Identification and characterization of novel SUMO genes in bread wheat

Identification of SUMO genes in wheat

Three wheat SUMO-genes have been reported (Wang et al., 2018) specifically TaSUMO1 (TraesCS3A02G424000.1, TraesCS3B02G460000.1, TraesCS3D02G419300.1), TaSUMO2 (TraesCS3B02G459900.1, TraesCS3D02G419200.1), and TaSUMO3 (TraesCS3A02G366700.1, TraesCS3D02G359600.1). We obtained their sequences from the Ensembl Plants (http://plants.ensembl.org/). To identify new SUMO genes in Triticum aestivum L. (TaSUMOs), SUMO sequences in Arabidopsis thaliana (AtSUMOs) were obtained from TAIR (http://www.arabidopsis.org, accessed on 16 January 2022) and rice SUMO sequences (OsSUMOs) derived from the Rice Annotation Project (https://rice.uga.edu/, accessed on 16 January 2022) were used. These sequences served as queries for exploring the wheat database within the Ensembl Plants datasets (http://plants.ensembl.org/Triticum_aestivum/Info/Index, accessed on 17 January 2022). To ensure the accuracy of subsequent analyses, stringent filtering criteria were applied (E-val < 1.0^e−5, %ID ≥ 50). This involved removing redundant genes and selecting the longest transcript representative of each gene family member. Further validation of the identified TaSUMO family members was conducted using the InterPro website (https://www.ebi.ac.uk/interpro/, accessed on 19 January 2022) by submitting the predicted protein sequences.

Plant material growth and DNA extraction

Seeds of Triticum aestivum L. cv. VEERY (Chilean variety) were germinated in a controlled environment chamber under the following conditions: 23 °C ± 2 °C throughout the day, 16 °C ± 2 °C at night, 70% relative humidity, 16 h of light and 8 h of darkness (long-day conditions), and a light intensity of approximately 60 µmol m⁻² s⁻¹ at the Plant Production Department, College of Food and Agriculture Sciences, King Saud University, Riyadh, Saudi Arabia. The cultivar grains were obtained from the International Maize and Wheat Improvement Center (CIMMYT). The molecular experiments were conducted at the Biotechnology Laboratory within the Plant Production Department, KSU. Total DNA was extracted from young wheat leaves via the Wizard^® Genomic DNA Purification Kit manufactured by Promega (Madison, WI, USA).

Candidate gene amplification and sequencing of putative TaSUMOs

The putative TaSUMO4-7 sequences were amplified by the definite primers presented in Table S1. These primers were designed based on the genomic data of the SUMO-region in the Triticum aestivum genome database. The PCR amplification involved the addition of 10 µL of GoTaq^® Green Master Mix (1 ×) (Promega Corporation, Madison, WI, USA), two µL of mixed primers, six µL of water, and two µL of DNA. The cycling procedure was 95 °C for 5 min (initial denaturation), followed by 35 cycles of 95 °C for 30 s (denaturation), 52−64 °C for 30 s (annealing), 72 °C for 1 min (extension), and a final extension at 72 °C for 7 min. Amplified DNA (10 µL) was visualized on a 3% agarose gel. The expected bands were purified with a QIAquick^® PCR Purification Kit and sequenced by Macrogen^® (Seoul, South Korea). The coding regions were analysed for SUMO family characteristics.

Bioinformatic characterization of TaSUMOs

Characterization of the TaSUMO gene family included features such as amino acid count, chromosomal location, gene ID, length of coding sequences (CDSs), and exon number extracted from the Ensembl Plants database. The biochemical characteristics of the SUMO proteins were analysed using the ExPASy ProtParam server tool (https://web.expasy.org/protparam/, accessed on 20 September 2025) (Gasteiger et al., 2003). This included calculating various parameters of the primary structure, like the amino acid composition, molecular weight (MW), and theoretical isoelectric point (T-pi). The grand average of hydropathicity (GRAVY) was calculated using the method described by Kyte & Doolittle (1982), while the instability index (Ii) was calculated using the method described by Guruprasad, Reddy & Pandit (1990). This latter analysis considers the frequency of specific dipeptides as an indicator of protein stability, with proteins having an Ii less than 40 considered stable and those above 40 considered unstable. Finally, the aliphatic index (Ai) was also calculated using the formula proposed by Ikai (1980).

The Protein Homology/analogY Recognition Engine V 2.0, Phyre2 web portal (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index/, accessed on 21 January 2022) (Kelley et al., 2015) was employed to computationally predict the secondary and three-dimensional (3D) structures of the proteins. Computational prediction of SUMO-binding sites within the TaSUMO genes was performed using the SUMOplot™ Analysis Program (https://www.abcepta.com/sumoplot/, accessed on 17 January 2022). Additionally, the pairwise sequence identity among the TaSUMO protein sequences was detected via Vector NTI^® Software (Invitrogen, Waltham, MA, USA).

TaSUMO sequence comparison

Phylogenetic analysis incorporated diverse data sets, including the three identified TaSUMO protein sequences from Wang et al. (2018), eight AtSUMO sequences reported by Kurepa et al. (2003), Novatchkova et al. (2004), and Hay (2005), and seven OsSUMO sequences described by Ikarashi et al. (2012), Rosa et al. (2018), Ibrahim et al. (2021), Teramura et al. (2021), and Ibrahim (2022). SUMO protein sequences from humans, yeast, and other species were got from the National Center for Biotechnology Information (NCBI) and the UniProt database. Multiple sequence alignments were performed through the UniProt database website (https://www.uniprot.org/align/, accessed on 17 January 2022) using the ClustalO function. The SUMO protein sequences were queried against the InterPro website (Finn et al., 2007; Larkin et al., 2007) to identify conserved domains characteristic of the SUMO family. A phylogenetic tree was constructed using the neighbor−joining method (Saitou & Nei, 1987) with the Poisson correction model and 1,000 bootstrap replicates (Felsenstein, 1985) implemented in MEGA X software (Kumar et al., 2018). This method was subsequently applied to generate a specific phylogenetic tree of TaSUMO protein sequences.

RNA extraction and RT–PCR

To determine the transcription levels of the TaSUMO4-7 genes, various parts of the plants were collected, frozen in liquid nitrogen, and ground. RNA was subsequently extracted with a commercial kit (RNeasy^® Plant Mini Kit, Qiagen, Hilden, Germany). The extracted RNA was used to create first-strand cDNA copies (SuperScript II, Invitrogen, Waltham, MA, USA). RT−PCR was subsequently performed by a specific enzyme (KOD Dash DNA polymerase, Toyobo, Osaka, Japan) and a thermal cycler (Veriti 96-well, Applied Biosystems, Waltham, MA, USA). Gene-specific primers for the putative TaSUMO genes (Table S1) were used to amplify their transcripts, and wheat actin served as an internal control for normalization.

Construction of expression vectors harboring TaSUMOs

We constructed expression vectors containing TaSUMO components using the pUC119 vector (Vieira & Messing, 1989) and protocols from Ikarashi et al. (2012). These vectors expressed fluorescent protein tags, either red (DsRFP) or green (GFP), under the regulation of the cauliflower mosaic virus 35S (CaMV35S) promoter (Fig. S1). All primers used are presented in Table S1. The new genes were introduced into pDsRFP via an In-Fusion HD Cloning Kit (Takara Bio) to create vectors named pDsRFP:TaSUMO4-7, which were used to study the localization and expression of the new TaSUMO proteins. Additionally, pDsRFP:TaSUMO4-7ΔGG vectors with deleted GG motifs were constructed to understand their role in protein localization. To study the coexpression and localization with TaSUMO4-7, the Arabidopsis thaliana SUMO-conjugation-enzyme (SCE1a) was inserted into the pGFP:SCE1a vector. Furthermore, cell organelle markers (peroxisome, mitochondria, plastid, cis-Golgi) were fused with GFP in vectors (pPTS2:GFP, pmt:GFP, pWxTP:GFP, pGFP:SYP31) and cotransfected with DsRFP:TaSUMO4-7 in onion cells to determine whether TaSUMO4-7 proteins localize to specific organelles (Fig. S1).

TaSUMOs, vectors transformation and protein expression visualization

The vectors containing the TaSUMO4-7 genes were introduced into onion epidermal cells with a particle bombardment technique (Biolistic^® PDS-1000/He, BioRad, Hercules, CA, USA). The bombarded cells were incubated in the dark for 24 h at room temperature. The expression of the TaSUMO4-7 proteins was then visualized by confocal laser scanning microscopy (FV300-BX61; Olympus, Tokyo, Japan) following protocols explored by Kitajima et al. (2009) and Ikarashi et al. (2012).

New TaSUMO genes molecular characterization

The sequences of the novel TaSUMO genes were amplified using PCR with specifically designed primers (presented in Table S1). These primers were created based on the DNA sequences of potential SUMO regions identified within the wheat genome database (Fig. 1A). Sequencing results revealed that the 318, 321, 354, and 372 bp fragments of the novel TaSUMO4-7 genes were located on the long arms of chromosomes 1A, 1B, 2A and 2D, respectively, encoding 105, 106, 117, and 123 amino acid SUMO proteins, respectively (Table 1, Fig. 1B).

RT-PCR was employed to assess the expression levels of the TaSUMO4-7 genes across various wheat tissues, encompassing young leaves, roots from young plants, mature leaves, flag leaves, spikelets, and seeds. The new TaSUMO-genes were found to be expressed in all of the wheat tested tissues (Fig. 1C), demonstrating that they are expressed in a constitutive manner.

Proteins with 105, 106, 117, and 123 amino acid residues were predicted for the unique TaSUMO4-7 genes according to a BLAST investigation performed built on protein sequences in the wheat genome database. All the identified proteins possessed a C terminal di.glycine (GG) motif and shared consensus motifs (ΨKXE/D), indicative of a high resemblance degree of sequence with other known TaSUMO members (Fig. 2A). These findings revealed that TaSUMO4-7 contains SUMO protein family features.

A phylogenetic tree was established utilizing SUMO protein sequences from diverse species, encompassing wheat (Triticum aestivum, Ta), Arabidopsis thaliana (At), rice (Oryza sativa, Os), Brachypodium distachyon (Bd), human (Homo sapiens, Hs), sorghum (Sorghum bicolor, Sb), foxtail millet (Setaria italica, Si), and baker’s yeast (Saccharomyces cerevisiae, Sc). The phylogenetic tree exposed a high similarity degree between the four novel TaSUMO genes since they were clustered in the same tree, and the degree of similarity was greater between each of the TaSUMO4 and TaSUMO5 proteins and between the TaSUMO6 and TaSUMO7 proteins because they were found in the same sub-tree (Figs. 2B, 3A).

These findings were supported by the results obtained from Vector NTI^® Software (Invitrogen, Waltham, MA, USA) for the percentage of amino acid identity between TaSUMO proteins; however, the percentages were 71% and 72% between TaSUMO4 and TaSUMO5 and between TaSUMO6 and TaSUMO7, respectively (Table 2). Monocotyledons including rice (Oryza sativa, Os) and sorghum (Sorghum bicolor, Sb) had similarity with TaSUMO4-7 proteins (Fig. 3A).

TaSUMOs and SUMOs from diverse species have similar protein sequences (Fig. 2A) and conserved domains (Fig. 3B), indicating that TaSUMO4-7 are evolutionarily conserved SUMO family members.

TaSUMO4-7 in silico characterization

TaSUMO4-7 proteins were characterized using bioinformatic analysis and compared with other TaSUMO, AtSUMO and OsSUMO proteins. The TaSUMO4 and TaSUMO5 genes were found on the long arm of chromosome one; the TaSUMO6 and TaSUMO7 genes on the long arm of chromosome two; and the TaSUMO1, TaSUMO2, and TaSUMO3 genes on chromosome three, according to the findings (Table 1). The results showed that the TaSUMO4-7 genes have one exon, similar to the TaSUMO3 gene, whereas the TaSUMO1 and TaSUMO2 genes have three exons (Table 1). Furthermore, the TaSUMO4 protein has two motifs for SUMOylation sites at the K72 and K50 positions, while TaSUMO5 has two motifs at K10, similar to TaSUMO1, 2 and K53 positions; however, TaSUMO6, 7 have only one motif at the K59 and K74 positions, respectively (Table 1).

Investigations of biochemical properties

Table 3 and Table S2 and Figs. 4 and 5 show the results of the biochemical features analysis of the SUMO proteins performed by ProtParam. The MWs of the TaSUMO4-7 proteins were determined to be 11.58, 11.84, 13.10, and 13.68 kDa. The MW of the TaSUMO6 protein was the same as the MW of the AtSUMO2 protein. The T-Pi values for the TaSUMO4-7 proteins were 5.85, 6.90, 4.91, and 6.98. The TaSUMO5 protein has a T-Pi value that is similar to that of TaSUMO7 and AtSUMO4.

Furthermore, the TaSUMO4-6 proteins had the same TNPCR (Arg + Lys) values (13) as OsSUMO1, OsSUMO2, AtSUMO1, and AtSUMO1. For TaSUMO5, seven proteins, TNNCR (Asp + Glu) and TNPCR (Arg + Lys), had equal values of 13 and 19, respectively. Moreover, the TaSUMO4 protein, like AtSUMO7, had a TNNCR (Asp + Glu) value (15), and the TaSUMO6 protein had a value (21) similar to that of TaSUMO3, OsSUMO3 and AtSUMO4 for the same parameter. Additionally, the TaSUMO7 protein had a value (19) identical to that of the OsSUMO6, AtSUMO2 and AtSUMO3 proteins (Table 3, Table S2).

The instability index (Ii) values for TaSUMO4 and five proteins were less than 40 (31.91, 27.71), suggesting that the proteins were stable; however, for TaSUMO6, seven proteins were more than 40 (50.80, 47.52), showing that the proteins were unstable, similar to the TaSUMO1-3 proteins. Moreover, the Ai values for the TaSUMO4-6 proteins were fairly comparable (74.19, 74.25, 73.25), whereas the TaSUMO7 protein was slightly different (81.54).

In terms of GRAVY, TaSUMO4 and TaSUMO5 proteins had equal values (−0.21), while TaSUMO6 and TaSUMO7 proteins had extremely close values (−0.40 and −0.49, respectively), as shown in Table 3 and Table S2. The amino acid composition analysis revealed that the TaSUMO5-7 proteins sequences had all 20 amino acids except cysteine (C), whereas the TaSUMO4 protein sequences contained all 20 amino acids except asparagin (N) (Fig. 4A, Table S3). Among the analysed SUMO proteins, TaSUMO4 had the highest percentage of the amino acid Pro (P, 6.7%), while the TaSUMO5 protein had the highest percentage of the amino acids Met (M, 9.4%) and Val (V, 13.2%). Additionally, TaSUMO6 had the largest amount of the amino acid Glu (E, 11.1%) and the lowest percentage of the amino acid Ile (I, 1.7%).

Additionally, the TaSUMO7 protein possessed high proportions of Arg (R, 10.6%) and Gly (G, 13%) amino acids and the lowest ratio of Pro (P, 1.6%) amino acid. Among the basic 20 amino acids, valine was the component with the largest percentage (10.2%, 13.2%, 12%) of the TaSUMO4-6 proteins, while it was a glycine (13%) in the TaSUMO7 protein (Fig. 4A, Table S4).

The amino acid’s atomic composition (AC) includes (carbon, C), (hydrogen, H), (nitrogen, N), (oxygen, O), and (sulfur, S). OsSUMO6 had the highest TNA value (2,034) among the analysed SUMO proteins, while TaSUMO3 had the highest TNA value (1,946) among the TaSUMO proteins, and TaSUMO7 had the highest TNA value among the four novel wheat SUMO proteins (Table S3). Analysis of the atomic composition using a radar graph (Fig. 4B) revealed a highly conserved elemental profile across all tested SUMO proteins from wheat, rice, and Arabidopsis, with only minor visual variations.

Secondary and tertiary structure prediction of TaSUMO4-7 proteins

The secondary structure was obtained using the PSIPRED method (Jones, 1999), which is based on sequence-based prediction. The predicted secondary structure of the protein contained three main states: α-helices, β-strands, and coils. These are visually represented by green helices, blue arrows, and faint lines, respectively. The confidence level in the prediction is also displayed, with red representing high confidence and blue demonstrating low confidence. The predicted secondary structure of all the novel TaSUMO proteins included six β-strands intervened by nine coils and three helixes, with the exception of TaSUMO7, which has four helixes (Fig. 6A). Using the Phyre2 engine, the three-dimensional model of TaSUMO5,6 were obtained based on the crystal structure of the human (H. sapiens) SUMO-1 domain (PDB code 1A5R) with a confidence of 99.9%, whereas the 3D structure of the TaSUMO4 protein was determined based on the crystal structure of the Trypanosoma brucei SUMO chain (PDB code 2K8H) with a confidence of 99.8%. However, for the TaSUOM7 protein, the 3D structural model was established by relying on the crystal structure of the Mus musculus SUMO chain (PDB code 3A4R) with a confidence of 99.7%. JSmol (JavaScript-Based Molecular Viewer From Jmol) (Hanson et al., 2013) was used to view 3D structural models of proteins (Fig. 6B).

TaSUMO4-7 proteins cellular localization and expression

DsRFP:TaSUMO4, DsRFP:TaSUMO5 and DsRFP:TaSUMO6 are found mainly in the cytoplasm and nucleus of onion cells, while DsRFP:TaSUMO7 is mostly localized in the nucleus (Fig. 7A). Additionally, the fusion proteins DsRFP:TaSUMO4ΔGG, DsRFP:TaSUMO5ΔGG, DsRFP:TaSUMO6ΔGG, and DsRFP:TaSUMO7ΔGG were observed in both the nucleus and cytoplasm, with significantly weaker signals in the nucleus (Fig. 7B). To further investigate the localization of these proteins, we used vectors expressing fluorescent markers (pPTS2:GFP, pmt:GFP, pWxTP:GFP, pGFP:SYP31) for different organelles (peroxisomes, mitochondria, plastids, and Golgi apparatuses). This allowed us to determine whether the SUMO proteins localize to any other cellular compartments. In onion epidermal cells, pDsRFP:TaSUMO4, pDsRFP:TaSUMO5, pDsRFP:TaSUMO6 and pDsRFPd:TaSUMO7 were transfected into cells. According to our findings, TaSUMO4-7 proteins were not found among any of the previously mentioned organelles under investigation (Fig. 8).

GFP:SCE1a and TaSUMO4-7 co localization and co expression

The GFP:SCE1a (SUMO-conjugation-enzyme) and DsRFP:TaSUMO4-7 fusion proteins colocalized and coexpressed in the nucleus, where they formed nuclear sub-domains. Unlike signals observed when expressed alone, signals were collected as dot-like structures inside the nucleus (Fig. 9A). In addition, high-magnification observation was performed to analyse the localization of the proteins in detail. High-magnification imaging (Fig. 9B) revealed colocalization of DsRFP-tagged TaSUMO4-7 proteins with the SUMO-conjugating enzyme GFP:SCE1a within the nucleus, suggesting that conjugation of SUMO proteins to their substrates likely occurs at these sites. However, it is important to note that the processing of the C-terminal di-glycine motif, essential for conjugation, may have already taken place in other cellular compartments prior to nuclear localization.