E2F/DP binding sites were found only in the promoters of the chromosome 5 genes (TaSIZ1-5A/5B/5D), hinting that these copies might be cell-cycle regulated [ 43 ]. Similarly, a plant-specific TF family, SRS (STY-related SHI gene regulators), is found only for TaSIZ1-4A/4B, and RAV, an AP2/B3 hybrid TF, is found only for those same two genes, suggesting a unique developmental or stress regulation of the chromosome-4 SIZ1 [ 44 ]. Further, the variety of cis-elements indicates that each TaSIZ1 gene integrates a distinct combination of upstream signals, including hormones such as ABA, gibberellin, and salicylic acid, as well as environmental cues such as heat shock or drought-induced dehydration. This regulatory divergence reconfirms that the expansion of SIZ1 copies has been accompanied by neo- or sub-functionalization of regulatory regions, enabling wheat to deploy SUMOylating capacity in a modular fashion under diverse stress conditions. Functional Implications of TaSIZ1 genes in Stress Adaptation In-silico and quantitative real-time PCR (qRT-PCR) expression analyses under various stress conditions demonstrate that the TaSIZ1 family is mostly stress-inducible, indicating a significant role in wheat’s adaptation to abiotic and biotic challenges. SUMOylating activity in plants is known to surge during stress as a protective response [ 45 , 46 ]and, correspondingly, we observed upregulation of multiple TaSIZ1 genes in response to heat, drought, and pathogen attack. The in-silico expression of TaSIZ1-1B, TaSIZ1-1D, TaSIZ1-3B and TaSIZ1-3D was markedly induced by heat stress as compared to drought, aligning with the need for enhanced SUMOylation to counteract protein destabilisation (Fig. 9 d) [ 9 ]. Additionally, qRT-PCR analysis of six genes, viz., TaSIZ1-1A, TaSIZ1-1B, TaSIZ1-1D, TaSIZ1-3B, and TaSIZ1-3D, showed significant increases in expression in drought-sensitive genotypes. However, the tolerant genotype C-306 showed higher expression levels than the drought-sensitive genotype HD2888 (Fig. 10 e). This suggests that efficient SIZ1 induction may correlate with better drought resilience. The Arabidopsis siz1 mutants show reduced SUMO conjugates and heat sensitivity [ 47 , 48 ], highlighting SIZ1’s role in thermotolerance. SUMOylation is dynamically regulated by heat stress and water status [ 49 , 50 ]. One homoeolog, TaSIZ1-1A, showed low in-silico expression, implying sub-functionalization with constitutive or alternative roles in the target genotype [ 12 ]. The role of SIZ1 in drought tolerance is well documented in Arabidopsis, where siz1 mutants are hypersensitive to drought and fail to induce ~ 300 drought-responsive genes [ 4 ]. In Arabidopsis, SIZ1 SUMOylates ABA INSENSITIVE 5 (ABI5), a bZIP transcription factor, attenuating ABA signalling during seed germination. Under drought, however, SIZ1 appears to promote stress-responsive gene expression, for instance, SIZ1 facilitates the expression of many ABA-responsive genes and proline biosynthesis genes ( P5CS ) that help in Osmo-protection [ 4 , 51 ]. Our finding that TaSIZ1 induction accompanies drought stress (and is stronger in a drought-hardy line) aligns with this protective function. It is likely that in wheat, SIZ1-mediated SUMOylation stabilises or activates positive regulators of drought tolerance, such as DREB transcription factors or enzymes of osmolyte pathways. SUMOylation might also suppress negative regulators of stress responses, striking a balance that favours survival under water limitation [ 9 , 52 ]. Therefore, the TaSIZ1 genes contribute to drought adaptation in wheat by boosting SUMOylation capacity, which in turn stabilises proteins and stress-response pathways that mitigate drought damage. Variation in TaSIZ1 response across genotypes may partly explain differences in drought resilience. This hypothesis could be tested by comparing SIZ1 allele function in tolerant vs. sensitive wheat varieties or differential SUMOylation patterns. Public transcriptome data and our infection assays indicate that one or more TaSIZ1 genes are induced during early infection by these pathogens. For instance, 48 hours after powdery mildew inoculation, transcripts of a TaSIZ1 on chromosome 5 were upregulated compared to an uninfected control (Fig. 9 b). Likewise, upon leaf rust infection, the rust-resistant cultivar (HD2888) showed a transient spike in TaSIZ1 expression at 24, 48 and 96 h post-inoculation, whereas a susceptible cultivar (C-306) had a low or non-significant expression folds (Fig. 10 d). In stark contrast, the stripe rust infection where there was no-significant induction over the infection time course (Fig. 9 b-c). This differential reaction suggests that wheat plants discriminate the pathogens at the signalling level, and that SIZ1-mediated SUMOylation is more engaged in the powdery mildew and leaf rust response than in the stripe rust response in a given genetic background. This pattern suggests TaSIZ1 may be involved in orchestrating or fine-tuning the defence response. The loss of SIZ1 leads to constitutive activation of salicylic acid (SA)-dependent defences and enhanced resistance to biotrophic pathogens [ 12 , 53 ]. This implies that AtSIZ1 normally restrains SA accumulation under non-stress conditions to prevent autoimmunity. However, SIZ1 also provides temperature-dependent disease resistance. At the elevated temperatures, siz1 mutants cannot effectively resist pathogens because SIZ1 is needed to stabilise specific NB-LRR immune receptors or other defence machinery [ 12 , 54 ]. In wheat, our observation of TaSIZ1 induction during pathogen challenge, especially in resistant backgrounds, suggests a positive contribution to defence under actual attack. One possible explanation is that upon pathogen detection, wheat elevates SIZ1 levels to enhance SUMOylation of defence regulators, thereby potentiating immune signalling or protecting key defence proteins from degradation [ 55 ]. SUMOylation in plant immunity can both suppress negative regulators and support positive regulators of defence, depending on context [ 11 , 56 ]. Further experiments, i.e. silencing or overexpressing TaSIZ1 genes in wheat will be required to clarify their exact role in disease resistance. Nonetheless, the induction patterns and the known functions of SIZ1 in model plants strongly suggest that TaSIZ1 proteins contribute to immune responses of wheat, likely by maintaining a balance between activating defences and preventing hyperactivation that could be detrimental to growth. Our current results highlight the biological significance of the TaSIZ1 family for stress adaptation. By having multiple copies of the SIZ1 genes, wheat can deploy the SUMOylation machinery across various stress conditions, with each TaSIZ1 optimised for specific signals. These multi-copies may provide combinatorial control and robustness, i.e. , if one pathway is compromised, alternative SIZ1 regulation can uphold the SUMOylation capacity. The fact that SIZ1 influences diverse processes in Arabidopsis [ 40 , 52 , 57 ] suggests that TaSIZ1 genes could have pleiotropic effects on wheat development and stress physiology. We observed basal expression of TaSIZ1 under non-stress conditions, suggesting roles in growth or reproduction warranting investigation. For instance, OsSIZ 1 in rice is involved in nitrogen and phosphate signalling and anther dehiscence [ 20 , 58 , 59 ]. Importantly, our results, together with prior studies, highlight TaSIZ1 genes as promising targets for crop improvement. SIZ1 is already considered a candidate gene for engineering stress-tolerant crops, as enhancing its expression can improve tolerance to drought, heat, and even multi-stress conditions without obvious downsides. In wheat, the presence of multiple homoeologs raises the possibility of modifying one copy using tools such as genome editing to boost stress responsiveness while retaining others for essential functions. One could envision editing TaSIZ1 promoters to create hyper-responsive TaSIZ1 alleles that turn on more strongly under stress, thereby ramping up protective SUMOylation when needed. Alternatively, breeding programs might exploit natural variation in TaSIZ1 regulatory regions that confer higher drought- or pathogen-induced SIZ1 expression. Any such strategies must consider the delicate balance SIZ1 strikes between growth and defence; however, the reports suggest that increasing SIZ1 activity tends to enhance stress resilience. Conclusion Our pangenome-wide analysis identified 15 TaSIZ1 genes in bread wheat, grouped into three major evolutionary clades, whose expansion was primarily driven by whole-genome and segmental duplications and maintained under strong purifying selection (Ka/Ks < 1). Despite high structural conservation, TaSIZ1 genes exhibit pronounced regulatory diversification, with promoter regions enriched in stress- and hormone-responsive cis-elements (ABRE, MBS, ARE, GT1) and binding sites for key transcription factor families, including ERF, WRKY, MIKC_MADS, and MYB, enabling fine-tuned transcriptional control. Expression profiling across tissues and stress conditions, together with validation in genotypes with contrasting phenotypic responses, revealed strong, genotype-dependent induction of TaSIZ1-1A, TaSIZ1-1D, TaSIZ1-3B, and TaSIZ1-3D under leaf rust infection in the resistant genotype HD2888 and under drought stress in the tolerant genotype C306. Thus, the results demonstrate that SIZ1-type SUMO E3 ligases have evolved into a small yet functionally powerful multigene family in wheat, acting as master regulators of stress-responsive SUMOylation to amplify stress-signalling pathways and protect the proteome. This study provides the first comprehensive genomic and regulatory framework for TaSIZ1 genes in wheat and establishes a foundation for dissecting TaSIZ1-mediated molecular mechanisms, prioritizing TaSIZ1 genes for multi-stress engineering, and accelerating the breeding of climate-resilient wheat cultivars. Abbreviations ABA Abscisic acid ABRE ABA-responsive element BLAST Basic Local Alignment Search Tool CDS Coding sequence GO Gene Ontology HMM Hidden Markov Model IWGSC International Wheat Genome Sequencing Consortium Ka/Ks Ratio of non-synonymous to synonymous substitution rates Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Competing Interest: The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this manuscript. Funding The work is supported by the Department of Biotechnology, Government of India, through a project entitled “Mapping and transfer of novel resistance genes for multiple biotic stresses in wheat ( Triticum aestivum L.) (BT/PR33041/AGIII/103/1166/2019)”. The funding agencies had no role in designing the study, data collection and analyses, the decision to publish, or the preparation of the manuscript. Author Contribution **Hemant Sharma:** Data curation, Formal Analysis, Writing- Original draft preparation. **Mallana Gowdra Mallikarjuna:** Conceptualization, Methodology, Formal Analysis, Supervision, Writing- Original draft preparation, Writing – review & editing. **Garudapalya Muniswamy Keerthi:** Methodology, Data curation. **Niharika Malik:** Data curation, Writing – review & editing. **Niranjana Murukan:** Data curation, Writing – review & editing. **Lekshmi Sathee:** Data curation, Writing – review & editing. **Shailendra Kumar Jha:** Conceptualization, Funding acquisition, Supervision, Writing – review & editing. Acknowledgement: We are thankful to “The National Phytotron Facility, Indian Agricultural Research Institute, New Delhi” for extending the controlled environment glasshouse facility. Data Availability All raw datasets were downloaded from publicly available databases. The remaining supporting data sets are included in the supplementary files. References Gill G. 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legend\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9114016/v1/66eba7825693168ae0580298.jpg"},{"id":106403875,"identity":"bdde0533-9ff0-498a-a2d1-4d86f7da461b","added_by":"auto","created_at":"2026-04-08 09:15:08","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2047921,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9114016/v1/86cc906ea2ebd5994efe5eb5.jpg"},{"id":106403629,"identity":"989407ab-eb26-4a54-933d-d34e1631d622","added_by":"auto","created_at":"2026-04-08 09:14:38","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":1183682,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Fig9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9114016/v1/00778ae5b21f990614e4942b.jpg"},{"id":106404751,"identity":"bd7ffd74-df22-4880-822f-32b630c5bf21","added_by":"auto","created_at":"2026-04-08 09:16:57","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1613852,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend\u003c/p\u003e","description":"","filename":"Fig10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9114016/v1/9792a860a362fb2b9c21ff2f.jpg"},{"id":106405962,"identity":"f589e6ad-495a-46d5-ada3-726a9a5665dc","added_by":"auto","created_at":"2026-04-08 09:29:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13570781,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9114016/v1/44f7f4e9-9121-43de-a129-8ef2c71628c3.pdf"},{"id":106319450,"identity":"cb565555-ac88-4675-a6f4-3e88d8c0d765","added_by":"auto","created_at":"2026-04-07 12:01:46","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":22069,"visible":true,"origin":"","legend":"","description":"","filename":"Table1SIZ1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9114016/v1/6993dc1d7821e5f4c8570fc7.docx"},{"id":106403956,"identity":"48b58335-c4a6-4d23-9a7a-addd814d0ce2","added_by":"auto","created_at":"2026-04-08 09:15:16","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":834362,"visible":true,"origin":"","legend":"","description":"","filename":"STablesR.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-9114016/v1/8e42224c4d766fd85cc70801.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pangenome-Wide Identification, Evolutionary Characterisation and Stress- Responsive Dynamics of SIZ1-Type SUMO E3 Ligase Gene Family in Bread Wheat (Triticum aestivum L.)","fulltext":[{"header":"Background","content":"\u003cp\u003ePost-translational modifications (PTMs) regulate the function, stability, and interaction networks of proteins, thereby influencing numerous biological processes. SUMOylation is one of the crucial PTMs, where a Small Ubiquitin-like Modifier (SUMO) protein is covalently attached to target proteins, modulating their activity, localisation, or stability. SUMOylation modifications are conserved among eukaryotes and are involved in critical cellular processes, including transcription, DNA repair, response to abiotic and biotic stresses, and cell cycle regulation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. SUMOylation involves a cascade of enzymatic reactions, including the activation (E1), conjugation (E2), and ligation (E3) of SUMO to target proteins. The E3 SUMO ligases facilitate substrate specificity and enhance the efficiency of SUMO transfer. Among known E3 ligases, the SAP and Miz1 (SIZ1)-type SUMO E3 ligase is particularly well-studied. SIZ1 plays a pivotal role in plant stress and developmental processes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, the SIZ1 mediate the SUMOylation of key proteins of pathways controlling photomorphogenesis, flowering, phosphate starvation, and freezing tolerance, showing responses to cold, drought, and heat stress conditions, highlighting its significance in stress adaptation [\u003cspan additionalcitationids=\"CR5 CR6\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Functional studies across species demonstrate that overexpression of SIZ1 homologs, such as \u003cem\u003eOsSIZ1\u003c/em\u003e in rice and cotton, significantly enhances tolerance to abiotic stresses, including drought, heat, and salt, while also improving photosynthesis, biomass accumulation, and yield under adverse conditions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Recently, alternative splicing of SIZ1 has been shown to generate distinct isoforms with stress-specific localisation and activity, underscoring the dynamic regulation of SUMOylation in response to environmental fluctuations [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurthermore, SIZ1 plays a key role in enhancing salicylic acid (SA) signalling, which is essential for systemic acquired resistance (SAR) against biotrophic pathogens. It facilitates the SUMOylation of key defence regulators, such as NPR1 (Non-Expressor of Pathogenesis-Related Genes 1), thereby stabilising NPR1 and promoting the expression of defence-related genes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, SIZ1 helps balance growth and immune responses, preventing the detrimental effects of constitutive defence activation. In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, the \u003cem\u003esiz1\u003c/em\u003e mutants exhibit enhanced resistance to pathogens, such as \u003cem\u003ePseudomonas syringae\u003c/em\u003e, but suffer from growth retardation due to excessive salicylic acid (SA) signalling [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. SIZ1 also interacts with the jasmonic acid (JA) pathway, which fine-tunes the crosstalk between SA- and JA-mediated defence to optimise plant immunity [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Similarly, SIZ1 provides temperature-sensitive immunity, where the SIZ1 function is essential for maintaining disease resistance across different environments [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These evidences underline SIZ1 as a key regulator of plant immunity, making it a valuable target for improving crop disease resistance.\u003c/p\u003e \u003cp\u003eBread wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) is a major staple crop of global importance for food and nutrition with a large and complex genome [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The identification and characterisation of SIZ1-like genes help in understanding the SUMOylation pathway, which is crucial for improving stress resilience and yield stability. Previous studies have identified components of the SUMOylation machinery in model plants, such as Arabidopsis and rice (\u003cem\u003eOryza sativa\u003c/em\u003e), but the comprehensive identification, evolutionary and functional characterization of SUMO E3 ligases in wheat remains limited [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRecent advancements in bioinformatics tools and the availability of high-quality wheat genome assemblies (IWGSC RefSeq v2.1) have facilitated the identification of gene families across the wheat genome [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Utilising wheat genomic resources, a genome-wide identification and analysis of SIZ1-type SUMO E3 ligases in wheat can provide insights into their gene structure, evolutionary and regulatory relationships and expression responses. This information can serve as a foundation for functional studies and the development of wheat varieties with enhanced stress tolerance through molecular breeding or genetic engineering. Thus, the present investigation aimed to identify and characterise SIZ1-like SUMO E3 ligase genes in the wheat genome, determine the evolutionary relationships between wheat SIZ1 homologs and those from other cultivars, and decipher the regulatory and functional response of the identified SIZ1 genes to various stresses.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSequence Retrieval and Identification of SIZ1 Genes and Proteins\u003c/h2\u003e \u003cp\u003eThe whole-genome sequences and annotation files of common wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e) and the sequences of SIZ1 from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (AtSIZ1, AT5G60410) and rice (OsSIZ1, Os05t0125000) were downloaded from the EnsemblPlants database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://plants.ensembl.org/\u003c/span\u003e\u003cspan address=\"https://plants.ensembl.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The wheat pangenomes were retrieved from the wheat pangenomes database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wheatgenomes.com/\u003c/span\u003e\u003cspan address=\"https://wheatgenomes.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The protein sequences (CDSs) of \u003cem\u003eAtSIZ1\u003c/em\u003e and \u003cem\u003eOsSIZ1\u003c/em\u003e were used to perform BLASTp search (E-Value\u0026thinsp;\u0026le;\u0026thinsp;1e-5) against the wheat proteome assembly (IWGSC RefSeq v2.1). HMMSearch (E-value\u0026thinsp;\u0026le;\u0026thinsp;1e-5) was performed across the whole wheat proteome for pfam profile PF02891. The non-redundant hits from both the BLASTp and HMMSearch were again subjected to HMMScan to confirm the presence of the PF02891 domain and finalise the number of SIZ1 genes in wheat. Further, to mine SIZ1 in related species for evolutionary analysis, the identified wheat SIZ1 (TaSIZ1) proteins were used as query sequences to perform BLASTp searches (E-Value\u0026thinsp;\u0026le;\u0026thinsp;1e-5) against the rice, sorghum, maize and barley proteomes downloaded from EnsemblPlants Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://plants.ensembl.org/\u003c/span\u003e\u003cspan address=\"https://plants.ensembl.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the HMMSearch and HMMScan were performed as explained above.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnalysis of Chromosome Location, Gene Structures, and Motifs\u003c/h3\u003e\n\u003cp\u003eThe physical locations and gene structures of the identified SIZ1 genes were retrieved from the GFF file downloaded from the EnsemblPlants database and mapped using TBtools (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/CJ-Chen/TBtools\u003c/span\u003e\u003cspan address=\"https://github.com/CJ-Chen/TBtools\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Conserved motifs within SIZ1 proteins were identified using the MEME Suite (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme-suite.org/tools/meme\u003c/span\u003e\u003cspan address=\"http://meme-suite.org/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and motif annotation was verified using the HMMER tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ebi.ac.uk/Tools/hmmer/search/phmmer\u003c/span\u003e\u003cspan address=\"https://www.ebi.ac.uk/Tools/hmmer/search/phmmer\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003ePhysico-chemical properties and Gene Ontology (GO) Terms Analysis\u003c/h3\u003e\n\u003cp\u003ePhysicochemical properties, including molecular weight, isoelectric point (pI), and amino acid composition, were calculated using the ProtParam tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://web.expasy.org/protparam\u003c/span\u003e\u003cspan address=\"http://web.expasy.org/protparam\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Subcellular localization and Gene Ontology (GO) annotations of SIZ1 proteins were predicted using the WoLFPSORT (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://wolfpsort.hgc.jp\u003c/span\u003e\u003cspan address=\"https://wolfpsort.hgc.jp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) server and the ShinyGO tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.sdstate.edu/go\u003c/span\u003e\u003cspan address=\"http://bioinformatics.sdstate.edu/go\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), respectively.\u003c/p\u003e\n\u003ch3\u003eEvolutionary Analyses of SIZ1 Family in Wheat and Related Species\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMultiple Sequence Alignment and Phylogenetic Analysis\u003c/h2\u003e \u003cp\u003eThe SIZ1 proteins from wheat, rice, maize, barley and sorghum were aligned with MUSCLE [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. The multiple alignment was trimmed with the trimAL v1.2 tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://trimal.cgenomics.org\u003c/span\u003e\u003cspan address=\"http://trimal.cgenomics.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The phylogenetic tree was constructed using the maximum likelihood method. The best model identified using Bayesian information criterion and phylogenetic tree was constructed using 5000 ultrabootstraps using IQ-TREE version 2 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] software. The tree was realised in iTOL [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of SIZ1 family expansion and selection pressure\u003c/h2\u003e \u003cp\u003eIdentification of TaSIZ1 paralogs was performed through self-BLASTp search (e-value\u0026thinsp;\u0026lt;\u0026thinsp;1e-5) within the wheat proteome using BLAST\u0026thinsp;+\u0026thinsp;2.10.1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Subsequently, DupGen_finder software was used to harvest the various duplications in the wheat genome [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. For identification of orthologs across the target species, viz., wheat, rice, maize, barley, and sorghum, the SIZ1 sequences were subjected to OrthoFinder software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/davidemms/OrthoFinder\u003c/span\u003e\u003cspan address=\"https://github.com/davidemms/OrthoFinder\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The nonsynonymous rate (Ka), synonymous rate (Ks) and evolutionary constraint (Ka/Ks) were calculated for each of the paralog and ortholog pairs of SIZ1 using KaKs calculator v.3.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ngdc.cncb.ac.cn/tools/kaks\u003c/span\u003e\u003cspan address=\"https://ngdc.cncb.ac.cn/tools/kaks\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSynteny and Collinearity Analysis\u003c/h3\u003e\n\u003cp\u003eThe whole proteomes of related cereals, \u003cem\u003eviz\u003c/em\u003e., rice, maize, barley and sorghum, were aligned with the wheat proteome using the BLASTp with an e-value\u0026thinsp;\u0026lt;\u0026thinsp;1e-10 and the best five hits per query. The MCScanX software was used to identify syntenic and collinear blocks [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] and visualised with TBtools v2.052 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bio.tools/tbtools\u003c/span\u003e\u003cspan address=\"https://bio.tools/tbtools\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eRegulatory analysis SIZ1 Genes\u003c/h3\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePrediction of Cis-acting Elements in SIZ1 Promoters\u003c/h2\u003e \u003cp\u003eSequences of 1.5 kb upstream regions of SIZ1 genes were retrieved and scanned for cis-acting elements using the PlantCARE server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Only response elements with a matrix value\u0026thinsp;\u0026gt;\u0026thinsp;5 on the sense strand were fetched for subsequent analysis and interpretation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSIZ1-Regulatory Network Analysis\u003c/h2\u003e \u003cp\u003ePutative microRNA (miRNA) targets in SIZ1 genes were predicted using the psRNATarget server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://plantgrn.noble.org/psRNATarget/\u003c/span\u003e\u003cspan address=\"https://plantgrn.noble.org/psRNATarget/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with default parameters. The Transcription factor (TF) was predicted using PlantRegMap (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://plantregmap.gao-lab.org/binding_site_prediction.php\u003c/span\u003e\u003cspan address=\"https://plantregmap.gao-lab.org/binding_site_prediction.php\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Further, the regulatory network of \u003cem\u003eTaSIZ1\u003c/em\u003es was visualised with Cytoscape [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGene Expression Analysis\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003eIn-silico Expression Analysis\u003c/h2\u003e \u003cp\u003eTranscriptome data from the Wheat Expression Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.wheat-expression.com/\u003c/span\u003e\u003cspan address=\"http://www.wheat-expression.com/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were retrieved to analyse the expression of SIZ1 genes across different tissues and stress conditions. The heatmaps were generated using TBtools.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative real-time PCR analysis of TaSIZ1 genes\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003ePlant material and treatments\u003c/h2\u003e \u003cp\u003eThe leaf rust\u0026ndash;susceptible and drought-tolerant cultivar C306 and the leaf rust\u0026ndash;resistant but comparatively drought-sensitive cultivar HD2888 were used to study the TaSIZ1s expression (Supplementary Table\u0026nbsp;1). Seeds were surface-sterilised with 0.1% HgCl₂ for 2 minutes, rinsed thoroughly with sterile distilled water, and treated with 0.5% Bavistin before sowing. Plants were grown in a controlled growth chamber under greenhouse conditions in soil-filled pots. For the leaf rust expression analysis, 14-day-old seedlings were inoculated with the 77\u0026thinsp;\u0026minus;\u0026thinsp;5 strain of \u003cem\u003ePuccinia triticina\u003c/em\u003e. Leaf samples were collected at 0, 24, 48, and 96 hours post-inoculation. For drought stress treatment, 14-day-old seedlings of both genotypes were transferred to a nutrient solution supplemented with 10% polyethene glycol (PEG-6000) to simulate osmotic stress. Leaf tissues were harvested after 7 days of treatment. All collected samples were immediately flash-frozen in liquid nitrogen, stored at \u0026minus;\u0026thinsp;80\u0026deg;C, and later used for total RNA isolation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and quantitative real-time PCR\u003c/h2\u003e \u003cp\u003eSix highly expressed TaSIZ1 gene copies (based on in-silico expression analysis) were selected for qRT-PCR validation. The primers used for RT-qPCR of TaSIZ1 genes were synthesised using Primer Express Software v3.0.1 (Thermo Fisher Scientific; Supplementary Table\u0026nbsp;2). Total RNA was isolated using the TRIzol reagent (Ambion, Naugatuck) as per the manufacturer\u0026rsquo;s specifications and treated with RNase-free DNase I (Invitrogen, California, United States) for 15 minutes to degrade any residual genomic DNA. First-strand cDNA was synthesised from DNase I-treated total RNA using Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, Massachusetts, United States) according to the manufacturer\u0026rsquo;s instructions. RT-qPCR was performed in optical 96-well plates using a CFX96 (Bio-Rad) and SYBR Green (Bio-Rad). The Wheat β-actin gene (accession number AB181991) was used as an endogenous control. The CT values were analysed through the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eMining and Copy Number of SIZ1 genes in Wheat Pangenome Cultivars and Related Species\u003c/h2\u003e \u003cp\u003eA pangenome-wide identification of the SIZ1 gene family, using the hexaploid wheat reference genome (cv. Chinese Spring) along with 12 high-quality cultivar assemblies from the pangenome project, revealed 15 \u003cem\u003eSIZ1\u003c/em\u003e genes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Supplementary Table\u0026nbsp;3; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Genes were systematically renamed based on their chromosomal positions in Chinese Spring as \u003cem\u003eTaSIZ1-1A\u003c/em\u003e to \u003cem\u003eTaSIZ1-7D\u003c/em\u003e, with corresponding copies in each cultivar assigned the same names. In Chinese Spring, 14 TaSIZ1 genes were identified with three homoeologs each on chromosomes 1, 4, 5, and 7 of the A, B, and D genomes, and two on chromosome 3 (\u003cem\u003eTaSIZ1-3B\u003c/em\u003e and \u003cem\u003eTaSIZ1-3D\u003c/em\u003e). Similarly, the genotypes Lancer, Landmark, and Mattis showed 14 TaSIZ1 genes on chromosomes 1, 4, 5, and 7 of the A, B, and D genomes, and two on chromosome 3 (\u003cem\u003eTaSIZ1-3A\u003c/em\u003e and \u003cem\u003eTaSIZ1-3B\u003c/em\u003e). The lowest number of TaSIZ1 genes (12) is reported in the Alchemy and Kariega genotypes, with non-significant hits on 1B, 4D, 7D, and 7A, 7B, 7D chromosomes, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Further, mining SIZ1s in related species, \u003cem\u003eviz\u003c/em\u003e., barley, maize, rice, and sorghum, yielded 5, 4, 3, and 4 SIZ1s, respectively (Supplementary Table\u0026nbsp;4). All the predicted SIZ1 proteins had the characteristic zinc-finger MIZ (zf-MIZ; PF02891) domain, with Zn\u0026sup2;⁺-coordinating Cys/His residues located in the central region of the protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), which confirms that the identified sequences represent SIZ1-type SUMO E3 ligases. Additionally, the target species, including wheat, showed non-uniform distributions of SIZ1 genes across their genomes (Supplementary Tables\u0026nbsp;3\u0026ndash;4; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe distribution and range of various physicochemical properties of TaSIZ1 sequences mined from thirteen pangenome species.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNo. of genomes*\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGene Length (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003ecDNA Length (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCDS Length (bp)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGene % GC content\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eProtein Length (aa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eGRAVY\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eAliphatic Index\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eInstability Index\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eMolecular Weight (kDa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003eAromaticity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eIsoelectric Point (pl)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaSIZ1-1A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e13/13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14874\u0026ndash;16412\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3105\u0026ndash;4826\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2619\u0026ndash;2625\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e41.12\u0026ndash;42.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e872\u0026ndash;874\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.61\u0026ndash;0.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e70.76\u0026ndash;71.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e45.69\u0026ndash;46.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e96.91\u0026ndash;97.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e4.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaSIZ1-1B\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e12/13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e14559\u0026ndash;16368\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3001\u0026ndash;4042\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2622\u0026ndash;2625\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.57\u0026ndash;41.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e873\u0026ndash;874\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.59\u0026ndash;0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e72.12\u0026ndash;72.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e44.97\u0026ndash;45.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e96.99\u0026ndash;97.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e4.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaSIZ1-1D\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e13/13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15318\u0026ndash;21517\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3070\u0026ndash;5037\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2622\u0026ndash;4512\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.82-47.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e873\u0026ndash;930\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.6\u0026ndash;0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e69.55\u0026ndash;73.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e45.00-48.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e96.91-103.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e4.92\u0026ndash;5.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaSIZ1-3A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e6/13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7520\u0026ndash;7878\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3057\u0026ndash;3572\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2787\u0026ndash;2805\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.48\u0026ndash;43.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e928\u0026ndash;934\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.54\u0026ndash;0.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e79.08\u0026ndash;81.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e46.39\u0026ndash;51.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e102.44-103.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e4.85\u0026ndash;4.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaSIZ1-3B\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e12/13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6580\u0026ndash;8015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2775\u0026ndash;3705\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2775\u0026ndash;2793\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.13\u0026ndash;42.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e924\u0026ndash;930\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.53\u0026ndash;0.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e78.47\u0026ndash;79.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e45.93\u0026ndash;46.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e102.26-103.04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e4.87\u0026ndash;4.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaSIZ1-3D\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e4/13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6490\u0026ndash;7634\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2787\u0026ndash;3363\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2787\u0026ndash;2901\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e42.06\u0026ndash;42.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e928\u0026ndash;966\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.5\u0026ndash;0.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e79.39\u0026ndash;80.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e45.68\u0026ndash;47.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e102.64-106.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e4.92\u0026ndash;4.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaSIZ1-4A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e13/13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8317\u0026ndash;9057\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2334\u0026ndash;3074\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2334\u0026ndash;2334\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.54\u0026ndash;41.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e777\u0026ndash;777\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.55\u0026ndash;0.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e73.87-74.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e38.93\u0026ndash;39.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e85.16\u0026ndash;85.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e5.16\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaSIZ1-4B\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e13/13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8764\u0026ndash;12143\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2334\u0026ndash;4667\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2334\u0026ndash;3972\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.67\u0026ndash;41.52\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e777\u0026ndash;837\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.57\u0026ndash;0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e72.79\u0026ndash;74.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e38.25\u0026ndash;43.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e85.12\u0026ndash;91.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e5.02\u0026ndash;5.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaSIZ1-4D\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e12/13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8358\u0026ndash;9442\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2334\u0026ndash;3021\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2334\u0026ndash;2517\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.63\u0026ndash;41.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e777\u0026ndash;838\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.58\u0026ndash;0.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e73.28\u0026ndash;73.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e38.57\u0026ndash;43.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e85.26\u0026ndash;91.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e5.20\u0026ndash;5.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaSIZ1-5A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e13/13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8258\u0026ndash;9238\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2379\u0026ndash;3359\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2379\u0026ndash;2583\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.99\u0026ndash;41.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e792\u0026ndash;860\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.61\u0026ndash;0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e79.03\u0026ndash;80.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e40.71\u0026ndash;48.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e87.73\u0026ndash;94.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e5.27\u0026ndash;5.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaSIZ1-5B\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e13/13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8939\u0026ndash;9504\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2986\u0026ndash;3154\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2385\u0026ndash;2661\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.59\u0026ndash;41.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e794\u0026ndash;886\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.63\u0026ndash;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e74.28\u0026ndash;80.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e41.88\u0026ndash;62.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e87.97\u0026ndash;97.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e5.15\u0026ndash;6.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaSIZ1-5D\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e13/13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8852\u0026ndash;10028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2904\u0026ndash;3613\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2355\u0026ndash;2622\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e41.50-42.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e784\u0026ndash;873\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.62\u0026ndash;0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e78.29\u0026ndash;79.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e40.74\u0026ndash;50.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e86.54\u0026ndash;96.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e5.20\u0026ndash;5.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaSIZ1-7A\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e12/13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8688\u0026ndash;10327\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2788\u0026ndash;3319\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2352\u0026ndash;2739\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e38.65\u0026ndash;40.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e783\u0026ndash;912\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.47\u0026ndash;0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e72.45\u0026ndash;74.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e62.29\u0026ndash;64.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e85.01\u0026ndash;98.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e6.36\u0026ndash;7.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaSIZ1-7B\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e11/13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9003\u0026ndash;9723\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2941\u0026ndash;3373\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2385\u0026ndash;2661\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e40.18\u0026ndash;41.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e794\u0026ndash;886\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.6\u0026ndash;0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e74.28\u0026ndash;80.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e41.88\u0026ndash;62.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e87.97\u0026ndash;96.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e5.15\u0026ndash;6.60\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eTaSIZ1-7D\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003e12/13\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8075\u0026ndash;8782\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2825\u0026ndash;3222\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2316- 2652\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e38.57\u0026ndash;40.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e771\u0026ndash;883\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e-0.45\u0026ndash;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e73.44\u0026ndash;74.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e60.3-61.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e83.9-95.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c12\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c13\"\u003e \u003cp\u003e6.11\u0026ndash;6.45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003ePhysico-Chemical Properties of SIZ1 Genes and Protein Sequences\u003c/h2\u003e \u003cp\u003eGenomic lengths range from about 6.6 kb (\u003cem\u003eTaSIZ1-3B\u003c/em\u003e in Kariega) to over 21 kb (\u003cem\u003eTaSIZ1-1D\u003c/em\u003e in Chinese Spring) owing to differences in intron number and size rather than changes in the core CDS. In contrast, cDNA lengths are generally 3.0-3.6 kb, with a few longer transcripts up to 5,037 bp (Alchemy: \u003cem\u003eTaSIZ1-1D\u003c/em\u003e), suggesting alternative splicing or longer UTRs in certain genotypes. CDS lengths are mostly 2,316\u0026ndash;2,811 bp, with some longer variants up to 4,512 bp, resulting in protein lengths of 771\u0026ndash;966 amino acids. TaSIZ1s from chromosome 4 (TaSIZ1-4A/4B/4D) consistently showed shorter proteins of about 777 amino acids (aa), while TaSIZ1s from chromosomes 3 and 5 code longer proteins (930\u0026ndash;966 aa), likely due to domain extensions or low-complexity region insertions. The GC content of TaSIZ1 genes is moderate and highly conserved, typically ranging from 38.5% to 47.0%. Chromosome-1D \u003cem\u003eTaSIZ1s\u003c/em\u003e have the highest GC content (46\u0026ndash;47%) compared to 7A and 7D \u003cem\u003eTaSIZ1s\u003c/em\u003e (38\u0026ndash;39% GC) (Supplementary Table\u0026nbsp;3).\u003c/p\u003e \u003cp\u003ePhysicochemical analysis showed that TaSIZ1 proteins are highly similar across all cultivars, reinforcing the strong functional conservation within the SIZ1 SUMO E3 ligases across the target pangenome species. Predicted molecular weights range from approximately 83.90 kDa to 106.7 kDa, consistent with CDS length variation. Aromaticity values for TaSIZ1 were low (about 0.05\u0026ndash;0.07), typical of regulatory proteins rich in charged and polar residues. All TaSIZ1 proteins have strongly negative GRAVY scores (\u0026minus;\u0026thinsp;0.63 to \u0026minus;\u0026thinsp;0.40), reflecting a hydrophilic nature, as observed in nuclear-localised proteins, and allowing extensive protein\u0026ndash;protein interactions. Despite this, they display relatively high aliphatic indices (69\u0026ndash;82), suggesting reasonable thermostability. Most proteins have instability indices above 40 and are predicted to be unstable, a common property of signalling and scaffold proteins. However, chromosome-4 homoeologs (TaSIZ1-4A, TaSIZ1-4B, and TaSIZ1-4D) have instability indices below 40 and are classified as stable, indicating this clade may form more rigid or long-lived structural components within the SIZ1 network. Predicted isoelectric points (pI) are mostly in the acidic to near-neutral range (pI 4.85\u0026ndash;7.02), so nearly all TaSIZ1 proteins carry a negative net charge at pH 7, with only a few group-7A/7D members approaching neutrality or slight basicity (Supplementary Table\u0026nbsp;3\u0026ndash;4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eGene structure and motif analysis of TaSIZ1 Genes\u003c/h2\u003e \u003cp\u003eStructural analysis of the TaSIZ1 genes showed that all members have multiple exons and introns, with a highly conserved exon-intron organisation across homoeologs. The genes are intron-rich, with exon counts ranging from 14 to 17 and intron counts from 13 to 16. \u003cem\u003eTaSIZ1-1A\u003c/em\u003e and \u003cem\u003eTaSIZ1-1B\u003c/em\u003e are the most complex, each with 17 exons and 16 introns, while \u003cem\u003eTaSIZ1-1D\u003c/em\u003e has 16 exons and 15 introns. Most other genes (\u003cem\u003eTaSIZ1-3B\u003c/em\u003e, \u003cem\u003eTaSIZ1-4A\u003c/em\u003e, \u003cem\u003eTaSIZ1-\u003c/em\u003e4B, \u003cem\u003eTaSIZ1-\u003c/em\u003e4D, \u003cem\u003eTaSIZ1-5A\u003c/em\u003e, \u003cem\u003eTaSIZ1-5B\u003c/em\u003e, \u003cem\u003eTaSIZ1-5D\u003c/em\u003e, \u003cem\u003eTaSIZ1-7B\u003c/em\u003e) share 16 exons and 15 introns. \u003cem\u003eTaSIZ1-3D\u003c/em\u003e and \u003cem\u003eTaSIZ1-7A\u003c/em\u003e have 15 exons and 14 introns, and \u003cem\u003eTaSIZ1-7D\u003c/em\u003e has 14 exons and 13 introns. Phase 0 introns are most common, accounting for about 43.48% of the total, followed by phase 2 with 32.61% and phase 1 with 23.91% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B). Motif analysis showed that TaSIZ1 proteins contain multiple conserved motifs, with each gene having 4 to 10 motifs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The majority of \u003cem\u003eTaSIZ1\u003c/em\u003e members possess nearly all motifs (9\u0026ndash;10), while TaSIZ1-3D, TaSIZ1-7A, and TaSIZ1-7D have fewer, indicating structurally simplified variants. Motif 1, along with Motifs 4, 6, and 9, was found across the TaSIZ1 genes and was the most conserved, whereas Motifs 8 and 10 were found less commonly in the TaSIZ1 family (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eGene Ontology (GO) Terms and Sub-Cellular Localisation Analysis\u003c/h2\u003e \u003cp\u003eGO annotation indicates that all 14 TaSIZ1 proteins from cv. Chinese spring was significantly enriched for the molecular function GO term zinc ion binding (GO:0008270). Further, the 12 of 14 TaSIZ1s (excluding TaSIZ1-3D; TaSIZ1-7A) were significantly enriched for Sumo ligase activity (GO:0061665), Sumo transferase activity (GO:0019789), Aminoacyltransferase activity (GO:0016755), Ubiquitin-like protein transferase activity (GO:0019787), Transition metal ion binding (GO:0046914) and Acyltransferase activity (GO:0016746). Similarly, for biological function category the these 12 TaSIZ1s were showed significant enrichments for Protein sumoylation (GO:0016925), Peptidyl-lysine modification (GO:0018205), Peptidyl-amino acid modification (GO:0018193), Protein modification by small protein conjugation (GO:0032446), Post-translational protein modification (GO:0043687), Protein modification by small protein conjugation or removal (GO:0070647) under the biological function category (Supplementary Table\u0026nbsp;5). Further, the subcellular localisation predictions indicate that TaSIZ1 proteins are predominantly nuclear localised, with confidence scores of 0.89\u0026ndash;0.96. (Supplementary Table\u0026nbsp;6).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eEvolutionary Analyses of the SIZ1 Family in Wheat and Related Species\u003c/h2\u003e \u003cdiv id=\"Sec24\" class=\"Section4\"\u003e \u003ch2\u003eMultiple Sequence Alignment and Phylogenetic Analysis\u003c/h2\u003e \u003cp\u003eThe multiple sequence alignment of SIZ1 proteins using MUSCLE produced an intact alignment of the PFAM domain PF02891 from 345 to 395 amino acids, indicating sequence conservation and suitability for phylogenetic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The IQ-Tree model find option identified JTT+G4 based on the Bayesian Information Criterion (BIC) as best model for phylogenetic tree construction. The phylogenetic analysis grouped SIZ1 proteins from wheat, barley, rice, maize, and sorghum into three major clades based on percent identity (\u0026gt;\u0026thinsp;50%) among the protein sequences (Supplementary Table\u0026nbsp;7), corresponding to wheat homoeologous chromosome groups 1/3, 4/5, and 7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eClade I contains wheat TaSIZ1 proteins from chromosomes 4A, 4B, 4D, 5A, 5B, and 5D, barley HvSIZ1-4H/5H, OsSIZ1b, ZmSIZ1b, SbSIZ1a, and SbSIZ1d from barley, rice, maize, and sorghum, respectively. Wheat chromosome 5 homoeologs show high per cent identity (95.42\u0026ndash;95.71%), and HvSIZ1-5H shares 92.29\u0026ndash;93.19% identity with them. Wheat chromosome-4 proteins are nearly identical (91.98\u0026ndash;99.05% identity), with HvSIZ1-4H showing 89.12\u0026ndash;93.55% identity. OsSIZ1b is moderately similar (67\u0026ndash;72%) to Triticeae SIZ1 proteins in the clade. The Panicoid pair ZmSIZ1b and SbSIZ1d is closely related (85.54%) but only 62.61\u0026ndash;63.38% identical to TaSIZ1-5A, TaSIZ1-5B and TaSIZ1-5D, indicating an earlier divergence between Triticeae and Panicoideae. SbSIZ1a has emerged as the most distant and outlier member of this phylogenetic tree. The clade II (yellow) includes wheat TaSIZ1 proteins from chromosomes 1A, 1B, 1D, 3B, and 3D, as well as HvSIZ1-1H/3H from barley and OsSIZ1a, ZmSIZ1a, ZmSIZ1c, and SbSIZ1c from rice, maize, and sorghum. Within this clade, \u003cem\u003eTriticeae\u003c/em\u003e proteins are highly conserved: HvSIZ1-1H shares 97.03\u0026ndash;98.05% identity with TaSIZ1-1A/1B/1D, and HvSIZ1-3H shares 91.41\u0026ndash;91.53% identity with TaSIZ1-3B/3D. Maize and sorghum sequences shared 73\u0026ndash;77% identity with wheat and barley but remained highly similar to each other (\u0026gt;\u0026thinsp;90.00%). Clade III (green) includes TaSIZ1-7A, 7B and 7D proteins from chromosome 7, HvSIZ1-7H from barley, OsSIZ1c and OsSIZ1d from rice, ZmSIZ1d from maize, and SbSIZ1b from sorghum. The three wheat homoeologs are again highly conserved (96.12\u0026ndash;97.40%) and share high identity (90.41\u0026ndash;92.45%) with HvSIZ1-7H (Supplementary Table\u0026nbsp;7, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eDuplication and selection pressure analysis of TaSIZ1\u003c/h2\u003e \u003cp\u003eGene duplications drive the expansion of gene families by generating new members that contribute to functional diversity and enable the evolution of new functions. We analysed the types of duplications and the selection pressures that influenced the expansion of \u003cem\u003eTaSIZ1\u003c/em\u003e genes in the wheat genome. The results indicate that single-gene duplications play a significant role in the expansion of SIZ1 (Supplementary Table\u0026nbsp;8 and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). In total, 24 paralog pairs were found among the 14 TaSIZ1s of the Chinese Spring cultivar, encompassing\u0026thinsp;\u0026gt;\u0026thinsp;50% of whole-genome duplications (13), followed by segmental (9) and dispersed duplications (2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSelection pressure was assessed by analysing the ratio of non-synonymous (Ka) to synonymous (Ks) substitutions, which indicates the direction and strength of natural selection on protein-coding genes. Ka/Ks analysis of 24 wheat paralogous pairs showed Ka/Ks\u0026thinsp;\u0026lt;\u0026thinsp;1, indicating strong purifying selection and functional conservation during the expansion of TaSIZ1 sequences in the wheat genome (Supplementary Table\u0026nbsp;8; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Pairs with very low Ks (\u0026lt;\u0026thinsp;0.10) among the 14 duplication pairs observed within chromosomal group but between the homoeologous genomes A, B, D, except the duplication pair TaSIZ1-3D-TaSIZ1-7D (Supplementary Table\u0026nbsp;8), suggesting their recent origin. On the other hand, the paralogs between the chromosomal groups showed higher synonymous mutation rates (Ks\u0026thinsp;\u0026gt;\u0026thinsp;0.45), suggesting that these pairs arose from ancient polyploidy events (Supplementary Table\u0026nbsp;8; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003eOrtholog analysis of SIZ1 in wheat and related species\u003c/h2\u003e \u003cp\u003eOrtholog analysis of TaSIZ1 genes and their counterparts in barley, rice, maize, and sorghum identified many-to-one relationships except two pairs of one-to-one orthologs between wheat and barley (TaSIZ1-3D\u0026ndash;HvSIZ1-3H; TaSIZ1-4A\u0026ndash;HvSIZ1-4H) and many-to-many ortholog clade group between wheat and maize (TaSIZ1-1A/1B/1D/3D\u0026ndash;ZmSIZ1a/c). Among the orthologs between wheat and barley, three many-to-one (TaSIZ1-1A/1B/1D\u0026ndash;HvSIZ1-1H; TaSIZ1-7A/7B/7D\u0026ndash;HvSIZ1-7H; TaSIZ1-5A/5B/5D\u0026ndash;HvSIZ1-5H) orthologs with mean Ks values of 0.11, suggesting their recent divergence as compared to the rest of the species (Supplementary Table\u0026nbsp;9; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The many-to-one orthologs observed between wheat-rice orthologs (TaSIZ1-1A/1B/1D/3D\u0026ndash;OsSIZ1a; TaSIZ1-7A/7B/7D\u0026ndash;OsSIZ1c; TaSIZ1-4A/5A/5B/5D\u0026ndash;OsSIZ1b) and the mean Ks value of 0.61 was found among these orthologs (Supplementary Table\u0026nbsp;9; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Interestingly, wheat-maize orthologs showed a mean Ks value of 0.74 for many-to-many and many-to-one (TaSIZ1-4A/5A/5B/5D\u0026ndash;ZmSIZ1b; TaSIZ1-7A/7B/7D\u0026ndash;ZmSIZ1d) orthologs (Supplementary Table\u0026nbsp;9; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The highest range (0.60\u0026ndash;4.30) and mean (2.56) of Ks values among all the many-to-one ortholog pairs (TaSIZ1-1A/1B/1D/3D\u0026ndash;SbSIZ1c; TaSIZ1-7A/7B/7D\u0026ndash;SbSIZ1b; TaSIZ1-4A/5A/5B/5D\u0026ndash;SbSIZ1d) of wheat-sorghum suggesting varying divergence times owing to various evolutionary events like episodic bursts of gene duplication or biased gene retention etc (Supplementary Table\u0026nbsp;9; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). All these wheat TaSIZ1s diverged under strong purifying selection (Ka/Ks\u0026thinsp;\u0026lt;\u0026thinsp;1) for all the orthologs from rice (0.31), barley (0.27), maize (0.29), and sorghum (0.30).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eSynteny and Collinearity Analysis\u003c/h2\u003e \u003cp\u003eTo further understand deep evolutionary insights, the syntenic and collinearity was worked out between the wheat SIZ1 genes with barley, rice, maize and sorghum (Supplementary Table\u0026nbsp;10; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Synteny analysis of the SIZ1 gene families in wheat (Ta), barley (Hv), rice (Os), and maize (Zm) showed a highly conserved collinear relationship (Supplementary Table\u0026nbsp;10; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Nineteen SIZ1-containing syntenic blocks harboured 19 pairs of collinear SIZ1s between wheat and barley, indicating the strongest syntenic signals owing to their evolutionary lineage. Following barley, maize, sorghum, and rice, wheat also showed synteny with SIZ1 genes, with wheat spanning 13, 7, and 6 synteny blocks, each containing a respective number of SIZ1 gene pairs (Supplementary Table\u0026nbsp;10; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eRegulatory analysis SIZ1 Genes\u003c/h2\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003eCis-regulatory elements analysis\u003c/h2\u003e \u003cp\u003eScanning of 1.5 kb upstream of the TaSIZ1 promoter identified numerous cis-acting regulatory elements, suggesting diverse regulatory potential (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e; Supplementary Tables S11\u0026ndash;S13). A total of 45 distinct cis-element types were detected across all TaSIZ1 promoters. Each promoter harboured multiple elements, including abundant core promoter motifs such as the \u003cem\u003eTATA and CAAT\u003c/em\u003e boxes. Light-responsive elements such as \u003cem\u003eG-box, Box 4\u003c/em\u003e, and \u003cem\u003eI-box\u003c/em\u003e occurred in many promoters. Among the hormone-responsive elements, the ABA-responsive \u003cem\u003eABRE motif\u003c/em\u003e was observed most frequently (71) across the sub-genomes. The MeJA-responsive \u003cem\u003eCGTCA-motif\u003c/em\u003e (37) and \u003cem\u003eTGACG-motif\u003c/em\u003e (13) were also found in the promoter sequences of TaSIZ1s across the sub-genomes. Gibberellin-responsive elements (\u003cem\u003eP-box, GARE-motif\u003c/em\u003e) and auxin-responsive elements (\u003cem\u003eAuxRR-core, TGA-element\u003c/em\u003e) were detected at lower frequencies. Multiple stress-related cis-elements were present as well, including \u003cem\u003eARE\u003c/em\u003e (\u003cem\u003eanaerobic response element\u003c/em\u003e), \u003cem\u003eLTR\u003c/em\u003e (\u003cem\u003elow-temperature response element\u003c/em\u003e), \u003cem\u003eMBS\u003c/em\u003e (\u003cem\u003eMYB-binding site\u003c/em\u003e), \u003cem\u003eWUN-motif\u003c/em\u003e (wound-response element), and \u003cem\u003eSTRE\u003c/em\u003e (stress response element) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e; Supplementary Tables S11\u0026ndash;S13). Interestingly, some \u003cem\u003ecis\u003c/em\u003e elements are confined to specific homoeologs or sub-genomes. The auxin-responsive \u003cem\u003eTGA-element\u003c/em\u003e was found only in B-genome promoters, and a \u003cem\u003ecircadian\u003c/em\u003e rhythm-related element appeared in just two promoters (\u003cem\u003eTaSIZ1-1B; TaSIZ1-7D\u003c/em\u003e). Genome-specific differences in cis-element abundance were also observed: D-genome promoters harboured roughly twice as many cis-elements (392 total) as A-genome (176) or B-genome (177) promoters (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e; Supplementary Tables S11\u0026ndash;S13). TaSIZ1-7D exhibited the highest motif density, containing 23 \u003cem\u003eABREs\u003c/em\u003e and 21 \u003cem\u003eG-box\u003c/em\u003e elements, 9 \u003cem\u003eAREs\u003c/em\u003e and 7 \u003cem\u003eLTRs\u003c/em\u003e in addition to \u003cem\u003eTATA\u003c/em\u003e (25) and \u003cem\u003eCAAT\u003c/em\u003e (28) boxes, indicating complex regulation in response to stresses. Genes such as \u003cem\u003eTaSIZ1-1B\u003c/em\u003e and \u003cem\u003eTaSIZ1-1D\u003c/em\u003e also showed rich promoter compositions. \u003cem\u003eTaSIZ1-1B\u003c/em\u003e had 12 \u003cem\u003eABREs\u003c/em\u003e and 12 G-boxes, while \u003cem\u003eTaSIZ1-1D\u003c/em\u003e harboured nine \u003cem\u003eCGTCA-motifs\u003c/em\u003e (MeJA-responsive) and six \u003cem\u003eLTRs\u003c/em\u003e (low-temperature responsive). Notably, TaSIZ1-5A featured a rare \u003cem\u003e3-AF3 binding site\u003c/em\u003e and a \u003cem\u003eGCN4 Motif, suggesting a specialised regulatory\u003c/em\u003e pattern. Rare or unique cis-elements were gene-specific. Auxin-responsive motifs (\u003cem\u003eTGA-element, AuxRR-core\u003c/em\u003e) appeared exclusively in \u003cem\u003eTaSIZ1-4B\u003c/em\u003e, while salicylic acid-related \u003cem\u003eTCA-elements\u003c/em\u003e and the \u003cem\u003eCAG-motif\u003c/em\u003e were confined to \u003cem\u003eTaSIZ1-4D\u003c/em\u003e. The \u003cem\u003eAE-box\u003c/em\u003e was observed only in \u003cem\u003eTaSIZ1-5D\u003c/em\u003e. Moreover, motifs related to \u003cem\u003ecircadian\u003c/em\u003e regulation (circadian), the \u003cem\u003eLAMP-element\u003c/em\u003e, and \u003cem\u003eGap-boxes\u003c/em\u003e were limited to \u003cem\u003eTaSIZ1-1B\u003c/em\u003e and \u003cem\u003eTaSIZ1-7D\u003c/em\u003e. The \u003cem\u003eWUN-motif\u003c/em\u003e (wound response) appeared only in \u003cem\u003eTaSIZ1-3B\u003c/em\u003e and \u003cem\u003eTaSIZ1-3D\u003c/em\u003e. This cis-element landscape revealed that, while specific motifs, such as \u003cem\u003eABRE\u003c/em\u003e, were universally distributed, many genes exhibited distinct combinations of hormone-, stress-, or development-related elements. These patterns suggest functional divergence in transcriptional regulation among TaSIZ1 genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e; Supplementary Tables S11\u0026ndash;S13).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eRegulation of TaSIZ1s through MicroRNAs and Transcription Factors\u003c/h3\u003e\n\u003cp\u003eA total of 63 unique miRNAs were predicted to regulate 14 TaSIZ1 genes in the Chinese Spring cultivar. After removing duplicates, the number of unique miRNA targets per TaSIZ1 gene ranges from 7 to 24. \u003cem\u003eTaSIZ1-1B\u003c/em\u003e and \u003cem\u003eTaSIZ1-1D\u003c/em\u003e have the highest number of unique miRNAs (24), while \u003cem\u003eTaSIZ1-3D\u003c/em\u003e has the fewest (7). The most common miRNAs across the family are tae-miR1120, tae-miR1122, tae-miR1127, tae-miR1130, tae-miR1137, tae-miR1139, tae-miR9655, tae-miR9773, tae-miR9780, and tae-miR6197, each targeting multiple TaSIZ1 copies in groups 1, 3, 4, 5, and 7. In contrast, low-frequency miRNAs include rare or single targets such as tae-miR9664 (TaSIZ1-1D), tae-miR9667 (TaSIZ1-1A), tae-miR1136 and tae-miR5175 (TaSIZ1-7A), and tae-miR1119 (TaSIZ1-1D). Other sparsely distributed miRNAs, such as tae-miR6201 and tae-miR7757, are limited to group 1 copies (Supplementary Table\u0026nbsp;14; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea,b,f).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe prediction of transcription factor binding sites for TaSIZ1 genes revealed broad transcriptional regulation by 37 transcription factors at 3684 binding sites (Supplementary Table\u0026nbsp;15). Among the predicted TFs, ERF (1327), BBR-BPC (388), MIKC_MADS (222), C2H2 (203), MYB (187), and bZIP (119) were found to regulate all 14 TaSIZ1 genes in the Chinese spring cultivar (Supplementary Table\u0026nbsp;15; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec,f). Interestingly, the TF E2F/DP were predicted only in the homoeologs of chromosome 5 (\u003cem\u003eTaSIZ1-5A; TaSIZ1-5B; TaSIZ1-5D\u003c/em\u003e), where the TF AAR-B was found in the homoeologs of chromosomes 3 (\u003cem\u003eTaSIZ1-53; TaSIZ1-3D\u003c/em\u003e) and five only (Supplementary Table\u0026nbsp;15; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec). Further, among the genes, \u003cem\u003eTaSIZ1-1A\u003c/em\u003e and \u003cem\u003eTaSIZ1-1D\u003c/em\u003e showed the highest number of transcription factors (491), followed by TaSIZ1-3B (465) and \u003cem\u003eTaSIZ1-1B\u003c/em\u003e (330). In contrast, \u003cem\u003eTaSIZ1-3D\u003c/em\u003e (102) showed a lower number of TFs (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed). The \u003cem\u003eTaSIZ1\u003c/em\u003e genes of all the sub-genomes showed a majority of the TFs (34) identified in one or other gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ee). Interestingly, TFs SRS and RAV were limited to A and B sub-genomes (\u003cem\u003eTaSIZ1-4A,4B\u003c/em\u003e), and TALE to sub-genome B (\u003cem\u003eTaSIZ1-4B\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec,e).\u003c/p\u003e \u003cp\u003eThe regulatory network was constructed using unique TFSs and miRNAs to capture a regulatory snapshot of TaSIZ1 genes in the Chinese Spring cultivar (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ef). The TaSIZ1 network comprised 114 nodes, 596 unique edges, and an average of 10.46 neighbours, with a density of 0.046. The network confirmed that TFs play a major regulatory role in the expression and regulation of TaSIZ1, compared with miRNAs, by targeting multiple TaSIZ1 genes, although there are more unique regulatory miRNAs than unique TFs (Supplementary Table\u0026nbsp;15; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ef).\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eGene Expression Analysis\u003c/h2\u003e \u003cp\u003e \u003cb\u003eIn-silico\u003c/b\u003e \u003cb\u003eExpression Analysis\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003eTissue-specific expression of TaSIZ1 genes\u003c/h2\u003e \u003cp\u003eTissue-specific expression analysis of TaSIZ1 genes revealed apparent variation among homoeologs. The root (The \u003cem\u003eTaSIZ1-1A, TaSIZ1-1B\u003c/em\u003e, and \u003cem\u003eTaSIZ1-1D\u003c/em\u003e copies displayed the highest expression levels, especially in roots (2.84\u0026ndash;4.05 FPKM), stems (1.91\u0026ndash;3.24 FPKM), grains (2.46\u0026ndash;3.66), followed by spike (2.15\u0026ndash;2.96). Similarly, \u003cem\u003eTaSIZ1-3B\u003c/em\u003e (root: 3.64; stem: 2.90; grain: 2.92; spike, 2.18)and \u003cem\u003eTaSIZ1-3D\u003c/em\u003e (root: 3.35; stem: 2.57; grain: 2.92; spike, 2.07) showed a similar trend of expression across all target tissues, except leaves. Furthermore, the remaining \u003cem\u003eTaSIZ1s\u003c/em\u003e also showed moderate expression. However, leaf tissue showed lower \u003cem\u003eTaSIZ1\u003c/em\u003e expression levels (0.34\u0026ndash;1.35) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section3\"\u003e \u003ch2\u003eThe expression analysis of TaSIZ1 genes under Powdery Mildew and stripe rust infection\u003c/h2\u003e \u003cp\u003eExpression analysis of TaSIZ1 genes based on FPKM values revealed apparent differences among homoeologs in response to pathogen infection. \u003cem\u003eTaSIZ1-3B\u003c/em\u003e showed high expression among all genes under non-inoculated conditions (3.12); higher expression was observed in response to powdery mildew infection at 48 h (4.13) and 72 h (4.17). However, under leaf rust inoculation, \u003cem\u003eTaSIZ1-3B\u003c/em\u003e expression did not increase with post-inoculation hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb,c). A similar expression pattern was observed for TaSIZ1-3D in response to powdery mildew and leaf rust infections (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb,c). Similarly, the homoeologs of chromosome 1 showed enhanced expression in response to powdery mildew infection over post-inoculation hours (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb), whereas no elevated or differential expression patterns were found for leaf rust infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ec). The expression patterns suggest that TaSIZ1 genes are found to be more responsive to powdery mildew infection rather than leaf rust.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec34\" class=\"Section3\"\u003e \u003ch2\u003eThe expression analysis of TaSIZ1 genes under Heat and Drought\u003c/h2\u003e \u003cp\u003eExpression profiling of TaSIZ1 genes under drought, heat, and combined stress conditions revealed strong, differential regulation among homoeologs. Under control conditions, \u003cem\u003eTaSIZ1-1A, TaSIZ1-1B, TaSIZ1-1D\u003c/em\u003e, and \u003cem\u003eTaSIZ1-3B\u003c/em\u003e exhibited moderate expression levels (2.0\u0026ndash;2.5 FPKM), whereas most other members showed low expression (\u0026lt;\u0026thinsp;1.0 FPKM). During drought stress, expression of these chromosome 1 and 3 copies increased slightly at one hour (2.5\u0026ndash;3.0 FPKM) and remained steady at six hours. Under heat stress, a sharp induction was observed, particularly in \u003cem\u003eTaSIZ1-3B\u003c/em\u003e reaching approximately 4.5 FPKM at six hours, while the chromosome 1 copies also showed moderate increases (2.5\u0026ndash;3.0 FPKM). Under combined drought and heat stress, expression patterns resembled those seen under heat alone, with \u003cem\u003eTaSIZ1-3B\u003c/em\u003e being the most highly expressed gene (4.0 FPKM), followed by moderate expression of chromosome 1 homoeologs (2.5 FPKM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eUnder control conditions, \u003cem\u003eTaSIZ1-1B\u003c/em\u003e (2.77), \u003cem\u003eTaSIZ1-1D\u003c/em\u003e (1.94), \u003cem\u003eTaSIZ1-3B\u003c/em\u003e (2.90), and \u003cem\u003eTaSIZ1-3D\u003c/em\u003e (2.30) showed relatively high expression (2.0\u0026ndash;2.9 FPKM) as compared to the rest of the \u003cem\u003eTaSIZ1\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed). However, these genes showed elevated expression in response to prolonged heat (2.63\u0026ndash;4.30), and combined heat and drought (2.22\u0026ndash;3.68) stresses of 6h (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed), suggesting that these TaSIZ1 genes are responsive to prolonged heat stress conditions in a given set of genotypes in the target experiment. Interestingly, \u003cem\u003eTaSIZ1-1A\u003c/em\u003e showed the least expression across the stress conditions and control. The remaining TaSIZ1 expression showed only minor changes, largely independent of stress treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eqRT-PCR Expression Analysis of TaSIZ1 genes in wheat\u003c/h3\u003e\n\u003cp\u003eThe genotypes C-306 and HD2888 showed contrasting responses to leaf rust resistance and drought tolerance (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ea-c), indicating the underlying changes in the expression patterns at the genome level. Expression profiling of six TaSIZ1 genes across three leaf rust post-inoculation time points (24 h, 48 h, and 96 h) revealed transcriptional differences between the resistant genotype HD2888 and the susceptible genotype C306. For \u003cem\u003eTaSIZ1-1A\u003c/em\u003e, C306 showed slight upregulation at 24 h (1.1-fold) and 96 h (1.35-fold), but downregulation at 48 h (\u0026ndash;1.95-fold). In contrast, HD2888 showed strong, consistent upregulation, reaching a 4.1-fold increase at 96 hours. For \u003cem\u003eTaSIZ1-1B\u003c/em\u003e, C306 maintained low expression, with a downregulation at 24 h (\u0026ndash;1.11-fold) and a marginal increase at later stages, whereas HD2888 showed pronounced upregulation at 48 h (3.6-fold). Similarly, \u003cem\u003eTaSIZ1-1D\u003c/em\u003e showed low expression in C306 (1.05\u0026ndash;1.2-fold), whereas HD2888 showed marked upregulation at 24 h (5.2-fold) and 96 h (6.0-fold). For \u003cem\u003eTaSIZ1-3B\u003c/em\u003e, C306 showed downregulation at both 24 h (\u0026ndash;0.25-fold) and 96 h (\u0026ndash;1.85-fold), while HD2888 consistently exhibited upregulation (2.0\u0026ndash;3.1-fold). Finally, \u003cem\u003eTaSIZ1-3D\u003c/em\u003e was weakly expressed in C306 (1.0\u0026ndash;1.45-fold), whereas HD2888 was strongly upregulated at all stages, with the highest fold change at 24 h (4.6-fold) (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExpression analysis of TaSIZ1 genes in the drought-stressed plants for seven days revealed distinct differences between the drought-tolerant genotype C306 and the comparatively drought-sensitive genotype HD2888. Drought stress resulted in upregulation of all target TASIZ1 genes in both tolerant and sensitive genotypes (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ee). However, the tolerant genotype C306 showed significantly higher fold changes for \u003cem\u003eTaSIZ1-1A\u003c/em\u003e (C306: 3.24; HD2888: 2.12), \u003cem\u003eTaSIZ1-1B\u003c/em\u003e (C306: 2.50; HD2888: 1.80), \u003cem\u003eTaSIZ1-3B\u003c/em\u003e (C306: 4.21; HD2888: 3.09), and \u003cem\u003eTaSIZ1-3D\u003c/em\u003e (C306: 4.60; HD2888: 2.45); whereas \u003cem\u003eTaSIZ1-1D\u003c/em\u003e showed the fold changes in C306 as compared to HD2888 (C306: 2.91; HD2888: 3.70 ) expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ee).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec37\" class=\"Section2\"\u003e \u003ch2\u003eExpansion and Evolution of TaSIZ1 Gene Family\u003c/h2\u003e \u003cp\u003eThe TaSIZ1 gene family in wheat has expanded significantly due to ancient genome duplications and polyploidy. We identified multiple TaSIZ1 loci distributed across five homoeologous chromosome groups (1, 3, 4, 5, and 7) in bread wheat. This distribution mirrors the presence of SIZ1 genes on five chromosomes in diploid barley (1H, 3H, 4H, 5H, 7H), suggesting that the ancestral grass genome already harboured several SIZ1 paralogs. Certainly, cereals have experienced at least two rounds of whole-genome duplication [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], which likely generated multiple SIZ1 loci that were retained over evolution. Subsequent allopolyploidisation in bread wheat (AABBDD) tripled these loci, resulting in a complement of homoeologous TaSIZ1 copies from each progenitor genome. Most of these homoeologs have been maintained, indicating purifying selection and an essential conserved function. Supporting this, the duplicated TaSIZ1 pairs showed Ka/Ks\u0026thinsp;\u0026lt;\u0026thinsp;1.0, suggesting strong selective constraint on TaSIZ1 coding sequences; none of the TaSIZ1 duplicates showed evidence of positive selection. Hence, the retention of TaSIZ1 copies is supported by the theory of polyploid redundancy, which buffers the loss of critical stress-responsive genes in wheat [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe presence of TaSIZ1 genes across the pangenome of species further indicates that they are an essential part of the wheat core gene set. However, we did observe subtle presence/absence and copy number variations. Notably, a SIZ1 homoeolog on chromosome 3A is missing or pseudogenized in the Chinese Spring reference, whereas it is present in other cultivars, reflecting intraspecific variation in gene content. Such presence/absence variation (PAV) is not uncommon in wheat and can arise from structural rearrangements during breeding programmes [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. For example, we detected a cultivar-specific duplication on 4A in one genotype, suggesting rare tandem duplication or gene copy fragmentation events. Nevertheless, the fact that all three sub-genomes contribute at least four TaSIZ1 genes emphasises the expansion of this family relative to diploid models. In a dicot model, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, only a single SIZ1 gene exists (\u003cem\u003eAtSIZ1\u003c/em\u003e) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Diploid cereals also generally carry more than one SIZ1-type gene. The rice genome encodes two to three SIZ1 homologs [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] (Supplementary Table S4) and sorghum and maize (paleotetraploids) possess\u0026thinsp;~\u0026thinsp;4 SIZ1 each (Supplementary Table S4). These findings indicate that SIZ1-type SUMO E3 ligases form a small multigene family in plants, with lineage-specific expansion. Phylogenetic analysis clustered wheat SIZ1 proteins with their cereal orthologs, which is consistent with speciation. The wheat SIZ1 proteins share\u0026thinsp;~\u0026thinsp;59\u0026ndash;81% identity with rice (Supplementary Table S7) and \u0026gt;\u0026thinsp;30% identity with Arabidopsis SIZ1, demonstrating that SIZ1 is highly conserved yet has diversified within the grass lineage. Interestingly, \u003cem\u003eOsSIZ1\u003c/em\u003e and \u003cem\u003eOsSIZ2\u003c/em\u003e can functionally complement an Arabidopsis \u003cem\u003esiz1\u003c/em\u003e mutant, indicating that the core SUMO ligase activity of SIZ1 has been conserved from 140\u0026ndash;150\u0026nbsp;million years of monocot-dicot divergence [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The expansion of TaSIZ1 copies in wheat, therefore, likely provides additional regulatory scope or specialisation rather than entirely novel functions. The presence of intact coding sequences across the TaSIZ1 paralogs, with no premature stop codons or frame-shifts, and their promoter regions showing no evidence of transposon-driven degeneration, supports the notion of conserved constraint with potential sub-functionalization. Further, maintenance of multiple TaSIZ1 genes despite potential redundancy implies that each copy may contribute in distinct contexts, \u003cem\u003eviz\u003c/em\u003e., developmental stage, tissue, or stress type (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea), buffering the plant against losing this critical SUMO E3 ligase function and offering an additional level of resilience in wheat [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec38\" class=\"Section3\"\u003e \u003ch2\u003eStructural Features and Regulatory Divergence of SIZ1 Sequences\u003c/h2\u003e \u003cp\u003eDespite their expansion, the TaSIZ1 proteins are structurally conserved, characteristic of the Siz/PIAS family of SUMO E3 ligases. All identified TaSIZ1 proteins contain the canonical domains known from Arabidopsis and yeast SIZ1, an N-terminal SAP domain (a DNA-binding module), the PINIT domain, a centrally located Siz/PIAS-specific RING (SP-RING) domain, and a C-terminal region harbouring SUMO-interaction motifs (SIMs) and nuclear localisation signals (NLS) [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. We confirmed the presence of the Cys_3His-Cys_4 SP-RING motif in every TaSIZ1 through assuring the whole domain of SIZ1, which is essential for recruiting the SUMO-conjugating enzyme and catalysing SUMO transfer [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Thus, each TaSIZ1 protein is biochemically capable of catalysing SUMOylation, much like \u003cem\u003eOsSIZ1\u003c/em\u003e or \u003cem\u003eAtSIZ1\u003c/em\u003e genes.\u003c/p\u003e \u003cp\u003eMost TaSIZ1 genes span large genomic regions, ranging from 7.62 to 21.51 kb, and contain 14\u0026ndash;17 exons per gene. The exon/intron structure is conserved mainly among homoeologs. The conserved gene structure further supports a common origin. Nonetheless, some divergence exists in non-coding regions and untranslated regions, which could influence gene regulation. We identified numerous \u003cem\u003ecis\u003c/em\u003e-regulatory elements within the 1.5 kb promoters of TaSIZ1 genes, pointing to divergent regulatory controls. All TaSIZ1 promoters are enriched in stress-and hormone-responsive motifs, but the composition and abundance of these elements vary by gene (Supplementary Table S11). For example, many TaSIZ1 promoters contain several ABRE motifs, MBS elements, AREs, and low-temperature-responsive elements. Elements associated with defence and pathogen response, such as the WUN motif, TGACG motifs (elicitor-responsive), and TC-rich repeats, are also present, indicating potential regulation by biotic stress signals. Such differential cis-element profiles imply that although TaSIZ1 genes may be functionally redundant at the protein level, they could be differentially regulated at the transcriptional level in response to specific cues. This kind of sub-functionalization is common in duplicated stress-responsive genes, allowing finer control over when and where each copy is expressed [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBeyond promoter motifs, we investigated post-transcriptional and transcriptional regulators of TaSIZ1 by predicting several microRNA (miRNA) binding sites in TaSIZ1 mRNAs and Transcription factors (Supplementary Table S14-15). Notably, some homoeologs have unique miRNA target sites that others lack, suggesting homoeolog-specific miRNA-mediated regulation. For example, a conserved miR9664 target site is present in TaSIZ1-D copy but not in its A or B counterparts, hinting that miR9664 might selectively attenuate that homoeolog\u0026rsquo;s expression under stress [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Similarly, 37 distinct TF families were predicted for 14 TaSIZ1 promoters (Supplementary Table S15). Among these, ERF/AP2, BBR-BPC, MIKC-type MADS, C2H2 zinc-finger, MYB, and bZIP transcription factors appear to bind all TaSIZ1 promoters, as anticipated, since these TF families include many master regulators of stress and development. For example, bZIP and MYB factors mediate ABA and drought responses, and AP2/ERF factors mediate ethylene/jasmonate and abiotic stress signals [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Differences in TF binding motifs also mirror the subgenome bias. E2F/DP binding sites were found only in the promoters of the chromosome 5 genes (TaSIZ1-5A/5B/5D), hinting that these copies might be cell-cycle regulated [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Similarly, a plant-specific TF family, SRS (STY-related SHI gene regulators), is found only for TaSIZ1-4A/4B, and RAV, an AP2/B3 hybrid TF, is found only for those same two genes, suggesting a unique developmental or stress regulation of the chromosome-4 SIZ1 [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Further, the variety of cis-elements indicates that each TaSIZ1 gene integrates a distinct combination of upstream signals, including hormones such as ABA, gibberellin, and salicylic acid, as well as environmental cues such as heat shock or drought-induced dehydration. This regulatory divergence reconfirms that the expansion of SIZ1 copies has been accompanied by neo- or sub-functionalization of regulatory regions, enabling wheat to deploy SUMOylating capacity in a modular fashion under diverse stress conditions.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec39\" class=\"Section2\"\u003e \u003ch2\u003eFunctional Implications of TaSIZ1 genes in Stress Adaptation\u003c/h2\u003e \u003cp\u003e \u003cem\u003eIn-silico\u003c/em\u003e and quantitative real-time PCR (qRT-PCR) expression analyses under various stress conditions demonstrate that the TaSIZ1 family is mostly stress-inducible, indicating a significant role in wheat\u0026rsquo;s adaptation to abiotic and biotic challenges. SUMOylating activity in plants is known to surge during stress as a protective response [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]and, correspondingly, we observed upregulation of multiple TaSIZ1 genes in response to heat, drought, and pathogen attack. The \u003cem\u003ein-silico\u003c/em\u003e expression of \u003cem\u003eTaSIZ1-1B, TaSIZ1-1D, TaSIZ1-3B\u003c/em\u003e and \u003cem\u003eTaSIZ1-3D\u003c/em\u003e was markedly induced by heat stress as compared to drought, aligning with the need for enhanced SUMOylation to counteract protein destabilisation (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Additionally, qRT-PCR analysis of six genes, viz., TaSIZ1-1A, TaSIZ1-1B, TaSIZ1-1D, TaSIZ1-3B, and TaSIZ1-3D, showed significant increases in expression in drought-sensitive genotypes. However, the tolerant genotype C-306 showed higher expression levels than the drought-sensitive genotype HD2888 (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ee). This suggests that efficient SIZ1 induction may correlate with better drought resilience. The Arabidopsis \u003cem\u003esiz1\u003c/em\u003e mutants show reduced SUMO conjugates and heat sensitivity [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], highlighting SIZ1\u0026rsquo;s role in thermotolerance. SUMOylation is dynamically regulated by heat stress and water status [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. One homoeolog, TaSIZ1-1A, showed low \u003cem\u003ein-silico\u003c/em\u003e expression, implying sub-functionalization with constitutive or alternative roles in the target genotype [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe role of SIZ1 in drought tolerance is well documented in Arabidopsis, where siz1 mutants are hypersensitive to drought and fail to induce\u0026thinsp;~\u0026thinsp;300 drought-responsive genes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In Arabidopsis, SIZ1 SUMOylates ABA INSENSITIVE 5 (ABI5), a bZIP transcription factor, attenuating ABA signalling during seed germination. Under drought, however, SIZ1 appears to promote stress-responsive gene expression, for instance, SIZ1 facilitates the expression of many ABA-responsive genes and proline biosynthesis genes (\u003cem\u003eP5CS\u003c/em\u003e) that help in Osmo-protection [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Our finding that TaSIZ1 induction accompanies drought stress (and is stronger in a drought-hardy line) aligns with this protective function. It is likely that in wheat, SIZ1-mediated SUMOylation stabilises or activates positive regulators of drought tolerance, such as DREB transcription factors or enzymes of osmolyte pathways. SUMOylation might also suppress negative regulators of stress responses, striking a balance that favours survival under water limitation [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Therefore, the TaSIZ1 genes contribute to drought adaptation in wheat by boosting SUMOylation capacity, which in turn stabilises proteins and stress-response pathways that mitigate drought damage. Variation in TaSIZ1 response across genotypes may partly explain differences in drought resilience. This hypothesis could be tested by comparing SIZ1 allele function in tolerant vs. sensitive wheat varieties or differential SUMOylation patterns.\u003c/p\u003e \u003cp\u003ePublic transcriptome data and our infection assays indicate that one or more TaSIZ1 genes are induced during early infection by these pathogens. For instance, 48 hours after powdery mildew inoculation, transcripts of a TaSIZ1 on chromosome 5 were upregulated compared to an uninfected control (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb). Likewise, upon leaf rust infection, the rust-resistant cultivar (HD2888) showed a transient spike in TaSIZ1 expression at 24, 48 and 96 h post-inoculation, whereas a susceptible cultivar (C-306) had a low or non-significant expression folds (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003ed). In stark contrast, the stripe rust infection where there was no-significant induction over the infection time course (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eb-c). This differential reaction suggests that wheat plants discriminate the pathogens at the signalling level, and that SIZ1-mediated SUMOylation is more engaged in the powdery mildew and leaf rust response than in the stripe rust response in a given genetic background. This pattern suggests TaSIZ1 may be involved in orchestrating or fine-tuning the defence response. The loss of SIZ1 leads to constitutive activation of salicylic acid (SA)-dependent defences and enhanced resistance to biotrophic pathogens [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. This implies that \u003cem\u003eAtSIZ1\u003c/em\u003e normally restrains SA accumulation under non-stress conditions to prevent autoimmunity. However, SIZ1 also provides temperature-dependent disease resistance. At the elevated temperatures, \u003cem\u003esiz1\u003c/em\u003e mutants cannot effectively resist pathogens because SIZ1 is needed to stabilise specific NB-LRR immune receptors or other defence machinery [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In wheat, our observation of TaSIZ1 induction during pathogen challenge, especially in resistant backgrounds, suggests a positive contribution to defence under actual attack. One possible explanation is that upon pathogen detection, wheat elevates SIZ1 levels to enhance SUMOylation of defence regulators, thereby potentiating immune signalling or protecting key defence proteins from degradation [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. SUMOylation in plant immunity can both suppress negative regulators and support positive regulators of defence, depending on context [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Further experiments, i.e. silencing or overexpressing TaSIZ1 genes in wheat will be required to clarify their exact role in disease resistance. Nonetheless, the induction patterns and the known functions of SIZ1 in model plants strongly suggest that TaSIZ1 proteins contribute to immune responses of wheat, likely by maintaining a balance between activating defences and preventing hyperactivation that could be detrimental to growth.\u003c/p\u003e \u003cp\u003eOur current results highlight the biological significance of the \u003cem\u003eTaSIZ1\u003c/em\u003e family for stress adaptation. By having multiple copies of the SIZ1 genes, wheat can deploy the SUMOylation machinery across various stress conditions, with each \u003cem\u003eTaSIZ1\u003c/em\u003e optimised for specific signals. These multi-copies may provide combinatorial control and robustness, \u003cem\u003ei.e.\u003c/em\u003e, if one pathway is compromised, alternative SIZ1 regulation can uphold the SUMOylation capacity. The fact that SIZ1 influences diverse processes in Arabidopsis [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e] suggests that TaSIZ1 genes could have pleiotropic effects on wheat development and stress physiology. We observed basal expression of TaSIZ1 under non-stress conditions, suggesting roles in growth or reproduction warranting investigation. For instance, OsSIZ\u003cem\u003e1\u003c/em\u003e in rice is involved in nitrogen and phosphate signalling and anther dehiscence [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Importantly, our results, together with prior studies, highlight TaSIZ1 genes as promising targets for crop improvement. SIZ1 is already considered a candidate gene for engineering stress-tolerant crops, as enhancing its expression can improve tolerance to drought, heat, and even multi-stress conditions without obvious downsides. In wheat, the presence of multiple homoeologs raises the possibility of modifying one copy using tools such as genome editing to boost stress responsiveness while retaining others for essential functions. One could envision editing TaSIZ1 promoters to create hyper-responsive TaSIZ1 alleles that turn on more strongly under stress, thereby ramping up protective SUMOylation when needed. Alternatively, breeding programs might exploit natural variation in \u003cem\u003eTaSIZ1\u003c/em\u003e regulatory regions that confer higher drought- or pathogen-induced SIZ1 expression. Any such strategies must consider the delicate balance SIZ1 strikes between growth and defence; however, the reports suggest that increasing SIZ1 activity tends to enhance stress resilience.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur pangenome-wide analysis identified 15 TaSIZ1 genes in bread wheat, grouped into three major evolutionary clades, whose expansion was primarily driven by whole-genome and segmental duplications and maintained under strong purifying selection (Ka/Ks\u0026thinsp;\u0026lt;\u0026thinsp;1). Despite high structural conservation, TaSIZ1 genes exhibit pronounced regulatory diversification, with promoter regions enriched in stress- and hormone-responsive cis-elements (ABRE, MBS, ARE, GT1) and binding sites for key transcription factor families, including ERF, WRKY, MIKC_MADS, and MYB, enabling fine-tuned transcriptional control. Expression profiling across tissues and stress conditions, together with validation in genotypes with contrasting phenotypic responses, revealed strong, genotype-dependent induction of TaSIZ1-1A, TaSIZ1-1D, TaSIZ1-3B, and TaSIZ1-3D under leaf rust infection in the resistant genotype HD2888 and under drought stress in the tolerant genotype C306. Thus, the results demonstrate that SIZ1-type SUMO E3 ligases have evolved into a small yet functionally powerful multigene family in wheat, acting as master regulators of stress-responsive SUMOylation to amplify stress-signalling pathways and protect the proteome. This study provides the first comprehensive genomic and regulatory framework for TaSIZ1 genes in wheat and establishes a foundation for dissecting TaSIZ1-mediated molecular mechanisms, prioritizing \u003cem\u003eTaSIZ1\u003c/em\u003e genes for multi-stress engineering, and accelerating the breeding of climate-resilient wheat cultivars.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eABA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAbscisic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eABRE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eABA-responsive element\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBLAST\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBasic Local Alignment Search Tool\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCDS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCoding sequence\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene Ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eHMM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHidden Markov Model\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIWGSC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInternational Wheat Genome Sequencing Consortium\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKa/Ks\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eRatio of non-synonymous to synonymous substitution rates\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eCompeting Interest:\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this manuscript.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe work is supported by the Department of Biotechnology, Government of India, through a project entitled \u0026ldquo;Mapping and transfer of novel resistance genes for multiple biotic stresses in wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.) (BT/PR33041/AGIII/103/1166/2019)\u0026rdquo;. The funding agencies had no role in designing the study, data collection and analyses, the decision to publish, or the preparation of the manuscript.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003e**Hemant Sharma:** Data curation, Formal Analysis, Writing- Original draft preparation. **Mallana Gowdra Mallikarjuna:** Conceptualization, Methodology, Formal Analysis, Supervision, Writing- Original draft preparation, Writing \u0026ndash; review \u0026amp;amp; editing. **Garudapalya Muniswamy Keerthi:** Methodology, Data curation. **Niharika Malik:** Data curation, Writing \u0026ndash; review \u0026amp;amp; editing. **Niranjana Murukan:** Data curation, Writing \u0026ndash; review \u0026amp;amp; editing. **Lekshmi Sathee:** Data curation, Writing \u0026ndash; review \u0026amp;amp; editing. **Shailendra Kumar Jha:** Conceptualization, Funding acquisition, Supervision, Writing \u0026ndash; review \u0026amp;amp; editing.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement:\u003c/h2\u003e\n\u003cp\u003eWe are thankful to \u0026ldquo;The National Phytotron Facility, Indian Agricultural Research Institute, New Delhi\u0026rdquo; for extending the controlled environment glasshouse facility.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eAll raw datasets were downloaded from publicly available databases. The remaining supporting data sets are included in the supplementary files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGill G. Something about SUMO inhibits transcription. Curr Opin Genet Dev. 2005;15:536\u0026ndash;41. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.gde.2005.07.004\u003c/span\u003e\u003cspan address=\"10.1016/j.gde.2005.07.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiura K, Hasegawa PM. Sumoylation and other ubiquitin-like post-translational modifications in plants. 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Rice \u003cem\u003eSIZ1\u003c/em\u003e, a SUMO E3 ligase, controls spikelet fertility through regulation of anther dehiscence. New Phytol. 2011;189:869\u0026ndash;82. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1469-8137.2010.03538.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1469-8137.2010.03538.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Post-translational modification, SUMOylation, SUMO E3 ligase, Evolution, Expression dynamics, Stress","lastPublishedDoi":"10.21203/rs.3.rs-9114016/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9114016/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground:\u003c/h2\u003e \u003cp\u003eSUMOylation, a key post-translational modification in plants, modulates diverse stress responses through SUMO E3 ligases (SIZ1). The SIZ1 gene family remains uncharacterized in bread wheat (\u003cem\u003eTriticum aestivum\u003c/em\u003e L.), despite its proven functional relevance in model systems.\u003c/p\u003e\u003ch2\u003eResults:\u003c/h2\u003e \u003cp\u003eThe pangenome survey of bread wheat identified 15 TaSIZ1 genes, unevenly distributed across the genomes. The reference genome Chinese Spring harboured 14 TaSIZ1 genes distributed across homoeologous groups 1, 3, 4, 5, and 7. These intron-rich TaSIZ1 genes with 14\u0026ndash;17 exons encode nuclear-localised, hydrophilic SIZ proteins (pI 4.85\u0026ndash;6.60) that carry a conserved zf-MIZ1 domain. The TaSIZ1 family expansion occurred predominantly through whole-genome and segmental duplications under purifying selection (Ka/Ks\u0026thinsp;\u0026lt;\u0026thinsp;1). The synteny and orthology among wheat, barley, rice, maize, and sorghum SIZ1 genes further confirmed evolutionary conservation, with strong purifying selection (Ka/Ks: 0.21\u0026ndash;0.46). Promoter analysis revealed abundant stress- and hormone-responsive cis-elements (ABRE, MBS, ARE, GT1), alongside major transcription factor binding sites for ERF, WRKY, MIKC_MADS, and MYB families. Expression profiling showed higher basal activity of TaSIZ1-1A, 1B, 1D, 3B, and 3D in vegetative tissues and induction by heat, drought, powdery mildew, and leaf rust.\u003c/p\u003e\u003ch2\u003eConclusions:\u003c/h2\u003e \u003cp\u003eOur study showed that TaSIZ1 genes are structurally conserved but show functional divergence across homoeologs and genotypes. The strong, specific induction of selected copies underscores their utility in enhancing stress resilience. This study provides the first comprehensive framework for understanding TaSIZ1 regulation in wheat and nominates candidate genes for multi-stress engineering and wheat breeding.\u003c/p\u003e","manuscriptTitle":"Pangenome-Wide Identification, Evolutionary Characterisation and Stress- Responsive Dynamics of SIZ1-Type SUMO E3 Ligase Gene Family in Bread Wheat (Triticum aestivum L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-07 12:01:35","doi":"10.21203/rs.3.rs-9114016/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-16T10:31:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T01:38:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"306206761520902604563218714227971364348","date":"2026-05-11T00:58:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T01:26:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"99457912796494839239925675614078198003","date":"2026-05-07T02:34:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4713347710318478974389811709164990618","date":"2026-05-06T11:04:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67599227253453595286095099017515654220","date":"2026-05-06T00:35:36+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-01T22:38:33+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-01T22:33:53+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-31T10:07:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-31T08:53:18+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2026-03-31T07:56:51+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3d618958-8009-4428-8743-f5de31da86cd","owner":[],"postedDate":"April 7th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-16T10:31:38+00:00","index":80,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T01:38:01+00:00","index":77,"fulltext":""},{"type":"reviewerAgreed","content":"306206761520902604563218714227971364348","date":"2026-05-11T00:58:28+00:00","index":76,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-08T01:26:20+00:00","index":73,"fulltext":""},{"type":"reviewerAgreed","content":"99457912796494839239925675614078198003","date":"2026-05-07T02:34:27+00:00","index":72,"fulltext":""},{"type":"reviewerAgreed","content":"4713347710318478974389811709164990618","date":"2026-05-06T11:04:19+00:00","index":70,"fulltext":""},{"type":"reviewerAgreed","content":"67599227253453595286095099017515654220","date":"2026-05-06T00:35:36+00:00","index":69,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-07T12:01:36+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-07 12:01:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9114016","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9114016","identity":"rs-9114016","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}