2023 年 98 巻 1 号 p. 1-7
Posttranslational modifications (PTMs) to proteins are regulatory mechanisms that play a critical role in regulating growth and development. The SUMO system is a rapid and dynamic PTM system employed by eukaryotic cells. Plant SUMOs are involved in many physiological processes, such as stress responses, regulation of flowering time and defense reactions to pathogen attack. In Arabidopsis thaliana and rice (Oryza sativa), eight and seven SUMO genes, respectively, were predicted by sequence analysis. Phylogenetic tree analysis of these SUMOs shows that they are divided into two groups. One consists of SUMOs that contain no SUMO acceptor site and are involved in monoSUMOylation of their target proteins. Rice OsSUMO1 and OsSUMO2 are in this group, and are structurally similar to each other and to Arabidopsis AtSUMO1. The other group is composed of SUMOs in which an acceptor site (ΨKXE/D) occurs inside the SUMO molecule, suggesting their involvement in polySUMOylation. Several studies on the rice SUMOs have been performed independently and reported. Individual names of rice SUMOs are confusing, because a unified nomenclature has not been proposed. This review clarifies the attribution of seven rice SUMOs and unifies the individual SUMO names.
Posttranslational modifications (PTMs) to proteins are a regulatory mechanism that can actively alter protein function or introduce additional levels of functional complexity by altering cellular and subcellular locations, protein interactions and biochemical reaction pathways. PTMs are fundamental alterations that are involved in most cellular signaling events and play a critical role in regulating growth and development through the modulation of a protein’s functionality and its interaction with its partners (Chen and Kashina, 2021).
Ubiquitin and ubiquitin-like modifier systems are conserved in eukaryotic organisms. Ubiquitin-like proteins function as critical regulators of many cellular processes, including transcription, DNA repair, signal transduction, autophagy and cell cycle control (Kerscher et al., 2006). Small ubiquitin-related modifiers (SUMOs) are low-molecular-weight proteins consisting of approximately 100 amino acid residues that show structural similarity to ubiquitin, although the functions of ubiquitin and SUMOs are largely different (Kim et al., 2002). SUMOylation is proposed to suppress the degradation and/or to change the intracellular localization of the target proteins (Mahajan et al., 1998; Gill, 2003).
The SUMO system is a rapid, dynamic posttranslational mechanism employed by eukaryotic cells to respond to stress. Budding yeast, Saccharomyces cerevisiae, and fission yeast, Schizosaccharomyces pombe, have only one form of SUMOs (encoded by SMT3 and pmt3, respectively) (Tanaka et al., 1999). Likewise, Drosophila melanogaster and Caenorhabditis elegans have only one form of SUMOs (Lehembre et al., 2000; Jones et al., 2002). In contrast, plants and vertebrates express many SUMO isoforms (eight SUMOs in Arabidopsis thaliana and four SUMOs in human), which are encoded by distinct genes (Kurepa et al., 2003; Hickey et al., 2012).
The process of SUMOylation typically involves an enzymatic cascade containing the activation (E1), conjugation (E2) and ligation (E3) of a SUMO to a target protein (Johnson, 2004). The SUMO precursor is initially cleaved in the C-terminal region by a SUMO-specific protease to expose its di-glycine motif (Nayak and Müller, 2014). Additionally, SUMOylation also requires a class of SUMO proteases that generate mature SUMOs from their precursors and cleave them from the target protein, a process termed deSUMOylation (Hickey et al., 2012) (Fig. 1). SUMO proteases display two key functions in the SUMO cycle: the generation of free mature SUMOs, which has an important role in regulating the SUMO cycle, and the removal of SUMOs from SUMOylated proteins (Morrell and Sadanandom, 2019).
The SUMOylation cycle. The cycle starts when SUMO peptidase cleaves the C-terminus from the SUMO precursor to generate the mature SUMO (SUMO-GG). The mature SUMO is activated by the heterodimeric E1 activating enzyme, and then conjugated to the target using an E2 conjugating enzyme and an E3 ligating enzyme. Target proteins are SUMOylated at the K residue in the target site. Some target proteins are polySUMOylated. In the deSUMOylation step, SUMO is removed from the target protein to generate free SUMO, which is then available for another SUMOylation cycle.
Many SUMOylated proteins are located in the nucleus, and SUMOylation alters many nuclear processes. SUMOs function in constitutive transcription and during the activation of inducible genes in yeast. Some promoter-bound factors are SUMOylated and involved in promoter activation (Rosonina et al., 2010). In fission yeast, it has been reported that SUMOylation is involved in the negative regulation of telomere extension by telomerase. Telomeres protect DNA ends of linear eukaryotic chromosomes from degradation and fusion and ensure complete replication of the terminal DNA through recruitment of telomerase. The regulation of telomerase includes cis regulation by the shelterin complex in mammals and fission yeast. The SUMOylation pathway cooperates with shelterin and Stn1–Ten1 complexes to regulate telomere length (Tanaka et al., 1999; Miyagawa et al., 2014).
Recent studies in yeast and animals have revealed various functions for SUMOs in diverse biological phenomena, such as the regulation of cell division, DNA repair and transcription. In contrast, only a limited number of SUMO proteins have been characterized in plants, although plant SUMOs are involved in many physiological processes.
SUMOylation may affect the crucial processes of plant growth and development. In plants, the SUMOylation system functions as a regulatory mechanism involved in cell expansion and division, flowering, hormonal signaling, and responses to abiotic and environmental stresses (Elrouby et al., 2013; Ghimire et al., 2020; Srivastava et al., 2021). In maize, the SUMO system is highly activated in developing seeds, and is thought to be important during plant reproduction (Augustine et al., 2016). Regulation of flowering time, cell growth and development, and nitrogen assimilation have recently been added to the list of SUMO functions.
Due to their sessile nature, plants are constantly subjected to various environmental stresses, such as drought, salinity and pathogen infections. Studies mostly with Arabidopsis have connected the plant SUMO system to nutrient acquisition and defense against both biotic and abiotic challenges (Castro et al., 2012). One of the remarkable roles of SUMO is as a rapid defense against environmental challenges, such as the stresses of drought, cold, heat, nutrient deficiency, phosphate deficiency, heavy metals and salt (Lois et al., 2003; Yoo et al., 2006; Wang et al., 2015; Srivastava et al., 2016; Morrell and Sadananadom, 2019; Ghimire et al., 2020).
Identification of SUMO targets is important to characterize the function of SUMOylation and deSUMOylation. Mining novel targets of SUMO proteins expands the field of SUMOylation/deSUMOylation, revealing its unexpected complexity. SUMO binding proteins identify regulators and/or effectors that are involved in a large number of diverse downstream pathways (Park et al., 2011).
In Arabidopsis, there are eight SUMO genes (AtSUMO1–8) (Kurepa et al., 2003). AtSUMO1/2 isoforms are indispensable in Arabidopsis and are involved in multiple molecular pathways that ensure plant phenotypic plasticity and viability (Saracco et al., 2007). AtSUMO3 is considered to be involved in plant immune responses because it interacts with and modifies the protein NPR1, a master regulator of basal and systemic acquired resistance (Saleh et al., 2015). AtSUMO3 has also been shown to act downstream of salicylic acid (SA) accumulation, while AtSUMO1 and AtSUMO2 are involved in suppressing SA accumulation (van den Burg et al., 2010). Although the biological function of AtSUMO5 is yet to be elucidated, it is evolutionarily conserved in plants, and its transcript has been detected (Hammoudi et al., 2016). For the other SUMOs, AtSUMO4, AtSUMO6, AtSUMO7 and AtSUMO8, their biological functions as well as their expression patterns have not been elucidated.
Arabidopsis SUMO paralogs form a regulatory network and differentially modulate target modifications in response to various environmental conditions. To date, a small number of SUMO target proteins, such as MYB30, GTE3/5, PHR1, ICE1, ABI5 and FLD, that harbor SUMOylation consensus motifs have been identified in Arabidopsis (Miura et al., 2005, 2007, 2009; Garcia-Dominguez et al., 2008; Jin et al., 2008; Okada et al., 2009). In addition, systematic approaches including yeast two-hybrid assays and affinity enrichment methods with mass spectrometry have been used for mapping SUMO target proteins (Park et al., 2011). The existence of multiple distinct isoforms of SUMO confers diversity on the process of SUMOylation of target proteins and contributes to the dynamics of the molecular consequences of SUMOylation. Modulation of the function of a target protein by a specific isoform of SUMO may act as a critical regulatory point in a molecular signaling pathway (Roy and Sadanandom, 2021).
Among the eight SUMO isoforms encoded by the Arabidopsis genome, AtSUMO1 and AtSUMO2 are closely related and share 83% amino acid sequence identity, while the others (AtSUMO3–8) show a lower degree of conservation (Castaño-Miquel et al., 2011). This diversification between the SUMO isoforms implies that SUMOylation in plants is functionally diverged to a higher extent than in vertebrates. This may relate to differences in residues that perform critical molecular functions involving interactions with the key enzymes of the SUMO conjugation system, E1 and E2, as well as with the SUMO-interacting motif (Roy and Sadanandom, 2021).
Although the SUMOylation process resembles ubiquitination, unlike ubiquitin ligation, target proteins typically include several SUMOylation sites consisting of a short consensus sequence, ΨKXE/D (Ψ, hydrophobic amino acid; K, the acceptor lysine; X, any amino acid; E/D, glutamate or aspartate) (Schmidt and Müller, 2003), which is predicted to be a SUMO acceptor site. Among the eight Arabidopsis SUMOs, AtSUMO1–3 are considered to be involved in monoSUMOylation because they have no ΨKXE/D sequence (Kurepa et al., 2003). In contrast, AtSUMO4 and AtSUMO6 have a VKME motif as the predicted SUMO acceptor site. In addition, the amino acid sequences of AtSUMO7 and AtSUMO8 contain an IKRD sequence that may act as their SUMO acceptor site. These SUMOs may be involved in polySUMOylation (Fig. 2). In AtSUMO4, AtSUMO6 and AtSUMO7, there is a single glycine in the C-terminal region instead of the typical di-glycine motif (Fig. 2).
Alignment of amino acid sequences of rice SUMOs (OsSUMO1–7), Arabidopsis SUMOs (AtSUMO1–8) and S. pombe SUMO (Pmt3). Amino acid residues identical to those of OsSUMO1 are indicated by blue letters. Di-glycine motifs at the end of the mature SUMO proteins are indicated by red letters. Potential SUMOylation sites (ΨKXE/D) are boxed. Names of SUMOs possessing one or more potential SUMOylation sites are also in red.
In rice (Oryza sativa L.), seven SUMOs (OsSUMO1–7) were predicted (Table 1). OsSUMO1, OsSUMO2 and OsSUMO3 were the first reported rice SUMOs. OsSUMO1 and OsSUMO2 have high structural similarity to each other, as do the Arabidopsis SUMOs AtSUMO1 and AtSUMO2 (Chaikam and Karlson, 2010; Rosa et al., 2018). They have been suggested to be involved in the response to heat stress by the modification of OsFKB20-1b, which is implicated in the regulation of pre-mRNA splicing under stress conditions (Park et al., 2021). These SUMOs have no SUMO acceptor site and are therefore considered to be involved in monoSUMOylation. OsSUMO3 shows low structural similarity to OsSUMO1 and OsSUMO2 and contains a SUMO acceptor site, which may be involved in polySUMOylation (Teramura et al., 2021).
| Name | RAP-DP (TAIR accession number) | LOC number | Chaikam and Karlson (2010) | Rosa et al. (2018) | Ikarashi et al. (2012) | Teramura et al. (2021) | Hammoudi et al. (2016) | Joo et al. (2020) | Ibrahim et al. (2022) |
|---|---|---|---|---|---|---|---|---|---|
| OsSUMO1 | Os01g0918300 | Loc_01g68950 | SUMO1 | OsSUMO1 | (OsSUMO1) | OsSUMO1 | – | OsSUMO1 (Loc_4324360) | OsSUMO1 |
| OsSUMO2 | Os01g0918200 | Loc_Os01g68940 | SUMO2 | OsSUMO2 | (OsSUMO2) | OsSUMO2 | rice SUMO | OsSUMO2 (Loc_4324359) | OsSUMO2 |
| OsSUMO3 | Os07g0574500 | Loc_Os07g38690 | SUMO3 | OsSUMO3 | (OsSUMO3) | OsSUMO3 | SUMO paralog | OsSUMO4 (Loc_107276773) | OsSUMO3 |
| OsSUMO4 | Os01g0852300 | no registration | – | – | (OsSUMO5)* | OsSUMO4 | – | – | OsSUMO4 |
| OsSUMO5 | Os07g0574200** | Loc_Os07g38650 | – | – | (OsSUMO4) | OsSUMO5 | SUMO paralog | OsSUMO3 (Loc_4343692) | OsSUMO5 |
| OsSUMO6 | Os07g0574200** | Loc_Os07g38660 | – | – | (OsSUMO5)* | OsSUMO6 | SUMO paralog | – | OsSUMO6 |
| OsSUMO7 | Os07g0466300*** | Loc_Os07g28280 | – | – | – | – | – | – | OsSUMO7 |
Annotation of the genes was performed using the phylogenetic tree data in this paper.
We have reported three additional rice SUMO genes, OsSUMO4, OsSUMO5 and OsSUMO6. All six of these SUMO genes can complement the phenotype of the SUMO-deficient pmt3Δ mutant of fission yeast (Teramura et al., 2021). In vivo SUMOylation using a bacterial expression system revealed that these six rice SUMOs can modify the heat shock protein HSF7, which is SUMOylated in Arabidopsis. Among the amino acid sequences of rice SUMO proteins, consensus motifs (ΨKXE/D) of the SUMO acceptor site were found in OsSUMO4, OsSUMO5 and OsSUMO6 as well as OsSUMO3. SUMOylation reactions using these SUMOs have indicated the generation of larger proteins that are attached to multiple SUMO molecules. These results suggest that OsSUMO3, OsSUMO4, OsSUMO5 and OsSUMO6 are involved in the polySUMOylation of such large proteins (Teramura et al., 2021). Recently, the existence of OsSUMO7 was reported (Ibrahim et al., 2022). This protein has ΨKXE/D potential SUMO acceptor sites.
Phylogenetic tree analysis of rice and Arabidopsis SUMOs shows that SUMOs are divided into two groups. Group 1 consists of SUMOs that have no SUMO acceptor site and are involved in monoSUMOylation of the target protein. Group 2 is composed of SUMOs that possess an acceptor site, suggesting their involvement in polySUMOylation (Fig. 3). Members of Group 2 show low sequence similarity to those in Group 1, and the two groups are therefore evolutionarily diverged. It is inferred that these groups of SUMOs diverged before the generation of dicotyledonous plants and monocotyledonous plants. In the Group 1 SUMOs, OsSUMO1 shows high similarity to OsSUMO2. Similarly, the sequences of AtSUMO1 and AtSUMO2 highly resemble each other. OsSUMO1/2 and AtSUMO1/2 may have been generated by evolutionary duplication of the ancestral SUMOs. On the other hand, members of the Group 2 SUMOs show more extensive sequence differences, implying that each has evolved separately.
Phylogenetic tree analysis of SUMO proteins of rice and Arabidopsis. Amino acid sequences of the unprocessed forms of SUMO proteins were used for this analysis. The S. pombe Pmt3 (Pmt3) sequence is included as an outgroup. Alignment of protein sequences was performed using Clustal X software (http://clustal.org/clustal2/). The phylogenetic tree was constructed using the PHYLIP version 3.69 neighbor-joining method (https://evolution.genetics.washington.edu/phylip.html) with bootstrap values from 1,000 neighbor-joining bootstrap replicates. The tree was visualized using the TreeView program (Page, 1996). Red asterisks indicate SUMOs possessing potential SUMOylation sites. The scale bar represents the number of substitutions per site. “Group 1” shows SUMOs that have no predicted SUMO acceptor site, and “Group 2” shows SUMOs containing one or more short consensus sequences of the internal SUMO acceptor site.
There have been several reports on rice SUMOs, the individual names of which are confusing because of the lack of a unified nomenclature (Table 1). Here, we propose to unify the nomenclature of rice SUMO genes according to Teramura et al. (2021). It is expected that adopting the unified SUMO names will settle the confusion on the rice SUMO names.
The first report on OsSUMO1–3 was by Chaikam and Karlson (2010). They showed the amino acid sequences of three rice SUMOs in their report, which were derived from the genes Os01g0918300, Os01g0918200 and Os07g0574500, encoding OsSUMO1, 2 and 3, respectively. OsSUMO1–3 have been reviewed in detail by Rosa et al. (2018).
Ikarashi et al. (2012) reported that there are five SUMOs in the rice genome. This seems to be the first report on rice SUMOs other than OsSUMO1–3. However, because no information on the nucleotide sequence or the accession number of any of these genes was included in the paper, it is difficult to recognize which of our OsSUMO1–7 (Table 1) these five SUMO genes correspond to. Inferring from the phylogenetic tree shown in that paper, it is presumed that their SUMO3, SUMO4 and SUMO5 are our OsSUMO3, OsSUMO5 and either OsSUMO4 or OsSUMO6, respectively (Table 1).
Hammoudi et al. (2016) predicted the existence of many SUMO genes in the rice and Arabidopsis genomes by in silico analysis. They showed ten rice SUMOs in their paper, two of which were discussed, and described the genes Loc_07g38650 and Loc_07g38660 as SUMO-like sequences. From their genetic information, we determined that these genes correspond to OsSUMO5 and OsSUMO6 (Table 1). However, in their paper, no genetic information on the other eight SUMOs was presented.
Joo et al. (2020) described the response to drought stress of four SUMOs, OsSUMO1–4. This paper showed that OsSUMO1 is involved in SUMOylation of OsRanGAP1 and determined the subcellular localization of the SUMOs using GFP-fusion proteins. However, the accession numbers of the individual SUMO genes described in that paper appear to be mistyped, because they do not correspond precisely to any SUMOs. Based on the amino acid sequences described in a figure, we consider that their OsSUMO1, OsSUMO2, OsSUMO3 and OsSUMO4 correspond to our OsSUMO1, OsSUMO2, OsSUMO5 and OsSUMO3, respectively. For the above-mentioned reason, OsSUMO3 and OsSUMO4 used in that paper should be renamed OsSUMO5 and OsSUMO3, respectively (Table 1).
Regarding the genes in the rice genome, different gene IDs for the individual genes are assigned based on the different organization structures in the genome project. Therefore, a single gene may have two different gene names. This ambiguity may lead to confusion in understanding a gene’s function. This review clarifies the attribution of seven rice SUMOs to unify the names of individual SUMOs.
