**1. Introduction**

Plants residing in their native environments often encounter a multitude of stress factors, including elevated salinity levels, prolonged drought periods, high temperatures, and the presence of heavy metals. These stressors exert notable influences on the plants' overall growth, development, and productivity [1,2]. Under stress, plants adapt their homeostatic mechanisms by generating an excess of reactive oxygen species (ROS) within their cells. ROS are primarily generated in various parts of the plant cell, including the plasma membrane, peroxisomes, apoplast, cell walls, endoplasmic reticulum, mitochondria, and chloroplasts [3]. These ROS are toxic free radicals that can oxidize proteins, damage cell membranes, and cause harm to DNA when formed in excessive amounts [4,5]. The occurrence of stresses in plants inevitably leads to the production of ROS, such as peroxide

**Citation:** Khan, W.U.; Khan, L.U.; Chen, D.; Chen, F. Comparative Analyses of Superoxide Dismutase (SOD) Gene Family and Expression Profiling under Multiple Abiotic Stresses in Water Lilies. *Horticulturae* **2023**, *9*, 781. https://doi.org/ 10.3390/horticulturae9070781

Academic Editor: Hongmei Du

Received: 24 May 2023 Revised: 11 June 2023 Accepted: 14 June 2023 Published: 8 July 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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radicals (HOO−), hydrogen peroxide (H2O2), and singlet oxygen (1O2). For instance, several potent scavengers of active oxygen have the ability to mitigate environmental stresses by regulating the expression of genes belonging to enzyme reaction families, such as superoxide dismutase (*SOD*), catalase (*CAT*), peroxidase (*POD*), glutathione peroxidase (*GPX*), and peroxidase (*PrxR*) [6–9]. Plants have developed effective and intricate antioxidant defense mechanisms comprising a variety of enzymatic and non-enzymatic antioxidants to manage the harmful effects of ROS. Among various antioxidant enzymes, *SOD*, a group of metalloenzymes, is predominantly present in living creatures. In managing environmental cues, *SOD*s show a vital part in the physio-biochemical processes of plants via acting as the primary defense against ROS [3]. In plants, *SOD* enzymes are encoded by a family of genes that are classified based on their metal cofactors: (*FeSOD*), (*Cu*/*ZnSOD*), (*MnSOD*), and (*NiSOD*) [10–12]. Among them *NiSOD* is predominantly found in cyanobacteria, streptomyces, and marine organisms, but has not yet been documented in plants [13,14]. Iron and manganese superoxide dismutase are mainly present in lower plants, whereas copper and zinc are found in higher plants [15]. Such *SODs* are usually dispersed in different cell parts [16]. In the main, *Cu*/*ZnSODs* are localized in the cytosol, peroxisomes, and chloroplasts. *MnSODs* are found inside mitochondria and *FeSODs* are usually found in the peroxisomes and chloroplasts [17,18].

*SODs* have been demonstrated in recent studies to secure plants against abiotic stress factors including cold, drought, heat, salinity, ethylene and abscisic acid [19–22]. Various findings have demonstrated that *SOD* genes might be transcribed and induced in many plants in different stress circumstances [23,24]. In recently published articles, the *SOD* gene family under various abiotic and hormones stress situations in *Brassica napus* [25], *Zostera marina* [26], *Salvia miltiorrhiza* [27], and *Hordeum vulgare* [28] were reported. Furthermore, different stress conditions can result in varied expression patterns of diverse forms of *SOD* genes. For example, tomatoes (*Solanum lycopersicum*) exhibit specific patterns of regulation in their *SOD* genes; for instance, under salt stress, *SlSOD1* is a single gene among the nine *SlSOD* genes that shows significant upregulation, while *SlSOD2, SlSOD5, SlSOD6*, and *SlSOD8* are also regulated. However, in drought conditions, the expression levels of four genes among the nine, namely "*SlSOD2, SlSOD5, SlSOD6*, and *SlSOD8*," are observed to be high [23]. Moreover, the expression profiles of the identical *SOD* gene type varied in the presence of stress. For instance, the studies revealed that the expression of *MnSODs* in *Arabidopsis* remained unchanged during oxidative stress, while scientists observed a considerable alteration in the expression of *MnSODs* in *Zostera marina*, peas (*Pisum sativum*), and wheat (*Triticum aestivum*) during salinity stress [26,29–31]. The findings imply that diverse *SOD* genes unveil distinct expression patterns in reaction to varying environmental stresses. In addition, scientists have revealed that the regulation of *SOD* expression may involve various miRNAs and alternative splicing mechanisms [32,33].

Water lilies are the most significant ornamental waterscape plants in the world. It is a perennial aquatic plant of the order Nymphaeales, genus *Nymphaea* in the family *Nymphaeaceae*. *Nymphaea* (*Nymphaeaceae*), also called flowering plants, are angiosperms with large and showy flowers. There are more than 60 species in the world, mostly distributed in tropical, subtropical, and temperate regions. They have curved or rounded and variously notched waxy-coated leaves on long stalks, usually grow on the water, and surround flowers. Each plant can grow approximately 70 to 80 flowers. The aquaculture of water lily, flowers can also be used as fresh cut flowers, in tea, in dried flower crafts, and in textile production. Water lilies have garnered significant attention from scientists, researchers, and entrepreneurs worldwide due to their immense economic, medicinal, and cultural value. While these plants hold great importance in phylogenetic research, the accessibility to comprehensive genetic and genomic information remains somewhat limited [34]. Since we released the first water lily (*Nymphaea colorata*) genome sequence in 2020 [35], the *SOD* gene family has not yet been discovered in any species of water lily.

To fill in this gap, the present study aims to conduct a comparative genome-wide study to discover *SOD* genes in representative water lily species genomes. Our analysis included the characterization of their phylogenetic connections, conserved motifs, cis-elements, gene structure, expression analysis, protein-protein interaction and 3D structures, in order to decode its structural characteristics and functions under stresses.
