*3.4. 3D Structure Analysis of Water Lily SOD Proteins*

The examination of a protein's structure holds a great importance in understanding its function. We used SWISS MODEL and SOPMA online tools with the default search options, to predict the 3D structures of proteins. This study involved the prediction of threedimensional models for four water lily proteins. The generated models were downloaded for the purpose of visualizing the 3D structures. The helices are represented by yellow, while the sheets or strands are represented by green (Figure 4). Proteins that fall under specific groups exhibit related structural evenness. The *MnSD* and *FSD* subfamilies share a nearly identical structure, with an equal amount of helices and sheets. Similarly, proteins in the *CSD* subfamily also possess an analogous structure.

**Figure 4.** The 3D structures of four water lilies' (*Nymphaea colorata*, *Nymphaea thermarum*, *Nymphaea minuta*, and *Nymphaea mexicana*) *SOD* proteins categorized based on their sub families. (**a**) Represents the 3D structures of *FSD* subfamily; (**b**) Represents the 3D structures of *MnSD* subfamily; (**c**) Represents the 3D structures of *CSD* subfamily in all species. The final models are displayed, with diverse colors representing various sheets, domains, and helices. Note: In group (**a**) the *Nymphaea minuta* has no *FSD* subfamily.

#### *3.5. Analysis of Exon-Intron Structure of NcSOD*

The analysis of exon-intron structure of *NcSOD* genes was performed to elucidate the structural characteristics of species (Figure 5). *NcSOD* genes displayed varied exon-intron organizational patterns, with introns ranging from 5 (*NcFSD5*) to 9 (*NcFSD4*). The number of exons in *NcSOD* differs from 1 (*NcFSD1*) to 9 (*NcFSD2*). In one *NcFSD1* gene, introns are absent, and there is only one exon. The gene structure investigation revealed that the *SOD* gene family displayed a relatively conserved exon/intron organization.

**Figure 5.** Gene structure of *NcSOD* shows conserved exon/intron organization.

#### *3.6. Expression Examination of NcSOD Genes in Reproductive Organs*

The *SOD* gene family has a crucial role in plant growth, development, and response to stress. In order to investigate their specific biological functions in *N. colorata*, we observed the expression patterns of the 9 *NcSOD* genes in pollen and ovules using our own unpublished RNA-seq raw data. Under normal growth conditions, not all predicted genes in the *N. colorata SOD* family were expressed. 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; Table S5). *NcCSD1* and *NcCSD2* were moderately expressed in ovules and pollen throughout all days. *NcFSD2* and *NcFSD4* showed a moderate expression in ovule but exhibited no expression in pollen. Both *NcFSD1* and *NcCSD3* showed no expression levels in both ovules and pollen. Generally, results exhibited that genes from all three subfamilies, i.e., Fe, Mn and Cu, play essential roles in *N. colorata* reproduction, growth, and development.

**Figure 6.** Expression of the *NcSOD* genes was analyzed in pollen and ovule samples at four different time-points: 0 d, 1 d, 2 d, and 3 d. The expression bar from light blue to dark blue shows less or no expression of *NcSOD* genes. The light yellow to a dark red color exhibited less to high level of expression of these genes.

#### *3.7. Potential NcSOD Protein–Protein Interaction*

The potential *NcSOD* protein–protein interaction was analyzed via "STRING"11.0 (https://string-db.org/cgi/input.pl accessed on 25 April 2023). As shown in Figure 7, among nine *NcSOD* genes, seven *SOD* proteins participate in strong interaction networks. Interestingly, we observed that different proteins co-regulate each other to respond to stress conditions. For example, *NcCSD3, NcFSD1* and *NcFSD4* are upregulated after 2 h under cold stress (Figure 8c). In water lilies, they potentially exert a regulatory function by forming protein complexes to improve cold tolerance and cope with various stresses.

**Figure 7.** Protein interaction linkage among the seven *SOD* genes from *Nymphaea colorata*. Different colored lines show the interaction of the genes.

#### *3.8. Real-Time Quantitative PCR (RT-qPCR) Analysis of NcSOD Genes under Abiotic Stresses*

In order to know the function of *SODs*, we employed RT-qPCR to examine the expression patterns of the *SOD* gene under various stress conditions like salinity, heat, cold, and heavy metals (copper sulphate and cadmium chloride). Substantial variations were perceived in the expression levels of the *NcSOD* genes across various treatments, indicating a complex and dynamic nature of their expression patterns.

Salt treatment strongly induced the expression of all *NcCSDs*, peaking at 2 and 4 h. Our study found high expression of *NcCSDs* at 6 h, suggesting its involvement in salt response in *N. 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 (Figure 8a).

During the heat stress condition, the levels of expression of all *NcSOD* genes were upregulated at both 2 h and 4 h, with the exception of *NcFSD4*, which initially showed a decrease at 2 h and then an increase at 4 h. Following the 6 h treatment, the expression levels of the various genes showed variation (Figure 8b).

Under the 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 and at 4 h, while *NcCSD1* and *NcCSD2* expression was slightly low. On the other hand, the remaining members showed downregulated expression (Figure 8c).

*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. *NcCSD3* and *NcFSD1* displayed high expression at 2, 4 and 6 h, while other genes were low-expressed and different levels of expression were recorded. Furthermore, the highest expression levels for all genes were observed at the 6 h treatment (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 shown in the figure. At the 4 h and 6 h treatment, all genes experienced a gradual increase and demonstrated robust expression levels (Figure 8e). These findings can enhance our comprehension of *NcSOD* genes across various environmental conditions.

**Figure 8.** RT-qPCR analysis of the expression patterns of *NcSOD* genes in the leaves under various abiotic stresses: (**a**) salt; (**b**) heat; (**c**) cold; (**d**) CuSO4; and (**e**) CdCl2 stress (0 (CK), 2, 4, and 6 h). Data presented as means, ± standard error, *n* = 3; statistically significant differences are exhibited by asterisks (*p* ≤ 0.05), according to the LSD test.
