Definition and regulatory analysis of the SUMOylation system in Caixin (Brassica rapa var. parachinensis) during Pectobacterium carotovorum infection

Definition and regulatory analysis of the SUMOylation system in Caixin (Brassica rapa var. parachinensis) during Pectobacterium carotovorum infection | 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 Definition and regulatory analysis of the SUMOylation system in Caixin (Brassica rapa var. parachinensis) during Pectobacterium carotovorum infection Shikang Lei, Guangguang Li, Ding Jiang, Fanchong Yuan, Yansong Zheng, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4844966/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Dec, 2024 Read the published version in BMC Plant Biology → Version 1 posted 11 You are reading this latest preprint version Abstract Background The modification of protein substrates by small ubiquitin-related modifier (SUMO) plays a vital role in plants subjected to biotic and abiotic stresses. However, its role in the stress responses of Brassica plants remains poorly understood. Results A genome-wide analysis revealed the presence of 30 SUMOylation genes in the Caixin genome. These results demonstrated that the Caixin genome contains all the necessary components for SUMOylation. Analysis of the cis -acting elements revealed that the promoters of SUMOylation genes presented diverse combinations of developmental and stress-related cis -regulatory elements. The RNA-seq data indicated that 23 SUMOylation genes presented relatively high expression levels under normal conditions and exhibited a notable decrease in expression following Pectobacterium carotovorum subsp. carotovorum ( Pcc ) infection. Additionally, dynamic alterations in SUMO conjugates were observed in response to Pcc infection. Conclusions The Caixin genome contains genes involved in SUMOylation. The majority of these genes presented multiple copies, and analyses of their transcription and protein profiles indicate that they may play a role in the response to Pcc infection. SUMOylation Caixin (Brassica rapa var. parachinensis) Soft rot Biotic stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Posttranslational modifications (PTMs) are chemical modifications of proteins after translation and are important regulatory mechanisms through which proteins perform their biological functions. After translation, various functional groups are added to the corresponding amino acid residues of proteins through covalent bonds, resulting in changes in the spatial conformation of proteins and triggering functional changes [ 1 ]. SUMOylation, also known as ubiquitination, is an important form of posttranslational protein modification that regulates many life activities in eukaryotic cells. The SUMO protein has a three-dimensional structure similar to that of the ubiquitin protein and is conserved and widely expressed in eukaryotic cells [ 2 ]. Like ubiquitination, SUMOylation requires a series of enzymatic reactions. First, the SUMO precursor is cleaved to expose the C-terminal glycine, which is subsequently activated and coupled to a cysteine of the heterodimeric SUMO-activating enzyme E1 (SAE1/SAE2) in an ATP-dependent reaction. The activated SUMO is subsequently transferred to the SUMO-conjugating enzyme E2 (SCE1) through transesterification and the formation of the SUMO-E2 complex. Finally, E2 recognizes target proteins at a specific sequence (ΨKXE/D) (Ψ, large hydrophobic amino acid; K, acceptor lysine; X, any amino acid; E/D, glutamate or aspartate) and attaches SUMO to target proteins with the assistance of the SUMO E3 ligase [ 3 – 6 ]. SUMOylation is a reversible process capable of releasing SUMO for further conjugation cycles via the hydrolysis of isopeptide bonds by SUMO proteases [ 7 ]. SUMOylation has been demonstrated to be essential for plants, with several studies indicating its importance in integrating environmental inputs and responding appropriately to stress conditions [ 8 ]. Previous studies have shown that the abundance of SUMO conjugates in plants can be significantly increased upon exposure to heat, cold, high salinity, and abscisic acid [ 9 ]. Alterations in the function of SUMOylation components can lead to changes in plant adaptation to environmental stresses. Additionally, several SUMOylation components have been connected to biotic responses [ 8 ]. In 1999, Hanania et al. first identified a link between SUMOylation and plant immune responses, and subsequent studies have gradually elucidated the mechanisms by which SUMOylation is involved in the immune responses of plants [ 10 ]. In Arabidopsis , the absence of the SUMO ligase gene AtSIZ1 resulted in excessive accumulation of salicylic acid and upregulation of the expression levels of disease resistance-related genes in vivo, leading to the production of a dwarfing phenotype and an autoimmune response phenotype [ 11 ]. Recent studies have demonstrated that the autoimmune response of atsiz1 mutants is a consequence of the suppression of the immune response of AtTPR1 by SUMOylation [ 12 ]. Like atsiz1 , the SUMO protease mutant atesd4 also exhibited autoimmune phenotypes with elevated expression of SA (salicylic acid) and NHP (N-hydroxypipecolic acid) biosynthesis-related genes [ 13 ]. In maize, ZmSIZ1a/ZmSIZ1b-mediated SUMOylation plays a pivotal role in the comprehensive defense response of maize against FER. This is achieved by mediating several signalling pathways, especially early defense responses and flavonoid synthesis [ 14 ]. During pathogen attack, SUMOylation components also serve as targets of pathogen effectors, providing an opportunity to inhibit SUMOylation at different stages. Tomato Golden Mosaic Virus (TGMV) and Tomato Yellow Leaf Curl Sardinia Virus (TYLCSV) encode a protein, RepAC1 (or Rep), which is essential for the replication of the viruses. In tobacco, the SUMO E2-binding enzyme NbSCE1 specifically interacts with the Rep/RepAC1 protein to affect viral replication [ 15 ]. In another study, yeast two-hybrid screening of SCE1 from Arabidopsis revealed a single interaction involving AtSCE1 and the viral RNA-dependent RNA polymerase Nlb, and the Arabidopsis atsce1 mutant also presented increased resistance to TuMV [ 16 ]. These findings offer new insights into the underlying role of SUMOylation components in virus infection in plants. Despite the importance of SUMOylation in the plant development process or under stress conditions [ 17 ], functional studies on this topic have been largely restricted to Arabidopsis . With the increase in the number of deciphered plant genomes, SUMOylation components have gradually been identified in apple [ 18 ], tomato [ 10 , 19 ], poplar [ 20 ], rice [ 9 , 21 ], soybean [ 22 ], maize [ 23 ] and other plants [ 24 ]. However, no available information on SUMOylation in Brassica species has been reported. In this study, we comprehensively characterized the core SUMOylation components of Caixin, a popular and widely cultivated leafy vegetable crop in southern China. We also investigated the transcriptional responses of SUMOylation genes during Pcc infection. Our findings provide an overview of the SUMOylation in Caixin and reveal the potential roles of SUMOylation in the soft rot stress response. Results Identification and characterization of SUMOylation genes in Caixin Whole-genome sequencing of Caixin was completed in 2023 [ 25 ]. Using known Arabidopsis SUMOylation component sequences as queries, we conducted a search for their respective orthologues in the Caixin genome via BLAST and domain confirmation methods. A total of 30 genes encoding the core components of SUMOylation were identified in this Brassica species (Table 1 ). Our list revealed that the Caixin genome contains nine SUMO genes, termed BrSUMO1 to BrSUMO9 . To further understand the evolutionary relationships among these SUMO isoforms, we searched a number of other plant genomes for related sequences, including those of Arabidopsis , rice, soybean and maize (Fig. 1 A). Phylogenetic analysis revealed that these SUMOs clustered into three groups: the “canonical SUMO”, “noncanonical SUMO” and “SUMO variant” groups. Members belonging to the “canonical SUMO” group were strongly conserved. Five members of the BrSUMO family (BrSUMO1 to BrSUMO5) are divided into this group and are evolutionarily most closely related to AtSUMO1. The “noncanonical SUMO” group, which contains four BrSUMOs (BrSUMO6 to BrSUMO9), shares low amino acid identity. No BrSUMOs belong to the “SUMO variant” group. The C-terminal di-Gly motif of SUMO is necessary for substrate conjugation. Alignment of SUMO sequences revealed that, with the exception of BrSUMO7, both canonical and noncanonical BrSUMOs have a C-terminal di-Gly motif, indicating their potential ability to covalently attach to target proteins. We also noted that the SUMO interaction motif in BrSUMO6 and BrSUMO7 is different from that in other BrSUMOs. Additionally, the other two noncanonical BrSUMOs (BrSUMO8 and BrSUMO9) did not contain the conserved Lys (Lysine) residue, which is required for the formation of SUMO chains (Fig. 1 B). Table 1 Characteristics of SUMOylation system genes in Caixin Group Gene Sequence ID Chromosome Protein length (aa) MW (Da) SUMO BrSUMO1 Bra_cxA08g015400 A08 101 11124.43 BrSUMO2 Bra_cxA03g012940 A03 98 10844.17 BrSUMO3 Bra_cxA01g030080 A01 113 12733.35 BrSUMO4 Bra_cxA10g021190 A10 140 15970.09 BrSUMO5 Bra_cxA02g036080 A02 109 12399.09 BrSUMO6 Bra_cxA03g050360 A03 121 13471.4 BrSUMO7 Bra_cxA05g032960 A05 152 16996.64 BrSUMO8 Bra_cxA09g065660 A09 92 10332.47 BrSUMO9 Bra_cxA06g009930 A06 116 13374.26 E1 BrSAE1 Bra_cxA01g031910 A01 322 35888.14 BrSAE2a Bra_cxA04g017510 A04 556 62585.22 BrSAE2b Bra_cxA09g010380 A09 552 62403.83 E2 BrSCE1a Bra_cxA04g035410 A04 160 17851.38 BrSCE1b Bra_cxA07g020470 A07 160 17843.4 BrSCE1c Bra_cxA04g037680 A04 265 30304.82 E3 BrSIZ1a Bra_cxA02g039720 A02 862 95055.24 BrSIZ1b Bra_cxA10g015860 A10 884 97190.92 BrSIZ1c Bra_cxA06g025660 A06 804 88269.61 BrMMS21 Bra_cxA05g011250 A05 160 17810.38 BrPIAL1a Bra_cxA08g002610 A08 986 108099 BrPIAL1b Bra_cxA09g003500 A09 636 70816.11 BrPIAL2a Bra_cxA04g019330 A04 723 79030.63 BrPIAL2b Bra_cxA07g023060 A07 640 69796.76 SUMO protease BrULP1A Bra_cxA01g003850 A01 439 51090.7 BrULP1B Bra_cxA03g019930 A03 617 70756.78 BrULP1C Bra_cxA06g038970 A06 569 65650.8 BrESD4 Bra_cxA08g024070 A08 404 46397.07 BrFUG1 Bra_cxA06g020780 A06 223 26836.84 BrSPF1 Bra_cxA08g003020 A08 638 71562.39 BrSPF2 Bra_cxA01g042650 A01 792 89798.89 SUMOylation requires an initial activation step in which the C-terminal di-Gly of mature SUMO is first adenylated by a heterodimeric SUMO-activating enzyme (SAE1/2) [ 26 ]. In contrast to Arabidopsis , the Caixin genome encodes one small-subunit SAE1 gene ( BrSAE1 ) and two large-subunit SAE2 genes ( BrSAE2a and BrSAE2b ). BrSAE1 encodes a protein of 322 amino acids that is conserved with AtSAE1a (90% identity) and AtSAE1b (82% identity). BrSAE2a and BrSAE2b both encode a protein of 625 amino acids that shares 87% and 93% identity with AtSAE2, respectively. Furthermore, functional domains were also identified in BrSAEs (Fig. S1 A, B). Phylogenetic analysis of SAEs from various plant species revealed that BrSAE1 and BrSAE2a/b were evolutionarily most closely related to AtSAE1a/b and AtSAE2, respectively (Fig. S2 A, B). We also predicted the 3D structures of SAE1/SAE2 heterodimers via SWISS-MODEL. The structures of both BrSAE1/BrSAE2a and BrSAE1/BrSAE2b were similar to those of AtSAE1a/AtSAE2 and AtSAE1b/AtSAE2 (Fig. S2 C). Upon activation by a SUMO-activating enzyme, the bound SUMO moiety is transferred via transesterification to an active site Cys (Cysteine) in the SUMO-conjugate enzyme SCE1 and then forms a SUMO-E2 thioester intermediate [ 27 ]. The Arabidopsis genome contains a single SCE1 gene ( AtSCE1 ) compared with three other SCE1 genes ( BrSCE1a , BrSCE1b and BrSCE1c ) in Caixin. The maize genome encodes a novel class II isotype of SCE1 , which is found only in the cereal branch of monocots. To further examine the origins of BrSCE1 isotypes, we phylogenetically analysed a collection of 17 SCE protein sequences from 4 plant genomes. As shown in Fig. 2 A, all three BrSCE1 genes were clustered into class I and presented high similarity with homologues in other species, especially Arabidopsis . We then identified conserved motifs in the SCE1 proteins via the MEME program. A total of three conserved motifs were found in most of the SCE1 proteins, and motif 2 was present in all of these SCE1 proteins. BrSCE1a, BrSCE1b and BrSCE1c also share high sequence identity with AtSCE1 and have a highly conserved Ub-conjugating enzyme catalytic (UBC) domain (spanning from 8 to 150 aa residues, Fig. 2 B), which is common to the UBC family. Although SUMOylation can occur without SUMO E3 ligases under certain conditions [ 28 ], increasing evidence has revealed that the absence of E3 ligases significantly reduces SUMO conjugate levels in plants. There are three types of SUMO E3 ligases in plants: SIZ/PIAS (SIZ1), methyl methane sulfonate-sensitive protein-21/high ploidy-2 (MMS21/HPY2) and protein inhibitor of activated STAT (PIAS)-like (PIAL). The genome of Caixin encodes three SIZ1 genes ( BrSIZ1a , BrSIZ1b and BrSIZ1c ), four PIAL genes ( BrPIAL1a , BrPIAL1b , BrPIAL2a and BrPIAL2b ) and one MMS21/HPY2 gene ( BrMMS21 ). A phylogenetic analysis of SUMO E3 ligases from various plant species revealed that different types of SUMO E3 ligases in Caixin are evolutionarily most closely related to those in Arabidopsis (Fig. 3 A). Among the members of the SUMO E3 ligase family, AtSIZ1 is the most extensively studied in plants. Its involvement in various biological processes, including flowering [ 29 – 31 ], plant immunity [ 32 ], abiotic stress tolerance [ 8 ], thermomorphogenesis [ 33 , 34 ], and phytohormone signalling [ 35 – 38 ], has been well documented. A common feature of known SIZ1 is the presence of five conserved domains (SAP, PHD, MIZ/SP-RING domain, PINIT and SXS motifs). Both BrSIZ1a and BrSIZ1b have five conserved domains, but a deletion of the SAP domain was observed in BrSIZ1c (Fig. 3 B, S3 ). Like Arabidopsis , Caixin contains only one MMS21/HPY2-type E3 (BrMMS21), which also has a conserved SP-RING domain and shares high sequence identity with AtMMS21 (Fig. S4). Previous studies have shown that AtPIAL1 and AtPIAL2 function as SUMO ligases capable of SUMO chain formation. Sequence alignment revealed that both AtPIALs and BrPIALs have the MIZ/SP-RING domain as well as SIM motifs, which have specific affinities for binding to SUMO chains. However, the absence of the N-terminal SIM motif was observed in BrPIAL1b (Fig. S5). SUMOylation is a reversible process that requires the specific action of SUMO proteases to process SUMO precursors and release SUMO from substrate conjugates [ 39 ]. A search of the protein sequence of Arabidopsis SUMO proteases revealed the presence of seven SUMO proteases in the Caixin genome. We phylogenetically analysed the selected plant species that encode potential orthologues of known Arabidopsis SUMO proteases. The phylogenetic tree revealed that the SUMO proteases in Caixin clustered into four groups, namely, the ESD4-type, OTS-type, FUG-type and SPF-type groups, which was consistent with the classification results in Arabidopsis (Fig. S6). Cis ‑acting element analysis of SUMOylation gene promoters To obtain information regarding the stimulus-induced, temporal, and spatial expression patterns of the SUMOylation genes in Caixin, 2.0 kb promoter regions upstream of these 30 genes were extracted and utilized for cis -acting element searches via the PlantCARE website. As shown in Fig. S7, a diverse range of cis -acting elements were present in the promoters of these genes. We classified these cis -acting elements into three groups: “plant growth and development”, “phytohormone responsiveness” and “stress responsiveness” (Fig. 4 A). Almost all the promoters of these genes contained many phytohormone responsive elements related to ABA (ABRE), auxin (TGA element and AuxRR core), gibberellin (TATC-box, GARE motif and P-box), salicylic acid (TCA element) and MeJA (TGACG motif and CGTCA motif) (Fig. 4 B). ABREs are the most common cis -acting elements involved in the plant hormone response, accounting for 32% of all ABREs. In addition, nine types of cis -acting elements related to the cell cycle (MAS-like motif), circadian control (circadian motif), zein metabolism regulation (O 2 -site), meristem expression (CAT-box and NON-box), root-specific expression (motif I), endosperm expression (GCN4 motif and AACA motif) and differentiation of palisade mesophyll cells (HD-Zip 1), which cover most plant developmental stages, were involved in plant growth and development (Fig. 4 B). Seven types of cis -acting elements were divided into stress-responsive groups, namely, mixed stress response (TC-rich repeats), low-temperature responsiveness (LTR motif), anaerobic induction (ARE), MYB binding site (MBS) and wound-responsive element (WUN motif) elements, and ARE elements appeared in most of the promoters of these SUMOylation system genes (Fig. 4 B). These results indicated that the expression of these genes might be associated with diverse signalling pathways during plant growth and stress responses. Transcriptional profiles of SUMOylation genes under soft rot stress Soft rot is a common disease of Brassica crops caused primarily by Pcc , a bacterial pathogen that infects plants through wounds at the base of the stem or petiole [ 40 ]. As the infection time increased, the infected area expanded, and the area from the water stains decreased (Fig. 5 A). To further understand the potential roles and transcriptional regulation of the SUMOylation in Caixin under biotic stress, the expression patterns of the associated genes were analysed via RNA-seq datasets. The FPKM data (Table S1 ) from the RNA-seq of all the SUMOylation system genes upon Pcc infection are shown in a heatmap. As shown in Fig. 5 B, a total of 30 SUMOylation system genes were clustered into four subgroups according to their expression patterns. Among these genes, 23 had a relatively high level of expression under normal conditions and were downregulated after Pcc infection. The expression of four genes, namely, BrSUMO3 , BrSUMO8 , BrSCE1b and BrSIZ1b , was continuously downregulated by Pcc infection. The expression of four other genes, namely, BrSUMO5 , BrSUMO7 , BrPIAL2a and BrESD4a , was continuously induced by Pcc infection. These results suggest that the transcription of SUMOylated genes is not only essential for plant development but also regulated by soft rot stress. Pcc infection regulated SUMO conjugates in Caixin Abiotic stresses, including heat, drought, and oxidative stress, have been shown to substantially increase the levels of SUMO conjugates [ 9 , 23 , 41 ]. To determine whether SUMOylation in Caixin responds to soft rot stress, the variation in the SUMO complex was investigated. The occurrence of heat-induced SUMO conjugates has been confirmed in several plant species, and high temperature is a key factor in outbreaks of soft rot disease in the field. Therefore, we first examined heat-induced SUMO conjugates in Caixin as a positive control. As shown in Fig. 6 A, during 37°C treatment, the amount of SUMO conjugates in leaves increased substantially after 30 min of heat stimulus compared with that in the unstressed conditions. We then detected SUMO conjugates in Caixin leaves upon Pcc infection. After inoculation with the Pcc strain for 12 hpi, the amount of SUMO conjugates remained unchanged. However, the conjugate levels increased substantially from 12 to 24 hpi (Fig. 6 B). These results suggest that SUMOylation is involved in the response of Caixin to soft rot stress. Discussion Plant SUMOylation has been implicated in nutrient acquisition and defense against biotic and abiotic challenges [ 8 ]. In recent years, the SUMOylation system has been identified in several plants, crops, and fruits. However, the components of the SUMOylation in Brassica plants remain unknown. In this study, a total of 30 SUMOylation genes were identified in Caixin (Table 1 ). The genome of Caixin contains complete SUMOylation components, including SUMO, E1-activating enzymes, E2-conjugating enzymes, E3 ligases and SUMO proteases. Brassicaceae plants underwent whole-genome triplication (WGT) between 13 and 17 million years ago (MYA) and gene loss following whole-genome duplication [ 42 , 43 ], which indicates that close homologues of Arabidopsis SUMOylation genes may be present in multiple copies or disappear in the Caixin genome. Compared with the eight putative SUMO copies in the Arabidopsis genome, the Caixin genome contains nine SUMO members, five of which (BrSUMO1 to BrSUMO5) cluster into the canonical SUMO group, and the other four (BrSUMO6 to BrSUMO9) cluster into the noncanonical SUMO group. In the canonical SUMO group, all five BrSUMOs were closely related to AtSUMO1 in terms of their evolutionary relationships (Fig. 1 A). In Arabidopsis , two noncanonical SUMOs, AtSUMO3 and AtSUMO5, have been proposed to have arisen from a duplication of SUMO2 and an ancient paneudicot palaeohexaploidy event, respectively. Moreover, previous studies have suggested that SUMO3 is frequently deleted, converted back to SUMO2 or pseudogenized in another Brassica spp. [ 44 , 45 ]. In this study, we found that there is no homologue of AtSUMO3 in the Caixin genome and that all the canonical BrSUMOs are closer to AtSUMO1 but not AtSUMO2 (Fig. 1 A). This means that the homologue of AtSUMO3 is most likely lost in Caixin. SUMO can not only be attached as a monomer to its targets but also form polymeric SUMO chains via N-terminal lysine residues via PIAL ligases [ 46 ]. We found that BrSUMO8 and BrSUMO9 contain a di-Gly motif in the C-terminus but no conserved Lys residue in the N-terminus (Fig. 1 B). This means that BrSUMO8 and BrSUMO9 may only be able to conjugate to substrates in monomeric form and cannot form SUMO chains. Many experiments have confirmed that the substitution of GG with AA in SUMO may cause insufficient conjugation [ 47 ]. There was a valine residue variation present in the di-Gly motif of BrSUMO7 (Fig. 1 B), indicating that it may lack the ability to conjugate targets. The E1-activating enzyme is a heterodimer comprising the SAE2 large subunit and the SAE1 small subunit that functions as a catalyst for the initial step in SUMO conjugation. Diversification of the large and small subunits into different conserved isoforms among different plants [ 48 ]. The Arabidopsis and peanut genomes encode two SAE1 genes and one SAE2 gene, but the Caixin, maize and potato genomes encode one SAE1 gene and two SAE2 genes [ 49 ]. The soybean and apple genomes encode two SAE1 genes and two SAE2 genes [ 18 , 22 ]. A recent study reported that there are four SAE1 and two SAE2 isoforms in pigeonpea [ 50 ]. However, it is unclear whether the diversity of SAE1-SAE2 pairs has an impact on the biological function of E1 since it is hypothesized that E1 plays a role in SUMO isoform selection [ 51 , 52 ]. Most of the E1 functional domains are located in the SAE2 subunit. A comparative structural analysis revealed that the cysteine domain, which is responsible for noncovalent interactions with SCE1, is the most conserved region between AtSAE2 and BrSAE2 (Fig. S1 B). Notably, the UFD domain of SAE2 has also been demonstrated to facilitate noncovalent interactions with SCE1. A notable distinction exists between the C-termini of the UFD domains in BrSAE2a and BrSAE2b (Fig. S1 B). However, it remains uncertain whether this variation impacts the selection of SCE1, given the existence of three distinct SCE1 isomers in Caixin. BrSCE1s belong to the conjugating enzyme family, which is defined by the presence of a conserved UBC domain that spans the majority of the protein. In Caixin, three SCE1 genes presented high amino acid sequence similarity (Fig. 2 C), suggesting that these orthologous genes may have arisen through duplication events. Previous studies have indicated that the RNA polymerase of the turnip mosaic virus engages in interaction with the host protein SCE1, thereby facilitating the process of infection [ 16 ]. Another study demonstrated that the S-nitrosylation of SCE1 is a critical factor in the immune response of plants [ 53 ]. These findings suggest that SCE1 plays a significant role in plant defense against pathogen invasion. However, our transcriptome data indicate that the three BrSCE1 genes display disparate expression patterns in response to infection by the Pcc pathogen (Fig. 5 B). This suggests the possibility of functional differentiation of BrSCE1s in response to Pc c invasion. The SUMO E3 family appears to have expanded in Caixin, with three SIZ1 isoforms, one MMS21 isoform, and four PIAL isoforms. The SAP domain in SIZ1 is important for nuclear retention and transcriptional regulation. There is no SAP domain present in BrSIZ1c (Fig. 3 B), which may imply that BrSIZ1c is involved in extracellular SUMOylation, as several extranuclear proteins have been shown to be directly SUMOylated [ 54 ]. Notably, it is also likely that it does not possess the function of a SUMO ligase. Previous reports in Arabidopsis and maize have shown that SIZ1 plays a more important role in the response to the invasion of plant pathogens, and ZmSIZ1a and ZmSIZ1b are considered to have functional redundancy [ 12 , 14 ]. Our transcriptome data revealed that three BrSIZ1 genes were assigned to different expression clusters (Fig. 5 B), suggesting that the three BrSIZ1 genes may play different roles in response to Pcc invasion. A similar expansion of MMS21 was not observed in Caixin. MMS21 has been shown to play a more important role in DNA damage repair and root development in Arabidopsis [ 55 , 56 ]. BrMMS21 shares high sequence identity with AtMMS21, suggesting its functional similarity. Previous work has reported that Arabidopsis plants exposed to ABA () accumulate increased levels of SUMOylated proteins in a manner dependent on the activity of MMS21 [ 57 ]. In soybean, GmMMS21 is induced by ABA and salt treatment and is thought to play a pivotal role in the salt stress response in an ABA-dependent manner [ 22 ]. Our promoter analysis revealed that the BrMMS21 promoter contains six ABREs, which are the most enriched cis -acting elements in the promoters of SUMOylation genes (Fig. 4 B, S7 ). These findings suggest a close relationship between SUMOylation and ABA signalling in Caixin. SUMO proteases with deSUMOylation activity also play important regulatory roles in plant development and stress responses. In this study, the transcription of five Caixin SUMO protease genes ( BrULP1A , BrULP1B BrSPF1 , BrSPF2 and BrFUG1 ) was significantly downregulated in response to Pcc infection (Fig. 5 B), indicating that these genes may participate in the regulation of soft rot stress responses. The presence of stress-responsive cis -regulatory elements in the promoter sequences of Caixin SUMOylation genes provides compelling evidence that SUMOylation plays an integral role in regulating plant growth and stress responses in Caixin (Fig. 4 ). A plethora of stressors, including elevated temperatures, salinity, drought, and pathogen infection, have been demonstrated to induce the accumulation of SUMO conjugates, which are crucial for plants to resist these threats. In this study, SUMO conjugates in Caixin were induced by heat stress and infection with Pcc (Fig. 6 ). Nevertheless, the biological functions and molecular mechanisms of SUMOylation in response to soft rot stress in Caixin remain largely uncharacterized. Conclusions In conclusion, we identified SUMOylation genes in the Caixin genome. The majority of these genes had multiple copies, and analyses of their transcription and protein profiles suggest that they play a role in the response to Pcc infection. The multiple copies likely arose from gene duplication events that subsequently underwent functional diversification, enabling the different gene copies to fulfil different roles in response to various stress conditions. Our data provide valuable new insights into the SUMOylation of Brassica species, which will contribute to further research on this topic. Methods Identification of SUMOylation components in Caixin The protein sequences of the Arabidopsis SUMOylation components were downloaded from the Arabidopsis Information Resource database ( http://www.Arabidopsis.org/ ) [ 58 ] and used as query sequences to preliminarily identify homologous protein sequences from Brassica rapa var. parachinensis genome via TBtools. The hidden Markov model (HMM) profiles of the SUMO, E1, E2, E3 and SUMO proteases were subsequently downloaded from the Pfam database ( http://pfam.xfam.org/ ) [ 59 ] to identify the target protein sequences in the Caixin protein database via the HMM search program of Tbtools-II [ 60 ]. The physicochemical properties of the SUMOylation components, including the number of amino acids and molecular weight (MW), were calculated via the ExPASy website ( https://web.ExPASy.org/protparam/ ) [ 61 ]. Multiple sequence alignment, phylogenetic and cis -acting elements analysis Amino acid sequence alignment was performed using Clustal X 2.0 program and the figures were generated using ESPript 3.0 ( https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi ) [ 62 ]. Functional domains of SUMOylation components were predicted using SMART online software ( http://smart.embl-heidelberg.de/ ) [ 63 ]. The phylogenetic tree was constructed using the neighbor-joining (NJ) method with MEGA 7.0 software [ 64 ]. The PlantCARE online program ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) [ 65 ] was employed to predict cis -acting elements in the Caixin SUMOylation gene promoters. The sequence of 2000 bp upstream of the start codon was utilized for a cis -acting elements scan. Plant materials and bacterial incubation Seeds of the Caixin cultivar ‘Youqing 49’ were sown in a soil mixture (peat moss, perlite, and vermiculite (8:1:1)) supplied by Shengsheng Agriculture Co., Ltd., Guangzhou, China. The seeds were then grown in a greenhouse at 20°C under a 16 h light/8 h dark cycle at 50% relative humidity. The Pcc pathogen, which was isolated and identified in our laboratory, was cultured in LB media overnight at 28°C. Next, the cells were harvested, resuspended in LB medium, and adjusted to an OD600 of 0.5. Six-leaf-stage plants were inoculated with Pcc pathogen mixture at the petiole, as previously described. The inoculated leaves were collected at 0, 12, and 24 h postinoculation (hpi), and three biological replicates were performed. The samples were immediately frozen in liquid nitrogen and stored at − 80°C for RNA isolation. RNA sequencing and data analysis Total RNA was isolated from inoculated leaves (0 hpi, 12 hpi, and 24 hpi) and used for RNA library construction. Transcriptome sequencing was performed on the Illumina HiSeq platform at Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China, with three biological replicates per treatment. The raw sequencing data were filtered to remove adapter sequences and obtain high-quality clean reads. The clean data were subsequently mapped to the Brassica rapa var. parachinensis reference genome. HTSeq (v0.6.1) was used to count the read numbers mapped to each gene, and the fragments per kilobase of transcript sequences per million sequenced base pairs (FPKM) were calculated on the basis of the length of the gene and read counts mapped to this gene. A clustering heatmap of the expression patterns of the SUMOylation system genes was generated on the basis of the FPKM values via TBtools software. Protein extraction and immunoblotting Total protein from Caixin leaves was isolated using a Plant Protein Extraction Kit (Solarbio, Beijing, China) according to the manufacturer’s instructions. The protein concentration was determined using a BCA protein assay kit (Solarbio, Beijing, China). For immunoblotting, 20 µg of purified protein was separated via sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and then transferred to a 0.45 µm PVDF membrane. The membrane was incubated with an anti- At SUMO1 antibody (Abcam, ab5316, China). The blot was developed via an enhanced chemiluminescent kit (Biosharp, Beijing, China) and detected by a chemiluminescence imaging system. Abbreviations SUMO: Small ubiquitin-related modifier; Pcc : Pectobacterium carotovorum subsp. carotovorum ; SAE: SUMO-activating enzyme; SCE: SUMO-conjugating enzyme; SA: Salicylic acid; NHP: N-hydroxypipecolic acid; FER: Fusarium ear rot; Cys: Cysteine; UBC: Ub-conjugating enzyme catalytic; MMS21/HPY2: methyl methane sulfonate-sensitive protein-21/high ploidy-2; PIAL: protein inhibitor of activated STAT (PIAS)-like; ABA: Abscisic acid. Declarations Funding This work was funded by the Key-Area Research and Development Program of Guangdong Province (2022B0202080001), the Seed Industry Revitalization Project of the Provincial Rural Revitalization Strategy Special Fund (2022-NJS-03-001, 2022-NPY-03-001), and the Guangzhou Scientific and Technological Projects (202206010173, 2023B03J1270), Guangzhou Agricultural Support Fund Project (Suinonghan [2024] No. 183). Author Contributions H.Z. and S.L. designed the research; S.L., G.L. F.Y. and D.J. performed the experiment and bioinformatics analysis; Y.Z. and B.C. analysed the data; S.L. and H.Z. wrote the manuscript. All the authors have read and agreed to the published version of the manuscript. Acknowledgements We extend our gratitude to all members of the laboratory for their contributions to the advancement of knowledge through their participation in discussions and provision of technical assistance. Data availability statement All the data analysed or generated in this study are available from the first or corresponding author upon reasonable request. The raw genome data were deposited in the China National GeneBank Data Base (CNGBdb) under Bioproject number CNP0001121. The RNA sequencing data were available in the NCBI SRA database (https://www.ncbi.nlm.nih.gov/sra) (Accession: PRJNA1144186). Conflicts of interest The authors have no conflicts of interest to declare. Ethics approval and consent to participate Not applicable. 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Supplementary Files Additionalfile.docx Supplementaryfigures.docx Figure S1. SUMO-activating enzymes (SAEs) in Caixin Figure S2. Multiple sequence alignment of SAEs from Caixin and Arabidopsis Figure S3. Amino acid sequence alignment of BrSIZ1s with AtSIZ1 Figure S4. Amino acid sequence alignment of BrPIALs with AtPIALs Figure S5. Amino acid sequence alignment of BrMMS21 with AtMMS21 Figure S6. Phylogenetic tree of SUMO proteases from Caixin and other plant species Figure S7. Analysis of cis -acting elements in the SUMOylation system gene promoter Supplementarytable.xlsx Table S1. The FPKM values of SUMOylation genes in Caixin Cite Share Download PDF Status: Published Journal Publication published 19 Dec, 2024 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 29 Oct, 2024 Reviews received at journal 19 Oct, 2024 Reviews received at journal 19 Sep, 2024 Reviewers agreed at journal 19 Sep, 2024 Reviewers agreed at journal 10 Aug, 2024 Reviewers agreed at journal 09 Aug, 2024 Reviewers invited by journal 08 Aug, 2024 Editor invited by journal 07 Aug, 2024 Editor assigned by journal 07 Aug, 2024 Submission checks completed at journal 07 Aug, 2024 First submitted to journal 01 Aug, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4844966","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":345651883,"identity":"8ba7030f-df2c-4f03-92d7-af281e2d455c","order_by":0,"name":"Shikang Lei","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYBACPgbGBmYGgwQgk/kAkDhAWAsbQgtbAlQLMyEtYCUg1TwGRGphb27+XFCQJm/Ov+abxIeaOwzm7P34XcfGc7BNeoZBjuHOGW+3Sc449ozBsucwAVskEtuYeQwqGDfcOLtNmoftMIPBjWQCWuQfNn8GarHfcOPMM+k//4Ba7j8mZAtjgzSPQU7ihvM9bNKMbSBbCHmfJ7ENqCUtecMNNmPL3r7DPAZnkg3wauFnP/74M8+fZNsN5w8/vPHj22E5g+MHH+C3Bg4kElgkgBQPkcrB9h1g/kCC8lEwCkbBKBhBAACUuUnI/n5lZQAAAABJRU5ErkJggg==","orcid":"","institution":"Guangzhou Academy of Agricultural Sciences","correspondingAuthor":true,"prefix":"","firstName":"Shikang","middleName":"","lastName":"Lei","suffix":""},{"id":345651884,"identity":"c16b8bd5-a59e-4ab8-98f2-ee7e4f6d323f","order_by":1,"name":"Guangguang Li","email":"","orcid":"","institution":"Guangzhou Academy of Agricultural and Rural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Guangguang","middleName":"","lastName":"Li","suffix":""},{"id":345651885,"identity":"1974a925-6e11-4ac9-bad4-eff95e4135d3","order_by":2,"name":"Ding Jiang","email":"","orcid":"","institution":"Guangzhou Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ding","middleName":"","lastName":"Jiang","suffix":""},{"id":345651886,"identity":"cd4ca242-a6ab-4b66-aed7-4c11a9547621","order_by":3,"name":"Fanchong Yuan","email":"","orcid":"","institution":"Guangzhou Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Fanchong","middleName":"","lastName":"Yuan","suffix":""},{"id":345651888,"identity":"7b323a56-8e75-4cd6-85e7-0b7da848441c","order_by":4,"name":"Yansong Zheng","email":"","orcid":"","institution":"Guangzhou Academy of Agricultural and Rural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yansong","middleName":"","lastName":"Zheng","suffix":""},{"id":345651890,"identity":"8226d464-67ab-4800-b098-f7e91fe2bc28","order_by":5,"name":"Bihao Cao","email":"","orcid":"","institution":"South China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Bihao","middleName":"","lastName":"Cao","suffix":""},{"id":345651891,"identity":"e1b98e8b-a023-4833-843f-cdbcdab3b957","order_by":6,"name":"Hua Zhang","email":"","orcid":"","institution":"Guangzhou Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-08-02 01:44:41","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4844966/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4844966/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-024-05807-w","type":"published","date":"2024-12-19T15:57:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63918931,"identity":"bc326a56-afa1-4b4d-9848-08deaacb7969","added_by":"auto","created_at":"2024-09-03 18:45:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":19178549,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSUMO-related proteins (SUMOs) in Caixin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003ePhylogenetic tree of BrSUMOs with SUMO proteins from Arabidopsis (\u003cem\u003eArabidopsis thaliana\u003c/em\u003e, \u003cem\u003eAt\u003c/em\u003e), rice (\u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eOs\u003c/em\u003e), maize (\u003cem\u003eZea mays\u003c/em\u003e, \u003cem\u003eZm\u003c/em\u003e) and soybean (\u003cem\u003eGlycine max\u003c/em\u003e, \u003cem\u003eGm\u003c/em\u003e). These proteins were classified into the canonical SUMO group (highlighted in green), the noncanonical SUMO group (highlighted in blue) and the SUMO variant group (highlighted in blue). \u003cstrong\u003eB: \u003c/strong\u003eAmino acid sequence alignment of BrSUMOs with AtSUMO1. Identical and similar amino acids are indicated by black and white boxes, respectively. The black triangle indicates the conserved Lys residue required for the formation of SUMO chains. The red line denotes the SUMO interactionmotif (SIM). The red triangles indicate the di-Gly motif essential for the conjugation of canonical SUMO proteins.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4844966/v1/d0285c2b220f327b6e99a0be.png"},{"id":63918936,"identity":"5a23c98c-9cd5-4487-a18f-adea33ae3b70","added_by":"auto","created_at":"2024-09-03 18:45:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8157801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSUMO-conjugating enzymes (SCEs) in Caixin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e Phylogenetic tree and conserved motifs of BrSCE1s from Caixin and other plant species. The phylogenetic relationships of the SCE1 protein sequences from Caixin, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (\u003cem\u003eAt\u003c/em\u003e), \u003cem\u003eZea mays\u003c/em\u003e (\u003cem\u003eZm\u003c/em\u003e), \u003cem\u003eOryza sativa\u003c/em\u003e (\u003cem\u003eOs\u003c/em\u003e) and \u003cem\u003eGlycine max\u003c/em\u003e (\u003cem\u003eGm\u003c/em\u003e) were assessed using the maximum likelihood (ML) method with MEGA 11. The variously colored boxes symbolize the motifs. The detailed sequence for each motif is provided in the supplementary file. \u003cstrong\u003eB:\u003c/strong\u003e Amino acid sequence alignment of BrSCE1s with AtSCE1. The white and black boxes indicatesimilar and conserved amino acids, respectively. The red line indicates the UBC domain.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4844966/v1/b2f68e459767df86b5d1551a.png"},{"id":63918932,"identity":"aa4f54d1-c67c-4a1a-9508-e9b6b3da47ea","added_by":"auto","created_at":"2024-09-03 18:45:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2872043,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe phylogenetic tree and schematic representation of three types of SUMO E3 ligases in Caixin\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e Phylogenetic tree of Caixin SUMO E3 ligases from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (\u003cem\u003eAt\u003c/em\u003e), \u003cem\u003eZea mays\u003c/em\u003e (\u003cem\u003eZm\u003c/em\u003e), \u003cem\u003eOryza sativa\u003c/em\u003e (\u003cem\u003eOs\u003c/em\u003e) and \u003cem\u003eGlycine max\u003c/em\u003e (\u003cem\u003eGm\u003c/em\u003e). These SUMO E3 ligases areclassified into SIZ1, MMS21, and PIAL types. \u003cstrong\u003eB:\u003c/strong\u003e Schematic representation of the functional domains of SIZ1, MMS21, and PIAL-type SUMO ligases.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4844966/v1/de02b38b11a35c505336d3a5.png"},{"id":63918929,"identity":"b59b3881-cd00-459f-9109-67fbf1c5e264","added_by":"auto","created_at":"2024-09-03 18:45:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2519088,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eClassification of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-acting regulatory elements in the promoters of SUMOylation system genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003eThe number of \u003cem\u003ecis\u003c/em\u003e-acting elements belonging to each functional group in the promoter regions of SUMOylation system genes. \u003cstrong\u003eB:\u003c/strong\u003e The ratio of each \u003cem\u003ecis\u003c/em\u003e-acting element in each functional group.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4844966/v1/1e6a356a6a4501e7fa341313.png"},{"id":63919152,"identity":"7c298ac7-26bc-486f-80a6-e164cde4e584","added_by":"auto","created_at":"2024-09-03 18:53:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":8539773,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTranscriptome analysis of SUMOylation system genes in Caixin under soft rot stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003ePhenotype of Caixin-infected patients with soft rot disease. Representative image of 4-week-old plant leaves infected with the \u003cem\u003ePcc\u003c/em\u003epathogen at 0, 12 and 24 hpi. \u003cstrong\u003eB:\u003c/strong\u003eExpression patternsof SUMOylation system genes upon \u003cem\u003ePcc \u003c/em\u003einfection. The FPKMvalues were log2transformed,and a heatmap was generated using TBtools software.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4844966/v1/3b818403e119d17ec0f89e9d.png"},{"id":63918933,"identity":"e7d49081-55ae-4cb1-ac41-5b551b73b6c5","added_by":"auto","created_at":"2024-09-03 18:45:38","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4782991,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSUMOylation profiles of Caixin in response to stress\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003eSUMO conjugates of Caixin plants exposed to 37 °C for 0, 15, 30, and 45 min. \u003cstrong\u003eB: \u003c/strong\u003eSUMO conjugates of Caixin plants inoculatedwith the \u003cem\u003ePcc\u003c/em\u003epathogen for 0, 6, and 12 hours. Total proteins were extracted from Caixin leaves, and subjected to SDS-PAGE and immunoblot analysis with anti-SUMO1 antibody. Coomassie blue-stained Rubisco large subunit (RBCL) was used as the loading control.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4844966/v1/b3585304fc90cb5aa3f10a13.png"},{"id":72202041,"identity":"222d654f-e600-457d-b7c9-5be1e219f38e","added_by":"auto","created_at":"2024-12-23 16:13:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":64877360,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4844966/v1/47aa2f84-61b1-4d8b-b1fa-1b933c5b0f1b.pdf"},{"id":63918927,"identity":"c0800595-ee9f-4527-bb91-f3d9d6b2eaaf","added_by":"auto","created_at":"2024-09-03 18:45:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":207092,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-4844966/v1/9fe644335f8da580637d45ed.docx"},{"id":63918928,"identity":"b94deb1c-ddb5-4802-8321-91b77595ba97","added_by":"auto","created_at":"2024-09-03 18:45:37","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2363011,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S1. SUMO-activating enzymes (SAEs) in Caixin\u003c/p\u003e\n\u003cp\u003eFigure S2. Multiple sequence alignment of SAEs from Caixin and \u003cem\u003eArabidopsis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFigure S3. Amino acid sequence alignment of BrSIZ1s with AtSIZ1\u003c/p\u003e\n\u003cp\u003eFigure S4. Amino acid sequence alignment of BrPIALs with AtPIALs\u003c/p\u003e\n\u003cp\u003eFigure S5. Amino acid sequence alignment of BrMMS21 with AtMMS21\u003c/p\u003e\n\u003cp\u003eFigure S6. Phylogenetic tree of SUMO proteases from Caixin and other plant species\u003c/p\u003e\n\u003cp\u003eFigure S7. Analysis of \u003cem\u003ecis\u003c/em\u003e-acting elements in the SUMOylation system gene promoter\u003c/p\u003e","description":"","filename":"Supplementaryfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-4844966/v1/cd37e97279c2392c44ff4e9b.docx"},{"id":63919151,"identity":"4312bd8c-1170-4a5f-b590-6862a7fae7f0","added_by":"auto","created_at":"2024-09-03 18:53:38","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":11323,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1. The FPKM values of SUMOylation genes in Caixin\u003c/p\u003e","description":"","filename":"Supplementarytable.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4844966/v1/1ac05d32f926545f31f91d6d.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Definition and regulatory analysis of the SUMOylation system in Caixin (Brassica rapa var. parachinensis) during Pectobacterium carotovorum infection","fulltext":[{"header":"Background","content":"\u003cp\u003ePosttranslational modifications (PTMs) are chemical modifications of proteins after translation and are important regulatory mechanisms through which proteins perform their biological functions. After translation, various functional groups are added to the corresponding amino acid residues of proteins through covalent bonds, resulting in changes in the spatial conformation of proteins and triggering functional changes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. SUMOylation, also known as ubiquitination, is an important form of posttranslational protein modification that regulates many life activities in eukaryotic cells. The SUMO protein has a three-dimensional structure similar to that of the ubiquitin protein and is conserved and widely expressed in eukaryotic cells [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Like ubiquitination, SUMOylation requires a series of enzymatic reactions. First, the SUMO precursor is cleaved to expose the C-terminal glycine, which is subsequently activated and coupled to a cysteine of the heterodimeric SUMO-activating enzyme E1 (SAE1/SAE2) in an ATP-dependent reaction. The activated SUMO is subsequently transferred to the SUMO-conjugating enzyme E2 (SCE1) through transesterification and the formation of the SUMO-E2 complex. Finally, E2 recognizes target proteins at a specific sequence (ΨKXE/D) (Ψ, large hydrophobic amino acid; K, acceptor lysine; X, any amino acid; E/D, glutamate or aspartate) and attaches SUMO to target proteins with the assistance of the SUMO E3 ligase [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. SUMOylation is a reversible process capable of releasing SUMO for further conjugation cycles via the hydrolysis of isopeptide bonds by SUMO proteases [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSUMOylation has been demonstrated to be essential for plants, with several studies indicating its importance in integrating environmental inputs and responding appropriately to stress conditions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Previous studies have shown that the abundance of SUMO conjugates in plants can be significantly increased upon exposure to heat, cold, high salinity, and abscisic acid [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Alterations in the function of SUMOylation components can lead to changes in plant adaptation to environmental stresses. Additionally, several SUMOylation components have been connected to biotic responses [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In 1999, Hanania et al. first identified a link between SUMOylation and plant immune responses, and subsequent studies have gradually elucidated the mechanisms by which SUMOylation is involved in the immune responses of plants [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In \u003cem\u003eArabidopsis\u003c/em\u003e, the absence of the SUMO ligase gene \u003cem\u003eAtSIZ1\u003c/em\u003e resulted in excessive accumulation of salicylic acid and upregulation of the expression levels of disease resistance-related genes in vivo, leading to the production of a dwarfing phenotype and an autoimmune response phenotype [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Recent studies have demonstrated that the autoimmune response of \u003cem\u003eatsiz1\u003c/em\u003e mutants is a consequence of the suppression of the immune response of AtTPR1 by SUMOylation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Like \u003cem\u003eatsiz1\u003c/em\u003e, the SUMO protease mutant \u003cem\u003eatesd4\u003c/em\u003e also exhibited autoimmune phenotypes with elevated expression of SA (salicylic acid) and NHP (N-hydroxypipecolic acid) biosynthesis-related genes [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In maize, ZmSIZ1a/ZmSIZ1b-mediated SUMOylation plays a pivotal role in the comprehensive defense response of maize against FER. This is achieved by mediating several signalling pathways, especially early defense responses and flavonoid synthesis [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. During pathogen attack, SUMOylation components also serve as targets of pathogen effectors, providing an opportunity to inhibit SUMOylation at different stages. Tomato Golden Mosaic Virus (TGMV) and Tomato Yellow Leaf Curl Sardinia Virus (TYLCSV) encode a protein, RepAC1 (or Rep), which is essential for the replication of the viruses. In tobacco, the SUMO E2-binding enzyme NbSCE1 specifically interacts with the Rep/RepAC1 protein to affect viral replication [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In another study, yeast two-hybrid screening of SCE1 from \u003cem\u003eArabidopsis\u003c/em\u003e revealed a single interaction involving AtSCE1 and the viral RNA-dependent RNA polymerase Nlb, and the \u003cem\u003eArabidopsis atsce1\u003c/em\u003e mutant also presented increased resistance to TuMV [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These findings offer new insights into the underlying role of SUMOylation components in virus infection in plants.\u003c/p\u003e \u003cp\u003eDespite the importance of SUMOylation in the plant development process or under stress conditions [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], functional studies on this topic have been largely restricted to \u003cem\u003eArabidopsis\u003c/em\u003e. With the increase in the number of deciphered plant genomes, SUMOylation components have gradually been identified in apple [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], tomato [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], poplar [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], rice [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], soybean [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], maize [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and other plants [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, no available information on SUMOylation in Brassica species has been reported. In this study, we comprehensively characterized the core SUMOylation components of Caixin, a popular and widely cultivated leafy vegetable crop in southern China. We also investigated the transcriptional responses of SUMOylation genes during \u003cem\u003ePcc\u003c/em\u003e infection. Our findings provide an overview of the SUMOylation in Caixin and reveal the potential roles of SUMOylation in the soft rot stress response.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIdentification and characterization of SUMOylation genes in Caixin\u003c/h2\u003e \u003cp\u003eWhole-genome sequencing of Caixin was completed in 2023 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Using known \u003cem\u003eArabidopsis\u003c/em\u003e SUMOylation component sequences as queries, we conducted a search for their respective orthologues in the Caixin genome via BLAST and domain confirmation methods. A total of 30 genes encoding the core components of SUMOylation were identified in this Brassica species (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Our list revealed that the Caixin genome contains nine SUMO genes, termed \u003cem\u003eBrSUMO1\u003c/em\u003e to \u003cem\u003eBrSUMO9\u003c/em\u003e. To further understand the evolutionary relationships among these SUMO isoforms, we searched a number of other plant genomes for related sequences, including those of \u003cem\u003eArabidopsis\u003c/em\u003e, rice, soybean and maize (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Phylogenetic analysis revealed that these SUMOs clustered into three groups: the \u0026ldquo;canonical SUMO\u0026rdquo;, \u0026ldquo;noncanonical SUMO\u0026rdquo; and \u0026ldquo;SUMO variant\u0026rdquo; groups. Members belonging to the \u0026ldquo;canonical SUMO\u0026rdquo; group were strongly conserved. Five members of the BrSUMO family (BrSUMO1 to BrSUMO5) are divided into this group and are evolutionarily most closely related to AtSUMO1. The \u0026ldquo;noncanonical SUMO\u0026rdquo; group, which contains four BrSUMOs (BrSUMO6 to BrSUMO9), shares low amino acid identity. No BrSUMOs belong to the \u0026ldquo;SUMO variant\u0026rdquo; group. The C-terminal di-Gly motif of SUMO is necessary for substrate conjugation. Alignment of SUMO sequences revealed that, with the exception of BrSUMO7, both canonical and noncanonical BrSUMOs have a C-terminal di-Gly motif, indicating their potential ability to covalently attach to target proteins. We also noted that the SUMO interaction motif in BrSUMO6 and BrSUMO7 is different from that in other BrSUMOs. Additionally, the other two noncanonical BrSUMOs (BrSUMO8 and BrSUMO9) did not contain the conserved Lys (Lysine) residue, which is required for the formation of SUMO chains (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\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\u003eCharacteristics of SUMOylation system genes in Caixin\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\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=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSequence ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChromosome\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProtein length (aa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eMW (Da)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003eSUMO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrSUMO1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBra_cxA08g015400\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e101\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e11124.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrSUMO2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBra_cxA03g012940\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e10844.17\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrSUMO3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBra_cxA01g030080\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e113\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e12733.35\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrSUMO4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBra_cxA10g021190\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e 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align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e121\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13471.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrSUMO7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBra_cxA05g032960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e152\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e16996.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrSUMO8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBra_cxA09g065660\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e 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colname=\"c2\"\u003e \u003cp\u003eBrSAE2b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBra_cxA09g010380\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e552\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e62403.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eE2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrSCE1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBra_cxA04g035410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e160\u003c/p\u003e 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colname=\"c5\"\u003e \u003cp\u003e265\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e30304.82\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e \u003cp\u003eE3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrSIZ1a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBra_cxA02g039720\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e862\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e95055.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrSIZ1b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e 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colname=\"c2\"\u003e \u003cp\u003eBrPIAL1b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBra_cxA09g003500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e636\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e70816.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrPIAL2a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBra_cxA04g019330\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA04\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e723\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e79030.63\u003c/p\u003e \u003c/td\u003e 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align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e439\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e51090.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrULP1B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBra_cxA03g019930\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e617\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e70756.78\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrULP1C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBra_cxA06g038970\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e569\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e65650.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrESD4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBra_cxA08g024070\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e404\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e46397.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrFUG1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBra_cxA06g020780\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e223\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e26836.84\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrSPF1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBra_cxA08g003020\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e638\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e71562.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBrSPF2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eBra_cxA01g042650\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eA01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e792\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e89798.89\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 \u003cp\u003eSUMOylation requires an initial activation step in which the C-terminal di-Gly of mature SUMO is first adenylated by a heterodimeric SUMO-activating enzyme (SAE1/2) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In contrast to \u003cem\u003eArabidopsis\u003c/em\u003e, the Caixin genome encodes one small-subunit \u003cem\u003eSAE1\u003c/em\u003e gene (\u003cem\u003eBrSAE1\u003c/em\u003e) and two large-subunit \u003cem\u003eSAE2\u003c/em\u003e genes (\u003cem\u003eBrSAE2a\u003c/em\u003e and \u003cem\u003eBrSAE2b\u003c/em\u003e). \u003cem\u003eBrSAE1\u003c/em\u003e encodes a protein of 322 amino acids that is conserved with AtSAE1a (90% identity) and AtSAE1b (82% identity). \u003cem\u003eBrSAE2a\u003c/em\u003e and \u003cem\u003eBrSAE2b\u003c/em\u003e both encode a protein of 625 amino acids that shares 87% and 93% identity with AtSAE2, respectively. Furthermore, functional domains were also identified in BrSAEs (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA, B). Phylogenetic analysis of SAEs from various plant species revealed that BrSAE1 and BrSAE2a/b were evolutionarily most closely related to AtSAE1a/b and AtSAE2, respectively (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA, B). We also predicted the 3D structures of SAE1/SAE2 heterodimers via SWISS-MODEL. The structures of both BrSAE1/BrSAE2a and BrSAE1/BrSAE2b were similar to those of AtSAE1a/AtSAE2 and AtSAE1b/AtSAE2 (Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUpon activation by a SUMO-activating enzyme, the bound SUMO moiety is transferred via transesterification to an active site Cys (Cysteine) in the SUMO-conjugate enzyme SCE1 and then forms a SUMO-E2 thioester intermediate [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The \u003cem\u003eArabidopsis\u003c/em\u003e genome contains a single \u003cem\u003eSCE1\u003c/em\u003e gene (\u003cem\u003eAtSCE1\u003c/em\u003e) compared with three other \u003cem\u003eSCE1\u003c/em\u003e genes (\u003cem\u003eBrSCE1a\u003c/em\u003e, \u003cem\u003eBrSCE1b\u003c/em\u003e and \u003cem\u003eBrSCE1c\u003c/em\u003e) in Caixin. The maize genome encodes a novel class II isotype of \u003cem\u003eSCE1\u003c/em\u003e, which is found only in the cereal branch of monocots. To further examine the origins of BrSCE1 isotypes, we phylogenetically analysed a collection of 17 SCE protein sequences from 4 plant genomes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, all three \u003cem\u003eBrSCE1\u003c/em\u003e genes were clustered into class I and presented high similarity with homologues in other species, especially \u003cem\u003eArabidopsis\u003c/em\u003e. We then identified conserved motifs in the SCE1 proteins via the MEME program. A total of three conserved motifs were found in most of the SCE1 proteins, and motif 2 was present in all of these SCE1 proteins. BrSCE1a, BrSCE1b and BrSCE1c also share high sequence identity with AtSCE1 and have a highly conserved Ub-conjugating enzyme catalytic (UBC) domain (spanning from 8 to 150 aa residues, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), which is common to the UBC family.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough SUMOylation can occur without SUMO E3 ligases under certain conditions [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], increasing evidence has revealed that the absence of E3 ligases significantly reduces SUMO conjugate levels in plants. There are three types of SUMO E3 ligases in plants: SIZ/PIAS (SIZ1), methyl methane sulfonate-sensitive protein-21/high ploidy-2 (MMS21/HPY2) and protein inhibitor of activated STAT (PIAS)-like (PIAL). The genome of Caixin encodes three \u003cem\u003eSIZ1\u003c/em\u003e genes (\u003cem\u003eBrSIZ1a\u003c/em\u003e, \u003cem\u003eBrSIZ1b\u003c/em\u003e and \u003cem\u003eBrSIZ1c\u003c/em\u003e), four PIAL genes (\u003cem\u003eBrPIAL1a\u003c/em\u003e, \u003cem\u003eBrPIAL1b\u003c/em\u003e, \u003cem\u003eBrPIAL2a\u003c/em\u003e and \u003cem\u003eBrPIAL2b\u003c/em\u003e) and one MMS21/HPY2 gene (\u003cem\u003eBrMMS21\u003c/em\u003e). A phylogenetic analysis of SUMO E3 ligases from various plant species revealed that different types of SUMO E3 ligases in Caixin are evolutionarily most closely related to those in \u003cem\u003eArabidopsis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Among the members of the SUMO E3 ligase family, AtSIZ1 is the most extensively studied in plants. Its involvement in various biological processes, including flowering [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], plant immunity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], abiotic stress tolerance [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], thermomorphogenesis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and phytohormone signalling [\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], has been well documented. A common feature of known SIZ1 is the presence of five conserved domains (SAP, PHD, MIZ/SP-RING domain, PINIT and SXS motifs). Both BrSIZ1a and BrSIZ1b have five conserved domains, but a deletion of the SAP domain was observed in BrSIZ1c (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Like \u003cem\u003eArabidopsis\u003c/em\u003e, Caixin contains only one MMS21/HPY2-type E3 (BrMMS21), which also has a conserved SP-RING domain and shares high sequence identity with AtMMS21 (Fig. S4). Previous studies have shown that AtPIAL1 and AtPIAL2 function as SUMO ligases capable of SUMO chain formation. Sequence alignment revealed that both AtPIALs and BrPIALs have the MIZ/SP-RING domain as well as SIM motifs, which have specific affinities for binding to SUMO chains. However, the absence of the N-terminal SIM motif was observed in BrPIAL1b (Fig. S5).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSUMOylation is a reversible process that requires the specific action of SUMO proteases to process SUMO precursors and release SUMO from substrate conjugates [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. A search of the protein sequence of \u003cem\u003eArabidopsis\u003c/em\u003e SUMO proteases revealed the presence of seven SUMO proteases in the Caixin genome. We phylogenetically analysed the selected plant species that encode potential orthologues of known \u003cem\u003eArabidopsis\u003c/em\u003e SUMO proteases. The phylogenetic tree revealed that the SUMO proteases in Caixin clustered into four groups, namely, the ESD4-type, OTS-type, FUG-type and SPF-type groups, which was consistent with the classification results in \u003cem\u003eArabidopsis\u003c/em\u003e (Fig. S6).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCis\u003c/b\u003e \u003cb\u003e‑acting element analysis of SUMOylation gene promoters\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo obtain information regarding the stimulus-induced, temporal, and spatial expression patterns of the SUMOylation genes in Caixin, 2.0 kb promoter regions upstream of these 30 genes were extracted and utilized for \u003cem\u003ecis\u003c/em\u003e-acting element searches via the PlantCARE website. As shown in Fig. S7, a diverse range of \u003cem\u003ecis\u003c/em\u003e-acting elements were present in the promoters of these genes. We classified these \u003cem\u003ecis\u003c/em\u003e-acting elements into three groups: \u0026ldquo;plant growth and development\u0026rdquo;, \u0026ldquo;phytohormone responsiveness\u0026rdquo; and \u0026ldquo;stress responsiveness\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Almost all the promoters of these genes contained many phytohormone responsive elements related to ABA (ABRE), auxin (TGA element and AuxRR core), gibberellin (TATC-box, GARE motif and P-box), salicylic acid (TCA element) and MeJA (TGACG motif and CGTCA motif) (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). ABREs are the most common \u003cem\u003ecis\u003c/em\u003e-acting elements involved in the plant hormone response, accounting for 32% of all ABREs. In addition, nine types of \u003cem\u003ecis\u003c/em\u003e-acting elements related to the cell cycle (MAS-like motif), circadian control (circadian motif), zein metabolism regulation (O\u003csub\u003e2\u003c/sub\u003e-site), meristem expression (CAT-box and NON-box), root-specific expression (motif I), endosperm expression (GCN4 motif and AACA motif) and differentiation of palisade mesophyll cells (HD-Zip 1), which cover most plant developmental stages, were involved in plant growth and development (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Seven types of \u003cem\u003ecis\u003c/em\u003e-acting elements were divided into stress-responsive groups, namely, mixed stress response (TC-rich repeats), low-temperature responsiveness (LTR motif), anaerobic induction (ARE), MYB binding site (MBS) and wound-responsive element (WUN motif) elements, and ARE elements appeared in most of the promoters of these SUMOylation system genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These results indicated that the expression of these genes might be associated with diverse signalling pathways during plant growth and stress responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eTranscriptional profiles of SUMOylation genes under soft rot stress\u003c/h2\u003e \u003cp\u003eSoft rot is a common disease of Brassica crops caused primarily by \u003cem\u003ePcc\u003c/em\u003e, a bacterial pathogen that infects plants through wounds at the base of the stem or petiole [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. As the infection time increased, the infected area expanded, and the area from the water stains decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). To further understand the potential roles and transcriptional regulation of the SUMOylation in Caixin under biotic stress, the expression patterns of the associated genes were analysed via RNA-seq datasets. The FPKM data (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) from the RNA-seq of all the SUMOylation system genes upon \u003cem\u003ePcc\u003c/em\u003e infection are shown in a heatmap. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, a total of 30 SUMOylation system genes were clustered into four subgroups according to their expression patterns. Among these genes, 23 had a relatively high level of expression under normal conditions and were downregulated after \u003cem\u003ePcc\u003c/em\u003e infection. The expression of four genes, namely, \u003cem\u003eBrSUMO3\u003c/em\u003e, \u003cem\u003eBrSUMO8\u003c/em\u003e, \u003cem\u003eBrSCE1b\u003c/em\u003e and \u003cem\u003eBrSIZ1b\u003c/em\u003e, was continuously downregulated by \u003cem\u003ePcc\u003c/em\u003e infection. The expression of four other genes, namely, \u003cem\u003eBrSUMO5\u003c/em\u003e, \u003cem\u003eBrSUMO7\u003c/em\u003e, \u003cem\u003eBrPIAL2a\u003c/em\u003e and \u003cem\u003eBrESD4a\u003c/em\u003e, was continuously induced by \u003cem\u003ePcc\u003c/em\u003e infection. These results suggest that the transcription of SUMOylated genes is not only essential for plant development but also regulated by soft rot stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePcc\u003c/b\u003e \u003cb\u003einfection regulated SUMO conjugates in Caixin\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAbiotic stresses, including heat, drought, and oxidative stress, have been shown to substantially increase the levels of SUMO conjugates [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. To determine whether SUMOylation in Caixin responds to soft rot stress, the variation in the SUMO complex was investigated. The occurrence of heat-induced SUMO conjugates has been confirmed in several plant species, and high temperature is a key factor in outbreaks of soft rot disease in the field. Therefore, we first examined heat-induced SUMO conjugates in Caixin as a positive control. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, during 37\u0026deg;C treatment, the amount of SUMO conjugates in leaves increased substantially after 30 min of heat stimulus compared with that in the unstressed conditions. We then detected SUMO conjugates in Caixin leaves upon \u003cem\u003ePcc\u003c/em\u003e infection. After inoculation with the \u003cem\u003ePcc\u003c/em\u003e strain for 12 hpi, the amount of SUMO conjugates remained unchanged. However, the conjugate levels increased substantially from 12 to 24 hpi (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). These results suggest that SUMOylation is involved in the response of Caixin to soft rot stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003ePlant SUMOylation has been implicated in nutrient acquisition and defense against biotic and abiotic challenges [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In recent years, the SUMOylation system has been identified in several plants, crops, and fruits. However, the components of the SUMOylation in Brassica plants remain unknown. In this study, a total of 30 SUMOylation genes were identified in Caixin (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The genome of Caixin contains complete SUMOylation components, including SUMO, E1-activating enzymes, E2-conjugating enzymes, E3 ligases and SUMO proteases. Brassicaceae plants underwent whole-genome triplication (WGT) between 13 and 17\u0026nbsp;million years ago (MYA) and gene loss following whole-genome duplication [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], which indicates that close homologues of \u003cem\u003eArabidopsis\u003c/em\u003e SUMOylation genes may be present in multiple copies or disappear in the Caixin genome. Compared with the eight putative SUMO copies in the \u003cem\u003eArabidopsis\u003c/em\u003e genome, the Caixin genome contains nine SUMO members, five of which (BrSUMO1 to BrSUMO5) cluster into the canonical SUMO group, and the other four (BrSUMO6 to BrSUMO9) cluster into the noncanonical SUMO group. In the canonical SUMO group, all five BrSUMOs were closely related to AtSUMO1 in terms of their evolutionary relationships (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In \u003cem\u003eArabidopsis\u003c/em\u003e, two noncanonical SUMOs, AtSUMO3 and AtSUMO5, have been proposed to have arisen from a duplication of SUMO2 and an ancient paneudicot palaeohexaploidy event, respectively. Moreover, previous studies have suggested that SUMO3 is frequently deleted, converted back to SUMO2 or pseudogenized in another \u003cem\u003eBrassica\u003c/em\u003e spp. [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In this study, we found that there is no homologue of AtSUMO3 in the Caixin genome and that all the canonical BrSUMOs are closer to AtSUMO1 but not AtSUMO2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This means that the homologue of AtSUMO3 is most likely lost in Caixin. SUMO can not only be attached as a monomer to its targets but also form polymeric SUMO chains via N-terminal lysine residues via PIAL ligases [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. We found that BrSUMO8 and BrSUMO9 contain a di-Gly motif in the C-terminus but no conserved Lys residue in the N-terminus (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). This means that BrSUMO8 and BrSUMO9 may only be able to conjugate to substrates in monomeric form and cannot form SUMO chains. Many experiments have confirmed that the substitution of GG with AA in SUMO may cause insufficient conjugation [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. There was a valine residue variation present in the di-Gly motif of BrSUMO7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), indicating that it may lack the ability to conjugate targets.\u003c/p\u003e \u003cp\u003eThe E1-activating enzyme is a heterodimer comprising the SAE2 large subunit and the SAE1 small subunit that functions as a catalyst for the initial step in SUMO conjugation. Diversification of the large and small subunits into different conserved isoforms among different plants [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The \u003cem\u003eArabidopsis\u003c/em\u003e and peanut genomes encode two \u003cem\u003eSAE1\u003c/em\u003e genes and one \u003cem\u003eSAE2\u003c/em\u003e gene, but the Caixin, maize and potato genomes encode one \u003cem\u003eSAE1\u003c/em\u003e gene and two \u003cem\u003eSAE2\u003c/em\u003e genes [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The soybean and apple genomes encode two \u003cem\u003eSAE1\u003c/em\u003e genes and two \u003cem\u003eSAE2\u003c/em\u003e genes [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A recent study reported that there are four SAE1 and two SAE2 isoforms in pigeonpea [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. However, it is unclear whether the diversity of SAE1-SAE2 pairs has an impact on the biological function of E1 since it is hypothesized that E1 plays a role in SUMO isoform selection [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Most of the E1 functional domains are located in the SAE2 subunit. A comparative structural analysis revealed that the cysteine domain, which is responsible for noncovalent interactions with SCE1, is the most conserved region between AtSAE2 and BrSAE2 (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). Notably, the UFD domain of SAE2 has also been demonstrated to facilitate noncovalent interactions with SCE1. A notable distinction exists between the C-termini of the UFD domains in BrSAE2a and BrSAE2b (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB). However, it remains uncertain whether this variation impacts the selection of SCE1, given the existence of three distinct SCE1 isomers in Caixin. BrSCE1s belong to the conjugating enzyme family, which is defined by the presence of a conserved UBC domain that spans the majority of the protein. In Caixin, three \u003cem\u003eSCE1\u003c/em\u003e genes presented high amino acid sequence similarity (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), suggesting that these orthologous genes may have arisen through duplication events. Previous studies have indicated that the RNA polymerase of the turnip mosaic virus engages in interaction with the host protein SCE1, thereby facilitating the process of infection [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Another study demonstrated that the S-nitrosylation of SCE1 is a critical factor in the immune response of plants [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. These findings suggest that SCE1 plays a significant role in plant defense against pathogen invasion. However, our transcriptome data indicate that the three \u003cem\u003eBrSCE1\u003c/em\u003e genes display disparate expression patterns in response to infection by the \u003cem\u003ePcc\u003c/em\u003e pathogen (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). This suggests the possibility of functional differentiation of \u003cem\u003eBrSCE1s\u003c/em\u003e in response to \u003cem\u003ePc\u003c/em\u003ec invasion.\u003c/p\u003e \u003cp\u003eThe SUMO E3 family appears to have expanded in Caixin, with three SIZ1 isoforms, one MMS21 isoform, and four PIAL isoforms. The SAP domain in SIZ1 is important for nuclear retention and transcriptional regulation. There is no SAP domain present in BrSIZ1c (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), which may imply that BrSIZ1c is involved in extracellular SUMOylation, as several extranuclear proteins have been shown to be directly SUMOylated [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Notably, it is also likely that it does not possess the function of a SUMO ligase. Previous reports in \u003cem\u003eArabidopsis\u003c/em\u003e and maize have shown that SIZ1 plays a more important role in the response to the invasion of plant pathogens, and ZmSIZ1a and ZmSIZ1b are considered to have functional redundancy [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Our transcriptome data revealed that three BrSIZ1 genes were assigned to different expression clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), suggesting that the three BrSIZ1 genes may play different roles in response to \u003cem\u003ePcc\u003c/em\u003e invasion. A similar expansion of MMS21 was not observed in Caixin. MMS21 has been shown to play a more important role in DNA damage repair and root development in \u003cem\u003eArabidopsis\u003c/em\u003e [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. BrMMS21 shares high sequence identity with AtMMS21, suggesting its functional similarity. Previous work has reported that \u003cem\u003eArabidopsis\u003c/em\u003e plants exposed to ABA () accumulate increased levels of SUMOylated proteins in a manner dependent on the activity of MMS21 [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. In soybean, \u003cem\u003eGmMMS21\u003c/em\u003e is induced by ABA and salt treatment and is thought to play a pivotal role in the salt stress response in an ABA-dependent manner [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Our promoter analysis revealed that the \u003cem\u003eBrMMS21\u003c/em\u003e promoter contains six ABREs, which are the most enriched \u003cem\u003ecis\u003c/em\u003e-acting elements in the promoters of SUMOylation genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, \u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003eS7\u003c/span\u003e). These findings suggest a close relationship between SUMOylation and ABA signalling in Caixin. SUMO proteases with deSUMOylation activity also play important regulatory roles in plant development and stress responses. In this study, the transcription of five Caixin SUMO protease genes (\u003cem\u003eBrULP1A\u003c/em\u003e, \u003cem\u003eBrULP1B BrSPF1\u003c/em\u003e, \u003cem\u003eBrSPF2\u003c/em\u003e and \u003cem\u003eBrFUG1\u003c/em\u003e) was significantly downregulated in response to \u003cem\u003ePcc\u003c/em\u003e infection (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), indicating that these genes may participate in the regulation of soft rot stress responses.\u003c/p\u003e \u003cp\u003eThe presence of stress-responsive \u003cem\u003ecis\u003c/em\u003e-regulatory elements in the promoter sequences of Caixin SUMOylation genes provides compelling evidence that SUMOylation plays an integral role in regulating plant growth and stress responses in Caixin (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A plethora of stressors, including elevated temperatures, salinity, drought, and pathogen infection, have been demonstrated to induce the accumulation of SUMO conjugates, which are crucial for plants to resist these threats. In this study, SUMO conjugates in Caixin were induced by heat stress and infection with \u003cem\u003ePcc\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Nevertheless, the biological functions and molecular mechanisms of SUMOylation in response to soft rot stress in Caixin remain largely uncharacterized.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, we identified SUMOylation genes in the Caixin genome. The majority of these genes had multiple copies, and analyses of their transcription and protein profiles suggest that they play a role in the response to \u003cem\u003ePcc\u003c/em\u003e infection. The multiple copies likely arose from gene duplication events that subsequently underwent functional diversification, enabling the different gene copies to fulfil different roles in response to various stress conditions. Our data provide valuable new insights into the SUMOylation of Brassica species, which will contribute to further research on this topic.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIdentification of SUMOylation components in Caixin\u003c/h2\u003e \u003cp\u003eThe protein sequences of the \u003cem\u003eArabidopsis\u003c/em\u003e SUMOylation components were downloaded from the Arabidopsis Information Resource database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.Arabidopsis.org/\u003c/span\u003e\u003cspan address=\"http://www.Arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] and used as query sequences to preliminarily identify homologous protein sequences from \u003cem\u003eBrassica rapa\u003c/em\u003e var. \u003cem\u003eparachinensis\u003c/em\u003e genome via TBtools. The hidden Markov model (HMM) profiles of the SUMO, E1, E2, E3 and SUMO proteases were subsequently downloaded from the Pfam database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org/\u003c/span\u003e\u003cspan address=\"http://pfam.xfam.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e] to identify the target protein sequences in the Caixin protein database via the HMM search program of Tbtools-II [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The physicochemical properties of the SUMOylation components, including the number of amino acids and molecular weight (MW), were calculated via the ExPASy website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://web.ExPASy.org/protparam/\u003c/span\u003e\u003cspan address=\"https://web.ExPASy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eMultiple sequence alignment, phylogenetic and\u003c/b\u003e \u003cb\u003ecis\u003c/b\u003e\u003cb\u003e-acting elements analysis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAmino acid sequence alignment was performed using Clustal X 2.0 program and the figures were generated using ESPript 3.0 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi\u003c/span\u003e\u003cspan address=\"https://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Functional domains of SUMOylation components were predicted using SMART online software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart.embl-heidelberg.de/\u003c/span\u003e\u003cspan address=\"http://smart.embl-heidelberg.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. The phylogenetic tree was constructed using the neighbor-joining (NJ) method with MEGA 7.0 software [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. The PlantCARE online program (\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) [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] was employed to predict \u003cem\u003ecis\u003c/em\u003e-acting elements in the Caixin SUMOylation gene promoters. The sequence of 2000 bp upstream of the start codon was utilized for a \u003cem\u003ecis\u003c/em\u003e-acting elements scan.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials and bacterial incubation\u003c/h2\u003e \u003cp\u003eSeeds of the Caixin cultivar \u0026lsquo;Youqing 49\u0026rsquo; were sown in a soil mixture (peat moss, perlite, and vermiculite (8:1:1)) supplied by Shengsheng Agriculture Co., Ltd., Guangzhou, China. The seeds were then grown in a greenhouse at 20\u0026deg;C under a 16 h light/8 h dark cycle at 50% relative humidity. The \u003cem\u003ePcc\u003c/em\u003e pathogen, which was isolated and identified in our laboratory, was cultured in LB media overnight at 28\u0026deg;C. Next, the cells were harvested, resuspended in LB medium, and adjusted to an OD600 of 0.5. Six-leaf-stage plants were inoculated with \u003cem\u003ePcc\u003c/em\u003e pathogen mixture at the petiole, as previously described. The inoculated leaves were collected at 0, 12, and 24 h postinoculation (hpi), and three biological replicates were performed. The samples were immediately frozen in liquid nitrogen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for RNA isolation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRNA sequencing and data analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from inoculated leaves (0 hpi, 12 hpi, and 24 hpi) and used for RNA library construction. Transcriptome sequencing was performed on the Illumina HiSeq platform at Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China, with three biological replicates per treatment. The raw sequencing data were filtered to remove adapter sequences and obtain high-quality clean reads. The clean data were subsequently mapped to the \u003cem\u003eBrassica rapa\u003c/em\u003e var. parachinensis reference genome. HTSeq (v0.6.1) was used to count the read numbers mapped to each gene, and the fragments per kilobase of transcript sequences per million sequenced base pairs (FPKM) were calculated on the basis of the length of the gene and read counts mapped to this gene. A clustering heatmap of the expression patterns of the SUMOylation system genes was generated on the basis of the FPKM values via TBtools software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eProtein extraction and immunoblotting\u003c/h2\u003e \u003cp\u003e Total protein from Caixin leaves was isolated using a Plant Protein Extraction Kit (Solarbio, Beijing, China) according to the manufacturer\u0026rsquo;s instructions. The protein concentration was determined using a BCA protein assay kit (Solarbio, Beijing, China). For immunoblotting, 20 \u0026micro;g of purified protein was separated via sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and then transferred to a 0.45 \u0026micro;m PVDF membrane. The membrane was incubated with an anti-\u003cem\u003eAt\u003c/em\u003eSUMO1 antibody (Abcam, ab5316, China). The blot was developed via an enhanced chemiluminescent kit (Biosharp, Beijing, China) and detected by a chemiluminescence imaging system.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eSUMO: Small ubiquitin-related modifier;\u0026nbsp;\u003cem\u003ePcc\u003c/em\u003e:\u003cem\u003e\u0026nbsp;Pectobacterium carotovorum\u0026nbsp;\u003c/em\u003esubsp.\u003cem\u003e\u0026nbsp;carotovorum\u003c/em\u003e; SAE: SUMO-activating enzyme; SCE: SUMO-conjugating enzyme; SA: Salicylic acid; NHP: N-hydroxypipecolic acid; FER: Fusarium ear rot; Cys: Cysteine; UBC: Ub-conjugating enzyme catalytic; MMS21/HPY2: methyl methane sulfonate-sensitive protein-21/high ploidy-2; PIAL: protein inhibitor of activated STAT (PIAS)-like; ABA: Abscisic acid.\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by the Key-Area Research and Development Program of Guangdong Province (2022B0202080001), the Seed Industry Revitalization Project of\u0026nbsp;the\u0026nbsp;Provincial Rural Revitalization Strategy Special Fund (2022-NJS-03-001, 2022-NPY-03-001), and the Guangzhou Scientific and Technological Projects (202206010173, 2023B03J1270), Guangzhou Agricultural Support Fund Project (Suinonghan [2024] No. 183).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH.Z. and S.L. designed the research; S.L., G.L. F.Y. and D.J. performed the experiment and bioinformatics analysis; Y.Z. and B.C. analysed the data; S.L. and H.Z. wrote the manuscript. All\u0026nbsp;the\u0026nbsp;authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extend our gratitude to all members of the laboratory for their contributions to the advancement of knowledge through their participation in discussions and provision of technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eavailability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data analysed or generated in this study are available from the first or corresponding author upon reasonable request. The raw genome data were deposited in the China National GeneBank Data Base (CNGBdb) under Bioproject number CNP0001121. The RNA sequencing data were available in the NCBI SRA database (https://www.ncbi.nlm.nih.gov/sra) (Accession: PRJNA1144186).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003einterest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\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"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVu LD, Gevaert K, De Smet I. Protein language: post\u0026ndash;translational modifications talking to each other. Trends Plant Sci. 2018;23(12):1068\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCube\u0026ntilde;as\u0026ndash;Potts C, Matunis MJ. 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New Phytol. 2016;211(1):172\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTomanov K, Zeschmann A, Hermkes R, Eifler K, Ziba I, Grieco M, Novatchkova M, Hofmann K, Hesse H, Bachmair A. \u003cem\u003eArabidopsis\u003c/em\u003e PIAL1 and 2 promote SUMO chain formation as E4\u0026ndash;type SUMO ligases and are involved in stress responses and sulfur metabolism. Plant Cell. 2014;26(11):4547\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQu GP, Li H, Lin XL, Kong X, Hu ZL, Jin YH, Liu Y, Song HL, Kim DH, Lin R, Li J, Jin JB. Reversible SUMOylation of FHY1 regulates phytochrome a signaling in \u003cem\u003eArabidopsis\u003c/em\u003e. Mol Plant. 2020;13(6):879\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNovatchkova M, Tomanov K, Hofmann K, Stuible HP, Bachmair A. Update on sumoylation: defining core components of the plant SUMO conjugation system by phylogenetic comparison. 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Mol Biol Evol. 2016;33(7):1870\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLescot M, D\u0026eacute;hais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, Rouz\u0026eacute; P, Rombauts S. PlantCARE, a database of plant cis\u0026ndash;acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002;30(1):325\u0026ndash;7.\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":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"SUMOylation, Caixin (Brassica rapa var. parachinensis), Soft rot, Biotic stress","lastPublishedDoi":"10.21203/rs.3.rs-4844966/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4844966/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe modification of protein substrates by small ubiquitin-related modifier (SUMO) plays a vital role in plants subjected to biotic and abiotic stresses. However, its role in the stress responses of Brassica plants remains poorly understood.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eA genome-wide analysis revealed the presence of 30 SUMOylation genes in the Caixin genome. These results demonstrated that the Caixin genome contains all the necessary components for SUMOylation. Analysis of the \u003cem\u003ecis\u003c/em\u003e-acting elements revealed that the promoters of SUMOylation genes presented diverse combinations of developmental and stress-related \u003cem\u003ecis\u003c/em\u003e-regulatory elements. The RNA-seq data indicated that 23 SUMOylation genes presented relatively high expression levels under normal conditions and exhibited a notable decrease in expression following \u003cem\u003ePectobacterium carotovorum\u003c/em\u003e subsp. \u003cem\u003ecarotovorum\u003c/em\u003e (\u003cem\u003ePcc\u003c/em\u003e) infection. Additionally, dynamic alterations in SUMO conjugates were observed in response to \u003cem\u003ePcc\u003c/em\u003e infection.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThe Caixin genome contains genes involved in SUMOylation. The majority of these genes presented multiple copies, and analyses of their transcription and protein profiles indicate that they may play a role in the response to \u003cem\u003ePcc\u003c/em\u003e infection.\u003c/p\u003e","manuscriptTitle":"Definition and regulatory analysis of the SUMOylation system in Caixin (Brassica rapa var. parachinensis) during Pectobacterium carotovorum infection","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-09-03 18:45:32","doi":"10.21203/rs.3.rs-4844966/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-29T08:22:27+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-10-19T14:53:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-19T15:52:15+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"71937911422309509671120006151613832907","date":"2024-09-19T09:29:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"205402086857632871303671017784036020250","date":"2024-08-10T12:15:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"114329875048799869141265550043140895189","date":"2024-08-09T10:15:38+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-08T11:05:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-08-07T07:10:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-08-07T07:08:49+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-08-07T07:08:43+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2024-08-02T01:43:16+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"399699c9-032b-4194-a213-65c15b0986b7","owner":[],"postedDate":"September 3rd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-23T16:06:22+00:00","versionOfRecord":{"articleIdentity":"rs-4844966","link":"https://doi.org/10.1186/s12870-024-05807-w","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2024-12-19 15:57:14","publishedOnDateReadable":"December 19th, 2024"},"versionCreatedAt":"2024-09-03 18:45:32","video":"","vorDoi":"10.1186/s12870-024-05807-w","vorDoiUrl":"https://doi.org/10.1186/s12870-024-05807-w","workflowStages":[]},"version":"v1","identity":"rs-4844966","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4844966","identity":"rs-4844966","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}