**4. Discussion**

Water lilies, with their significant ornamental, economic, medicinal, and cultural value, face challenges stemming from various abiotic stressors. However, through a combination of scientific research, technological innovations, and sustainable practices, we can optimize the growth and production of water lilies while preserving their aesthetic and functional benefits [45]. *SODs* have been demonstrated in recent studies to secure plants against abiotic stress factors including cold, drought, heat, salinity, ethylene, and abscisic acid [20–25]. In last several years, various plant species have been found to contain *SOD* family genes. For example, the aquatic sea grass (*Zostera marina)* has five *SOD* genes [26], *Medicago truncatula* [17] and barley (*Hordeum vulgare*) contain seven genes [28], sorghum (*Sorghum bicolor*) has eight genes [9], tomato (*Solanum lycopersicum*) has nine genes, and grapevine (*Vitis vinifera*) has ten genes [46]. Thus, we explored this family in four representative water lily species and checked the expression analysis in *Nymphaea colorata*.

In the present study, 43 *SOD* genes were identified in four water lily species, including 19 *Fe-SODs*, 15 *Cu*/*Zn-SODs*, and 9 *Mn-SODs* in all species (Table 1). The genes were classified into three major groups according to their binding domain (Figure 1). The number of genes in various water lily species was similar to that in cucumbers (*Cucumis sativus*) (9) and grapes (*Vitis vinifera*) (10), but fewer genes than the polyploidy crops cotton (*Gossypium hirustum*) (18) and wheat (*Triticum aestivum*) (26). However, the number of genes that encode Fe, Cu/Zn, and *Mn-SOD* differ among various species. For instance, *N. colorata* has three *Cu*/*Zn-SODs*, five *Fe-SODs*, and one *Mn-SOD*. The variation in *SOD* family member number could be due to the changes in genome sizes among species.

Previous research has shown that *Cu*/*ZnSODs* are consistently acidic, while *Fe-MnSODs* can be acidic or basic [42]. Most of the species investigated in this study displayed acidic properties. The results of subcellular localization of *SOD* proteins revealed that *Cu*/*Zn-SODs* are likely to be expressed in the cytoplasm, but *Mn-SODs* and *Fe-SODs* are expressed in mitochondria and chloroplasts, respectively, consistent with previous studies on *SODs* [18]. These distinct cellular locations enable *Fe-SODs*, *Cu*/*Zn-SODs*, and *Mn-SODs* to collaborate with one another to maintain the balance of free radicals in cells by functioning in different cellular parts.

Previous studies have indicated that the majority of cytoplasmic and chloroplast *SODs* comprise seven introns [13]. However, in our study it was revealed that *NcSOD* had a variable amount of exons ranging from 1 to 9. Furthermore, the number of introns in *NcSOD* varied from 5 to 9 (Figure 5). Notably, Figure 5 indicates that *NcFSD1* comprises only one exon and lacks introns. The variability in the gene structure of *SODs* may arise from the mechanisms involving the insertion or deletion of exons and introns [47].

Various research studies have demonstrated that *SOD* genes from distinct species are divided into three subfamilies [12]. In our study, we examined the evolutionary connections of *SOD* proteins in *N. colorata*, *N. thermarum*, *N. minuta*, *N. mexicana*, and *A. trichopoda* which categorized within three subfamilies (Figure 2a): *Fe*, *Cu*/*Zn*, and *Mn-SOD*. Within the phylogenetic tree, the three subfamilies were classified into two distinct groups: *Cu*/*ZnSODs* and *Fe-MnSODs*. *FeSODs* and *MnSODs* were clustered together, and a high bootstrap value separated them. The water lily *SODs* exhibited a strong clustering relationship with closely related species, while showing less affinity with outgroup. This suggests that this gene family has undergone relatively conserved evolution. The presence of specific domains suggests a basis for classifying these genes and the possibility of shared ancestral genes. In cotton (*Gossypium hirustum)*, the *MSD* and *FSD* families were found to have originated from a common ancestor, while the *CSD* subfamily developed independently. As a result, the two major groups expanded separately, as reported by Wang [48].

The analysis of promoters unveiled the existence of three main kinds of cis-components related to light, abiotic stress, and hormones response. Additionally, there were cis-elements associated with tissue-specific expression and developmental processes. Significant quantities of light-responsive cis-components were identified within *SOD* gene promoters, indicating the potential involvement of *SODs* in the abiotic stress response. Numerous investigations indicated the participation of *SOD* genes in the abiotic stress response across diverse plant species, including maize (*Zea mays*), *Pennisetum glaucum*, *Dendrobium catenatum*, and *Arabidopsis* [36,49–51]. Moreover, *SOD* gene promoters were found to contain a range of cis-elements linked to abiotic stress responses, including ARE, ABRE, MBS, ERE, Box-4, and TC-rich repeats. These cis-elements potentially contribute to the regulation of gene expression under diverse stress conditions. Among plant species like *Arabidopsis*, banana (*Musa paradisiaca*), rice (*Oryza sativa*), tomato (*Solanum lycopersicum*), poplar (*Populus angusti-folia*), and cotton (*Gossypium herbaceum*), the majority of *SOD* genes exhibit inducibility in response to various abiotic stresses [4,36,52–55]. Under various abiotic and hormones stress situations the *SOD* gene family were recently identified by many researchers in various different types of plants like in *Brassica napus* [25], *Zostera marina* [26], *Salvia miltiorrhiza* [27], and *Hordeum vulgare* [28].

The 3D structures of water lily *SOD* proteins remain relatively conserved, similar to conserved domains, gene structure, and phylogeny. The findings indicate that water lily *SODs* genes potentially perform diverse functions across various tissues and genotypes. These results supported earlier anticipated three dimensional structures of *SODs* in *G. arboretum* [54], sorghum (*Sorghum bicolor*) [9], rice (*Oryza sative*) [52], and in *Gossypium raimondii*. The preceding investigation demonstrated that metal ion binding active sites and the formation of conserved disulfide bonds within individual subunits contribute to protein stability, specificity, and dimerization [56].

To determine the specific expression profiles of *NcSOD* genes during various stages of development, we utilized RNA-seq data from ovules and pollen at various developmental stages. By analyzing the RNA-sequencing data from *N. colorata*, and examined the expressions of the 9 *NcSOD* genes in pollen and ovules at different days post-anthesis. Our analysis revealed that *NcFSD3*, *NcFSD5*, and *NcMnSD1* were highly expressed in ovules at 0, 1, 2, and 3 days, while showing relatively lower expression in pollen on day 1 (Figure 6). While *NcFSD1* and *NcCSD3* showed no expression levels in both ovules and pollen that are in agreement with earlier findings [46].

The RT-qPCR analyses offer valuable insights into the potential role of *NcSODs* in reaction to diverse stresses. Our research revealed significant changes in the expression levels of nine *NcSODs* in varied stress environments, suggesting their crucial regulatory role in response to stress and possible functional interconnections. Overexpressing *Cu*/*ZnSODs* improved salinity stress resistance in *Triticum aestivum, Oryza sativa, Puccinellia tenuiflora*, and *Arabidopsis* [57,58]. Salt treatment strongly induced the expression of all *NcCSDs*, peaking at 2 h and 4 h. Our study found high expression of *NcCSD* at 6 h, suggesting its involvement in salt response in *Nymphaea colorata*. Additionally, *NcMnSD1*, *NcFSD1*, *NcFSD2*, and *NcFSD5* were strongly induced and highly expressed under salt stress, implying their potential participation in the salt stress response, similar to *NcCSDs*, in *N. colorata* (Figure 8a). Particularly, most *NcSOD* genes exhibited upregulation throughout heat treatment, with some displaying analogous expression patterns (Figure 8b). During cold treatment, distinct expression profiles were observed among all *NcSODs. NcCSD3, NcFSD1*, and *NcFSD4* exhibited upregulated expression at almost all time points, and reached their maximum expression at 2 h and at 4 h, while *NcCSD1* and *NcCSD2* were slightly expressed. On the other hand, the remaining members showed down-regulated expression (Figure 8c). These conclusions are consistent with previous findings, which reported a notable increase in *SOD* activity in rapeseed (*Brassica napus*) under cold stress conditions [59]. *NcSOD* genes showed a positive response against heavy metals. In response to the copper sulphate treatment, the expressions of *NcSOD* genes exhibited variations at different time points (Figure 8d). During the cadmium chloride treatment, all *NcSOD* genes were upregulated at 2 h. Notably, *NcCSD3* exhibited consistently high expression levels across all time points, as showed in the (Figure 8e). Nevertheless, certain genes within the nine *NcSODs* exhibited a pattern of initially increasing and subsequently decreasing expressions in response to both heavy metal treatments. Similar results were also reported in reaction to heavy metals treatment in several plants [27]. However, experimental verification is still needed to fully elucidate the roles of *NcSODs* regulatory networks, and their interaction mechanism, under different abiotic stresses.

## **5. Conclusions**

In conclusion, this study conducted a comprehensive genome-wide analysis of the *SOD* gene family in four representative water lily species, resulting in the identification of 43 water lily *SODs*. The gathered information, encompassing exon-intron structure, cis-components, protein features, phylogenetic relations, and expression profiles of *N. colorata*, has shed light on the significant roles played by *NcSOD* genes in responding to salt, heat, cold, and heavy metal stresses. Findings of this systematic investigation provide a valuable resource for future functional research on *NcSOD* proteins in biological processes and lay a solid foundation for stress-resistant breeding of *N. colorata*. To further deepen our understanding of *NcSODs*' functions, our future studies will focus on gene engineering and comprehensive analysis, integrating genomics, transcriptomics, proteomics, and metabolomics.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/horticulturae9070781/s1, Table S1: the list of primer was used for gene expression analysis by RT-qPCR, Table S2: the protein sequences of SOD family genes in *Arabidopsis thaliana*, Table S3: the protein sequences of SOD family genes in four representative water lilies, Table S4: the information of identified 10 motifs in water lily SOD proteins, Table S5: The transcriptome data of *Nymphaea colorata* from ovules and pollen at various developmental stages.

**Author Contributions:** F.C. and W.U.K. conceived and designed this project. W.U.K. performed the analyses. L.U.K. and D.C. participated in the data analyses. W.U.K. carried out the experimental study and wrote the draft manuscript. F.C. and W.U.K. checked and revised the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work is supported by a grant from National Natural Science Foundation of China (32172614), a grant from Hainan Province Science and Technology Special Fund (ZDYF2023XDNY050), a fund from collaborative Innovation Center of Nanfan and Tropical High Efficient Agriculture, XTCX2022NYB04, and a start-up fund from Hainan Institute of Zhejiang University.

**Data Availability Statement:** The original contributions presented in the study are included in the article/Supplementary Materials, further inquiries can be directed to the corresponding author.

**Acknowledgments:** The authors thank the editor and anonymous reviewers for their valuable and insightful comments on this manuscript.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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