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

doi:10.21203/rs.3.rs-4844966/v1

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Research Article

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

https://doi.org/10.21203/rs.3.rs-4844966/v1

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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

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.

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. 1A). 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. 1B).

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. S1A, 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. S2A, 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. S2C).

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. 2A, 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. 2B), 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. 3A). 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. 3B, 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. 4A). 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. 4B). 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 (O₂-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. 4B). 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. 4B). 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. 5A). 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. 5B, 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. 6A, 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. 6B). These results suggest that SUMOylation is involved in the response of Caixin to soft rot stress.

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. 1A). 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. 1A). 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. 1B). 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. 1B), 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. S1B). 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. S1B). 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. 2C), 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. 5B). This suggests the possibility of functional differentiation of BrSCE1s in response to Pcc 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. 3B), 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. 5B), 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. 4B, 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. 5B), 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.

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.

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-AtSUMO1 antibody (Abcam, ab5316, China). The blot was developed via an enhanced chemiluminescent kit (Biosharp, Beijing, China) and detected by a chemiluminescence imaging system.

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.

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.

Consent for publication

Not applicable.

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No competing interests reported.

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

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

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Abstract

Background

Results

Conclusions

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Background

Results

Identification and characterization of SUMOylation genes in Caixin

Transcriptional profiles of SUMOylation genes under soft rot stress

Discussion

Conclusions

Methods

Identification of SUMOylation components in Caixin

Plant materials and bacterial incubation

RNA sequencing and data analysis

Protein extraction and immunoblotting

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

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Version 1