Genome-Wide Identification and Expression Analysis of Respiratory Burst Oxidase Homolog (RBOH) Gene Family in Eggplant (Solanum melongena L.) under Abiotic and Biotic Stress

Abstract

Respiratory burst oxidase homologs (RBOHs) are important proteins that catalyze the production of reactive oxygen species (ROS), which play important roles in growth and stress response. For a comprehensive analysis of SmRBOH genes, we conducted genome-wide identification of the SmRBOH gene family in eggplant (Solanum melongena L.) and analyzed the expression of SmRBOHs under abiotic (salt, high-temperature, and low-temperature) and biotic stress (Verticillium dahliae inoculation) by quantitative real-time PCR (qRT-PCR). The result showed that a total of eight SmRBOH members were identified from the genome database of eggplant, and they were relatively evenly distributed across seven chromosomes. The analysis of Motif and the conserved domain showed that SmRBOHs have high similarity in protein sequences and functions. Based on phylogenetics, SmRBOHs were classified into three distinct clades. Furthermore, the promoter regions of SmRBOHs were found to contain different cis-elements. Additionally, the results of the qRT-PCR demonstrated differential expression patterns of SmRBOHs in different tissues (the roots, stems, and leaves) and stress conditions. SmRBOHB, SmRBOHD, SmRBOHH1, and SmRBOHH2 showed significant upregulation (>20-fold) under at least one stress condition. Subcellular localization analysis of the above four members further confirmed that they localized on the plasma membrane. This study provides a theoretical foundation for understanding the functions of SmRBOHs in eggplant.

1. Introduction

Reactive oxygen species (ROS) are signaling molecules that include singlet oxygen (¹O₂), superoxide anion (O₂⁻), hydrogen peroxide (H₂O₂), and a hydroxyl radical (HO⁻). Plants produce low concentrations of ROS to maintain normal growth and metabolism under normal conditions. However, plants generate a large amount of ROS under stress conditions. On the one hand, the accumulation of ROS can cause oxidative damage to the plants themselves. On the other hand, under stress conditions, ROS, especially H₂O₂, act as signaling molecules and participate in the process of programmed cell death [1,2]. Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs), also known as respiratory burst oxidase homologs (RBOHs), are the key enzymes responsible for the generation of ROS [3]. RBOHs are predominantly localized to the plasma membrane and function as part of the plasma membrane redox system, transferring electrons from intracellular NADPH/NADH to oxygen, leading to the production of a large amount of O²⁻. Subsequently, the O²⁻ is disproportionated to produce H₂O₂ and other ROS [4]. In animals, NOXs consist of six subunits, with gp91^phox being the central functional subunit. Based on sequence alignment with gp91^phox, multiple RBOHs have been identified in plants [5]. The C-terminal region of RBOHs exhibits a high similarity to human gp91^phox, possessing conserved domains such as the FAD and NADPH binding domains. Unlike gp91^phox, the N-terminal of plant RBOHs typically contains two EF-hand Motifs associated with Ca²⁺ binding and phosphorylation sites, indicating the potential regulation of RBOH by Ca²⁺ binding and phosphorylation [6]. The first RBOH gene in plants was identified from rice (Oryza sativa L.), and subsequently, multiple RBOH genes have been cloned from other plants such as tomato (Solanum lycopersicum L.), potato (Solanum tuberosum L.), and tobacco (Nicotiana tabacum L.) [7,8,9]. The RBOH-dependent ROS burst can be effectively inhibited by the specific inhibitor of the animal NADPH oxidase, diphenyleneiodonium (DPI), suggesting functional similarities between plant RBOHs and animal gp91^phox [10]. In addition to higher plants, RBOH genes have also been identified in algae, and compared to higher plants, RBOHs in diatoms Thalassiosira pseudonana and the brown alga Phaeodactylum tricornutum are more closely related to animal gp91^phox [11,12], indicating that RBOHs are ancient genes that appeared in eukaryotic unicellular organisms as well.

The RBOH gene family has also been identified in several species. Members of this family have different physiological functions, which are important in stress response, growth and metabolism, and phytohormone regulation [13,14,15]. The ROS generated by RBOH proteins have profound effects on plant morphogenesis. For instance, AtRBOHC is involved in root hair growth in A. thaliana (Arabidopsis thaliana L.), while AtRBOHD and AtRBOHF lead to the local accumulation of superoxides in the roots of A. thaliana, thereby inhibiting lateral root development [16]. Moreover, OsRBOHA participates in the germination process of rice seeds and pollen, and its knockout results in reduced pollen viability and seed fertility [17].

In the face of abiotic stress, RBOH-dependent ROS are speculated to serve as signaling molecules, initiating cascade reactions that activate various defense mechanisms. Among the seven RBOH genes in citrus (Citrus reticulata Blanco), five CsRBOH genes have been found to respond to cold stress, and the knockout of CsRBOHD reduces plant cold tolerance [18]. Furthermore, studies have confirmed the involvement of RBOHs in the maintenance process of cold acclimation in cucumber (Cucumis sativus) [19]. H₂O₂ generated by AtRBOHD and AtRBOHF promotes the influx of Ca²⁺, a crucial step in signal transduction, which to some extent, maintains the Na⁺/K⁺ balance under salt stress. The double mutant of AtRBOHD and AtRBOHF shows significant inhibition of Ca²⁺ influx under salt stress, resulting in decreased salt tolerance in A. thaliana [20]. In rice, drought stress induces the activity of RBOH oxidases. Overexpression of OsRBOHA and OsRBOHB enhances plant drought tolerance. OsRBOHB is mainly involved in ROS production and abscisic acid (ABA) signal transduction. Mutants of OsRBOHB not only show reduced ROS production but also exhibit lower ABA content and larger stomatal apertures, leading to decreased drought resistance [21]. Under biotic stress conditions, RBOH-dependent ROS participate in plant immune responses, reinforcing cell walls, enhancing defense against pathogens, and playing a role in signal transduction, thereby increasing plant resistance to pathogens [22,23,24]. Verticillium wilt is a soil-borne disease primarily caused by V. dahliae (Verticillium dahlia). Virus-induced gene silencing (VIGS) and overexpression confirmed that GbRBOH5/18 was involved in the resistance of cotton to verticillium wilt [22].

Eggplant (S. melongena L.) is an important vegetable crop with rich nutrition and significant economic value. However, its growth cycle is lengthy, and its yield is often influenced by various adverse environmental factors [25]. Although numerous studies have demonstrated the regulatory roles of RBOHs in plant growth, development, and responses to stress [14,15,26], there have been no reports on SmRBOHs. In this study, we identified and conducted a bioinformatic analysis of the SmRBOH gene family in eggplant. We also analyzed the expression patterns of SmRBOHs in different tissues of eggplant and investigated their expression profiles under simulated salt, high-temperature and low-temperature stresses, and V. dahliae inoculation in eggplant seedlings. Additionally, we performed subcellular localization analysis for SmRBOH members that showed significant upregulation (>20-fold) under different stress treatments. This study aims to analyze the physicochemical properties, chromosome localization, Motif and conserved structure, phylogenetic evolutionary, cis-acting elements, the expression of SmRBOHs gene family, and identify SmRBOHs that play an important role under stress according to their expression under different stresses, providing a basis for subsequent research and utilization of SmRBOH genes.

2. Materials and Methods

2.1. Identification, Physicochemical Characterization, and Chromosomal Localization of SmRBOHs

In order to obtain the eggplant protein sequences, the genome data were downloaded from the eggplant Genome Database (http://eggplant-hq.cn/ accessed on 17 February 2023). TBtools v 1.120 [27] was utilized to extract the eggplant protein sequences. These sequences were then aligned with the already identified amino acid sequences of A. thaliana RBOHs through a double alignment [7]. To validate the alignments and identify conserved domains, further verification was conducted using the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/ accessed on 12 March 2023). Subsequently, the final eggplant RBOH gene family members were identified and named. The physicochemical properties of the SmRBOHs were analyzed on Expasy (https://web.expasy.org/protparam/ accessed on 12 March 2023), which provided information on the isoelectric point, instability index, hydrophilicity, and other relevant properties. Additionally, the subcellular localization of SmRBOH family members was predicted using WOLFSORT (https://wolfpsort.hgc.jp/ accessed on 12 March 2023). Chromosome localization analysis and visualization of the SmRBOHs were performed using the eggplant genome annotation files and the Tbtools [27].

2.2. Motif and Conserved Domain Analysis of the SmRBOHs Family

Using the amino acid sequences of SmRBOHs, we analyzed the Motifs of SmRBOHs in the MEME Suite v 5.5.2 (https://meme-suite.org/ accessed on 18 April 2023) website. We performed the analysis of conserved domains in the SmRBOH family using the CD (Conserved Domain) Search available on NCBI and visualized the results.

2.3. Phylogenetic Analysis of SmRBOHs

According to previous research, we obtained the protein sequences of SlRBOHs, AtRBOHs, and OsRBOHs in tomato, Arabidopsis, and rice [7,8]. Based on the SlRBOHs, AtRBOHs, and OsRBOHs protein sequences, combined with the identified SmRBOH proteins, a multiple sequence alignment was performed using the ClustalW method of MEGA 11 (detailed information in Tables S1 and S2), and we constructed a phylogenetic evolutionary tree using the Neighbor-Joining (NJ) method with the Bootstrap method set to 1000 replications. Additionally, we used Evolview (http://www.evolgenius.info/evolview/#/ accessed on 30 April 2023) to visualize the phylogenetic tree.

2.4. Analysis of SmRBOHs Cis-Acting Elements in Plants

To predict the potential cis-acting element, the genomic DNA sequences 2000 bp upstream of the initiation codons of SmRBOHs were extracted Using TBtools [27]. Then we used PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ accessed on 12 May 2023) to analyze the putative cis-regulatory elements of SmRBOHs and used TBtools to visualize the results.

2.5. Plant Materials and Treatments

In this study, the materials were the inbred eggplant variety “JS221,” which had been self-pollinated for several years. Ripe eggplant seeds were enclosed in gauze, soaked in water at 55 °C for 15 min, and then removed and kept moist. Once the seed embryos exposed their radicles, they were transferred to 7 × 7 small pots filled with nutrient soil, with one seedling per pot. The pots were then placed in a controlled growth chamber (Lishigao Instrument Equipment Co., Ltd., Nanjing, China) with a photoperiod of 16 h light/8 h dark, a temperature of 25 °C/18 °C, light intensity of 800 µmol m⁻² s⁻¹, and relative humidity of 70%. When the seedlings reached the stage of 4–6 true leaves, four healthy eggplant seedlings were selected for analyzing the relative expression patterns of SmRBOHs in different tissues. Roots, stems, and leaves were collected, flash-frozen in liquid nitrogen, and stored at −80 °C for later analysis. To simulate salt stress, a 150 mmol/L NaCl solution was prepared and applied by watering each pot with 50 mL of the NaCl solution, with a tray placed under the pots to prevent salt leakage [28]. For the heat stress treatment, eggplant seedlings were exposed to a high temperature of 42 °C, while other conditions remained the same. To induce low-temperature stress, eggplant seedlings at the 4–6 true leaf stage were placed at 4 °C while other conditions remained constant. The treatment with V. dahliae involved watering the soil around the roots of the eggplant with a suspension of V. dahliae “VdLS17” spores, with a concentration of 10⁷ spores/mL, and 20 mL of the spore suspension was added to each pot [29,30]. Samples were then collected at different time points (0, 3, and 6 h) for each treatment. For NaCl and V. dahliae treatments, the root samples of eggplants were collected, while for temperature stress treatment, leaf samples were collected. Four randomly selected seedlings were used as biological replicates for each treatment.

2.6. qRT-PCR Assay

The cDNA preparation method and reagents used were referenced from previous research reports [28,31]. ChamQ SYBR qPCR Master Mix (Q311, Novogene, Nanjing, China) was utilized for qRT-PCR with SmActin as the reference gene. The qRT-PCR program and reaction system were consistent with those described in previous research reports [28,31]. The primer sequences are provided in Table S3.

2.7. Statistical Calculations

We used the 2^−ΔΔCt method to calculate relative expression levels and used a one-way analysis of variance and Tukey’s test (p < 0.01) to analyze the differences between samples. The statistical calculations were performed using Microsoft Office Excel 2021 and IBM SPSS Statistics v 22, and the graphical representation was processed using GraphPad Prism v 9.5.0. [31].

2.8. Subcellular Localization

The vector used in this experiment is pBinGFP2, and the material used is N. benthamiana (Nicotiana benthamiana L.). We selected significantly upregulated members of SmRBOHs (SmRBOHB, SmRBOHD, SmRBOHE1, SmRBOHH2) under at least one stress condition. Following the methods established by previous researchers [28,31], SmRBOHs were cloned and inserted into the pBinGFP2 vector. The constructed vectors, along with the empty pBinGFP2 vector (used as a control), were transformed into Agrobacterium tumefaciens strain GV3101. The Agrobacterium containing the desired vectors was adjusted to an OD₆₀₀ of 0.8 using the infection solution, and the formula of the infection solution is reported in previous reports [28,31]. The OD₆₀₀ = 0.8 suspension of Agrobacterium was aseptically injected into the leaves of N. benthamiana using 1 mL syringes. After 48 h, the fluorescence signals in the leaves were observed using a laser scanning confocal microscope (LSM 880NLO; Leica Microsystems, Wetzlar, Germany). The primer sequences required for the cloning of SmRBOHs are provided in Table S4

3. Results

3.1. Identification, Physicochemical Characterization, and Chromosomal Localization of SmRBOHs

Using bioinformatics methods, eight SmRBOH genes were identified from the genome database of eggplant. These eight SmRBOH genes are relatively evenly distributed across seven chromosomes. Among them, chromosome 6 contains two members, namely SmRBOHD and SmRBOHH1. Additionally, chromosomes 1, 3, 5, 7, 8, and 12 each harbor one SmRBOH member (Figure 1).

The length of SmRBOH proteins ranges from 718 (SmRBOHD) to 963 amino acids (SmRBOHA), with relative molecular weights ranging from 82.136 to 109.25 KDa, suggesting potential structural differences. The isoelectric points are 8.72 (SmRBOHE1) to 9.41 (SmRBOHD), all of which are greater than 7.00, indicating an alkaline nature. SmRBOHC, SmRBOHD, and SmRBOHH2 exhibit instability coefficients ranging from 37.44 to 39.24, indicating stable proteins, while the rest have coefficients ranging from 41 to 49.15, indicating unstable proteins. Analysis of the hydrophilicity of SmRBOH revealed negative values, indicating that all SmRBOHs are hydrophilic proteins but with varying degrees of hydrophilicity. Subcellular localization prediction analysis suggests that all eight members of the SmRBOH gene family are most likely located on the plasma membrane. These results indicate that SmRBOHs share certain physicochemical properties, such as being alkaline, hydrophilic proteins, and most likely localized on the plasma membrane (

Table 1. Basic Information and features of the SmRBOH gene family members.

Gene Name	Protein Length/aa	Molecular Weight/KDa	Theoretical Isoelectric Point	Instability Index	Hydrophilia	Subcellular Localization 1
SmRBOHA	963	109.25	9.2	49.15	−0.237	plas:11, chlo:1, nucl:1, E.R.:1
SmRBOHB	866	98.81	8.19	41.93	−0.275	plas:12, nucl:2
SmRBOHC	938	105.40	9.13	37.44	−0.296	plas:14
SmRBOHD	718	82.14	9.41	38.24	−0.136	plas:9, E.R.:2, nucl:1, cyto: 1, mito: 1
SmRBOHE1	843	96.07	8.72	41.98	−0.174	plas:8, E.R.:3, nucl:1, mito:1, pero:1
SmRBOHE2	944	106.40	8.79	46.59	−0.153	plas:8, mito:3, chlo:1, nucl:1, pero:1
SmRBOHH1	792	90.79	8.83	41	−0.219	plas:13, nucl:1
SmRBOHH2	779	89.53	8.95	39.24	−0.142	plas:12, nucl:1, cyto:1

¹ plas: plasma membrane; ER: endoplasmic reticulum; chlo: chloroplast; nucl: nuclear; cyto: cytoplasmic; pero: peroxisome; mito: mitochondria.

).

3.2. Motif and Conserved Structural Domain Analysis of the SmRBOH Family

This study utilized the protein sequences of SmRBOHs to predict 10 Motifs (Figure 2a). Except for Motif 7, which is absent in SmRBOHH1, the other members contain all the Motifs, and the arrangement of the Motifs is entirely consistent. This indicates a high conservation of protein sequences among SmRBOH family members, suggesting potential functional similarities. The conserved domains analysis of SmRBOH revealed multiple conserved domains in this gene family (Figure 2b). All SmRBOHs have the conserved domain NADPH_Ox at the 5′(N-) terminal, and except for SmRBOHE1, other SmRBOH members have the NAD-binding domain NAD_binding_6 at the 3′(C-) terminal. With the exception of SmRBOHC, other SmRBOH members contain EF-hand_7, and except for SmRBOHA, SmRBOHD, and SmRBOHH1, the other SmRBOH members contain EFh. Both EF-hand_7 and EFh are recognized as EF-hand conserved domains associated with calcium ion binding. Except for SmRBOHE1 and SmRBOHE2, others contain Ferric_reduct, while SmRBOHE1 and SmRBOHE2 possess the PLN02631 superfamily; Ferric_reduct and the PLN02631 superfamily are related to ferric reductase. SmRBOHC, SmRBOHE2, and SmRBOHH1 contain FAD_binding_8, whereas other family members at this position have NOX_Duox_like_FAD_NADP. Both FAD_binding_8 and NOX_Duox_like_FAD_NADP are Ferredoxin reductase (FNR) domains involved in the photosystem electron transfer process [32,33,34].

3.3. Phylogenetic and Evolutionary Analysis of RBOHs

Based on the phylogenetic and evolutionary analysis of the RBOH protein sequences from eggplant, Arabidopsis, tomato, and rice, the SmRBOHs are relatively evenly divided into three evolutionary branches, each of which contains RBOHs from eggplant, Arabidopsis, tomato, and rice. RBOHs clustered in the same branch are more likely to exhibit functional similarities. SmRBOHA, SmRBOHE1, and SmRBOHE2 form one evolutionary branch, while SmRBOHB, SmRBOHC, and SmRBOHD form another evolutionary branch. Additionally, SmRBOHH1 and SmRBOHH2 constitute a separate evolutionary branch. Except for SmRBOHA, which is genetically closest to AtRBOHF, the other members of the SmRBOH family are genetically closest to SlRBOH gene family members. The results suggest that the evolutionary relationship between SlRBOHs and SmRBOHs is the closest, followed by AtRBOHs. Therefore, there might be functional similarities between SmRBOHs and tomatoes, as well as Arabidopsis RBOHs (Figure 3).

3.4. Analysis of SmRBOHs Cis-Acting Elements in Functional Regulation

To predict the potential functions of SmRBOHs, we extracted a 2000 bp upstream of the initiation codons of the SmRBOHs for cis-acting element analysis. The results indicate that SmRBOHs contain numerous cis-acting elements, primarily including binding sites for various transcription factors such as MYB, MYC, and W-box; light-responsive elements like Box 4, G-Box, and Sp1; phytohormone-responsive elements such as ABRE, TATC-box, and TGACG-Motif; as well as stress-responsive elements, including STRE, WUN-Motif, and LTR. All SmRBOH members possess light-responsive elements (Box 4, G-Box, Sp1, etc.), MYB transcription factor binding sites (MYB), and MYC transcription factor binding sites (MYC). SmRBOHE1 and SmRBOHH1 promoter sequences contain the most MYB and MYC binding sites, suggesting that RBOHs may respond to light and act in combination with MYB and MYC. Additionally, W-box is only present in SmRBOHA, SmRBOHB, and SmRBOHC, implying that WRKY transcription factors only bind to a subset of SmRBOH members. ABA response elements (ABREs) are found in all SmRBOH members, with SmRBOHH2 containing the highest number of ABREs. Except for SmRBOHH1, other members contain ethylene (ETH)-responsive cis-acting elements (EREs), with SmRBOHC having the most EREs. With the exception of SmRBOHE2 and SmRBOHH1, other members contain methyl jasmonate (MeJA) response elements (CGTCA-Motif, TGACG-Motif), with SmRBOHD containing the highest number of MeJA response elements. Except for SmRBOHC, SmRBOHH1, and SmRBOHH2, cis-acting elements (P-box and TATC-box) related to gibberellin (GA) response were present in other members. In addition, SmRBOHB, SmRBOHE2, SmRBOHH1, and SmRBOHH2 contain salicylic acid (SA) response elements (TCA-elements). This indicates that SmRBOHs can respond to different phytohormones, and SmRBOHB contains all the above-mentioned phytohormone response elements, potentially responding to all these phytohormones. Except for SmRBOHC, all SmRBOH members contain an anaerobic response element (ARE) associated with anaerobic induction. SmRBOHB, SmRBOHD, SmRBOHE2, and SmRBOHH1 contain stress-responsive element (STRE) cis-acting elements, and apart from SmRBOHA, SmRBOHD, and SmRBOHE1, other SmRBOHs members contain mechanical damage response elements (Wun-Motif). SmRBOHC, SmRBOHE2, and SmRBOHH1 have low-temperature-responsive LTR elements, while SmRBOHE1, SmRBOHH1, and SmRBOHH2 contain drought-responsive MBS elements. Furthermore, some members contain CAT-box, related to meristematic tissue expression, O2-site, related to corn protein metabolism regulation, and elements associated with circadian rhythm regulation. These findings suggest that SmRBOHs may have different expression levels and functions under various environmental conditions, potentially participating in the regulation of eggplant growth, development, and multiple stress response pathways (Figure 4).

3.5. Expression Analysis of SmRBOHs in Different Tissues

We analyzed the relative expression patterns of SmRBOHs in the roots, stems, and leaves of eggplant using qRT-PCR. The results revealed differential expression of SmRBOH gene members across various tissues. SmRBOHA, SmRBOHE1, SmRBOHE2, and SmRBOHH1 exhibited the highest expression levels in the roots. SmRBOHE2 showed relatively lower expression in the leaves, while SmRBOHH1 displayed reduced expression in both the stems and leaves compared to the roots. SmRBOHB showed the highest expression level in the stems. SmRBOHC and SmRBOHD exhibited the highest expression in the leaves. Therefore, this study suggests that SmRBOHs play distinct roles in different tissues of eggplant (Figure 5).

3.6. Expression Pattern Analysis of SmRBOHs under Different Stress Conditions

To study the response of SmRBOH genes under different stress conditions, we subjected 4–6 leaf stage eggplant seedlings to simulated salt stress, temperature stress, and V. dahliae treatment. Subsequently, we used qRT-PCR to analyze the relative expression levels of SmRBOHs under the aforementioned stress conditions. The results showed differential expression levels of different SmRBOH genes under various treatments. Under salt stress, the expression level of SmRBOHA showed no significant change, while SmRBOHE2 and SmRBOHH1 were downregulated. Conversely, the remaining SmRBOH genes showed varying degrees of upregulation. Notably, SmRBOHB showed the most substantial upregulation under salt stress, with its expression level increasing over 400-fold at 6 h compared to 0 h. Regarding high-temperature treatment, the expression levels of SmRBOHA, SmRBOHC, and SmRBOHE2 were downregulated. SmRBOHE2 reached its lowest expression level at 3 h and showed a slight upregulation at 6 h, but it was still significantly lower than its expression level at 0 h. SmRBOHE1 displayed an initial upregulation followed by downregulation, while the other four SmRBOH genes showed upregulation under high-temperature conditions, with SmRBOHD demonstrating the most significant upregulation. Under low-temperature treatment, the expression levels of SmRBOHA, SmRBOHC, and SmRBOHE2 were significantly downregulated, while the expression levels of the remaining SmRBOH members were significantly upregulated. Notably, SmRBOHB reached its peak expression level at 3 h. During V. dahliae inoculation within 6 h, the expression level of SmRBOHC showed no significant change, while SmRBOHB and SmRBOHE1 were upregulated in response to V. dahliae induction, with SmRBOHB showing the most pronounced upregulation (>160-fold). However, the expression of most SmRBOH genes decreased after V. dahliae inoculation. This study indicates that SmRBOHs exhibit different expression patterns under various treatments, suggesting functional diversity among SmRBOHs. Additionally, SmRBOHB consistently showed significant upregulation under different stress conditions, suggesting its potentially pivotal role in plant response to stress (Figure 6).

3.7. Plasma Membrane Localization of SmRBOHs

According to the WOLFSORT online prediction (Section 2.1), SmRBOHs are most likely located on the plasma membrane. To verify the above prediction, this study selected several SmRBOH members (SmRBOHB, SmRBOHD, SmRBOHE1, and SmRBOHH2) that showed a significant upregulation (>20-fold) under stress conditions to construct pBinGFP235S-SmRBOHs-GFP expression vectors. Then, we performed transient transformation experiments in the lower epidermal cells of N. benthamiana leaves using Agrobacterium-mediated transformation. The fluorescence of the above four SmRBOHs-GFP fusions was observed on the plasma membrane, indicating that SmRBOH proteins primarily localize to the plasma membrane. This finding is consistent with the previously predicted results and suggests that SmRBOHs mainly function on the plasma membrane (Figure 7).

4. Discussion

ROS, as signaling molecules, play essential roles in mediating plant growth and development, environmental adaptation, and programmed cell death processes. RBOHs are crucial enzymes involved in ROS generation. Previous studies have identified ten, nine, and seven members of the RBOH gene family in Arabidopsis, rice, and tomato, respectively [7,8]. In this study, we identified eight RBOH gene family members in eggplant, which are relatively evenly distributed across seven chromosomes. Studies have indicated that RBOH proteins are primarily located on the membrane, catalyzing NADPH in the cytoplasm while transferring electrons to generate ROS [9]. Topological structure analysis of RBOH proteins revealed the presence of transmembrane domains in all RBOHs, implying their classification as membrane proteins [35]. By utilizing specific antibodies against RBOHs to screen different subcellular fractions, only the fractions containing the plasma membrane exhibited positive hybridization bands [36]. In this study, the localization of SmRBOHs was predicted to be predominantly on the plasma membrane. We confirmed the accuracy of our predictions through the transient expression of some SmRBOHs (SmRBOHB, SmRBOHD, SmRBOHE1, and SmRBOHH2) in N. benthamiana leaves.

In wheat (Triticum aestivum L.), the 46 members of the NOX (NADPH oxidase) family are classified into three subgroups based on their conserved domains. The first subgroup consists of typical TaNOXs, which possess four conserved domains: NADPH_Ox, Ferric_reduct, FAD_binding_8, and NAD_binding_6. The second subgroup, TaNOX-likes, includes members with NADPH_Ox but lack one to two of the other conserved domains. The third subgroup, FRO [37], comprises members with the other three conserved domains but lack NADPH_Ox. In this study, the eight SmRBOHs all contain the NADPH_Ox domain. SmRBOHE2 is missing the Ferric_reduct domain, while SmRBOHE1 lacks both the Ferric_reduct and NAD_binding_6 domains. Except for SmRBOHC, SmRBOHE2, and SmRBOHH1, the rest of the SmRBOHs are devoid of the FAD_binding_8 domain, which is replaced by the NOX_Duox_like_FAD_NADP domain, and the FAD_binding_8 domain and NOX_Duox_like_FAD_NADP domain serve similar functions [33]. Additionally, SmRBOHs possess 1–2 EF-hand domains (Ef-hand_7 and EFh). These conserved domains have different functions [15]. The NADPH_Ox domain generates ROS, and the C-terminal region containing NAD_binding_6, NOX_Duox_like_FAD_NADP, FAD_binding_8, and PLN02631 superfamily domains play crucial roles in electron transfer and ROS production [33]. The EF-hand domains have a typical helix-loop-helix (HLH) structure and are widely regarded as calcium-binding regions. In the pathway of RBOH-mediated ROS production, small GTPases act as regulators, and the N-terminal region is considered a bridge connecting GTPases and RBOHs [38]. P. tricornutum’s RBOHs lack the EF-hand domain at the N-terminus and do not have homologs that regulate RBOH [12], further suggesting the critical role of EF-hand in the interaction between RBOHs and small GTPases [12].

SmRBOHs can be classified into three clades in terms of phylogenetic and evolutionary analysis. The evolutionary relationship between SmRBOHs and RBOHs in tomatoes and Arabidopsis is relatively close. Based on the results of phylogenetic tree analysis, genes that cluster together are more likely to share similar functions and structures. AtRBOHD and AtRBOHF play an active role under various stresses [23]. Studies have indicated that TaNOX8 and TaNOX12 exhibit high sequence similarity to AtRBOHD and AtRBOHF, respectively, and display similar expression patterns under stress conditions [37]. In this study, the relationship between SmRBOHD and AtRBOHD is found to be closer in terms of evolutionary relationship. AtRBOHD generates ROS under stress conditions by binding to Ca²⁺ and undergoing phosphorylation. SmRBOHD is significantly upregulated under temperature stress, especially high-temperature stress, suggesting a potential similarity with AtRBOHD in regulating temperature stress responses. On the other hand, SmRBOHA, which is evolutionarily closer to AtRBOHF, shows significant downregulation under all stresses except for salt stress. This indicates possible functional divergence between SmRBOHA and AtRBOHF.

Cis-regulatory elements play a crucial role in plant stress and phytohormone responses. In this study, through the analysis of SmRBOH promoter regions, numerous stress-related cis-regulatory elements were discovered, such as ARE, Wun-Motif, and STER. These elements are induced by different stressors [39] to regulate the production of ROS, leading to cell death or influencing antioxidant signal transduction in response to various biotic stresses, salt damage [40], drought [41], low-temperature [14], and other abiotic stresses. Previous studies have shown that ROS signaling pathways are closely related to phytohormone regulatory pathways. For example, ABA can induce the expression of the SlRBOH1 gene, enhance RBOH enzyme activity, and improve tomato tolerance [39]. Jasmonic acid compounds (JAs), as important stress signaling molecules, regulate the plant’s antioxidant enzyme system and also participate in the regulation of RBOH activity [42]. ETH and SA regulate the production of ROS dependent on AtRBOHD [43]. In this study, all SmRBOHs contain the ABA-responsive element (ABRE), and most family members also contain MeJA (CGTCA-Motif, TGACG-Motif)-, ETH (ERE)-, and SA (TCA-element)-responsive elements. The presence of these cis-regulatory elements suggests that the promoter regions of SmRBOHs can respond to the regulation of multiple signals.

The expression patterns of genes in different tissues reflect the functional characteristics. In Arabidopsis, 10 RBOH genes are distributed across various tissues. Both AtRBOHD and AtRBOHF are expressed throughout the whole plant, while AtRBOHH and AtRBOHJ are exclusively present in pollen tubes, and the remaining AtRBOH genes are mainly expressed in the roots [44]. Extensive research has also demonstrated the multifunctionality of AtRBOHD and AtRBOHF, while AtRBOHH and AtRBOHJ are involved in regulating pollen tube growth [13,23]. In this study, SmRBOHs are expressed in different parts of the plant, showing variations in expression levels. SmRBOHB exhibits the highest expression level in the stem, while SmRBOHC, SmRBOHD, and SmRBOHH2 show the highest expression in leaves. The remaining SmRBOH genes show the highest expression levels in the roots. These results imply that SmRBOHs play distinct roles and functions in different plant tissues.

The NADPH oxidases encoded by RBOHs on the plasma membrane mediate the production of ROS, which enhances plants’ adaptation to adverse environments. Under abiotic stress, the H₂O₂ generated by NADPH oxidases is considered the initial signal of the antioxidant pathway [8,26]. H₂O₂ produced by AtRBOHD and AtRBOHF can trigger an increase in Ca²⁺ levels, thereby regulating the homeostasis of Na⁺/K⁺. Double mutants of AtRBOHD and AtRBOHF exhibit increased sensitivity to salt stress, with significantly higher Na⁺/K⁺ levels compared to other mutants [26]. In this study, SmRBOHB showed a significant upregulation in expression under salt stress, while the expression of other members changed only slightly, indicating that SmRBOHB plays a crucial role in the response to salt stress (Figure 6). Changes in temperature can lead to alterations in membrane proteins and membrane lipid composition, affecting membrane permeability and causing cellular damage to plants [45]. Under high-temperature conditions, the expression of SmRBOHD was significantly upregulated, and the expression levels of SmRBOHB, SmRBOHH1, and SmRBOHH2 also increased notably. This suggests that these genes are involved in eggplant’s heat tolerance process (Figure 6). Under low-temperature conditions, SmRBOHB and SmRBOHE1 showed a substantial increase in expression levels, and compared to the 6 h time point, SmRBOHB exhibited even higher expression levels at 3 h. This indicates that SmRBOHB plays a role in the early cold tolerance process, while SmRBOHE1 demonstrates a more sustained effect (Figure 6). Plants generate ROS to trigger immune responses, limiting the invasion of pathogens or enhancing the cell wall to bolster the plant’s defense system under biotic stress. In A. thaliana, AtRBOHD and AtRBOHF respond to various pathogenic infections, with AtRBOHD primarily involved in ROS formation and AtRBOHF playing a role in cell death [46]. Additionally, the double mutant of AtRBOHD and AtRBOHF exhibits extreme sensitivity to bacterial AG8, whereas single mutations in AtRBOH do not show this effect, suggesting the potential interplay and mutual involvement of RBOHs under specific circumstances [47]. OsRBOHB expression is induced by rice blast pathogens, and the high concentration of ROS it produces effectively inhibits the hyphal growth and protein secretion of the pathogens, thus restricting further infection by the rice blast fungus [48]. As the causal agent of Verticillium wilt, V. dahliae has a wide range of hosts, such as cotton, tomato, and eggplant. The occurrence of Verticillium wilt inflicts irreversible damage to plants. In cotton, GbRboh5/18 participates in the anti-Verticillium wilt process by mediating ROS production [22]. In this study, SmRBOHB expression was significantly upregulated upon V. dahliae treatment (Figure 6). Based on previous research, we speculate that SmRBOHB, induced by V. dahliae, generates a substantial amount of ROS, which might be involved in cellular immune responses, restricting the growth of the pathogen. Furthermore, most SmRBOHs exhibit downregulated expression under V. dahliae infection, and their specific mechanisms of action require further investigation.

Some RBOHs exhibit specificity in their functions, while others display versatility. For instance, AtRBOHD and AtRBOHF not only play a positive role in response to various stresses such as diseases, salt, and cold stress [23] but also render plants more susceptible to stress, as observed in increased vulnerability to cyst nematode infestation in Arabidopsis [49]. In this study, SmRBOHB shows significantly increased expression under different stress conditions, suggesting its multi-functional nature in activating diverse signaling pathways to respond to various stresses.

This research identified RBOH genes in eggplant that respond to both biotic and abiotic stresses at the transcriptional level, revealing their involvement in eggplant disease resistance and stress response. This has important implications for molecular breeding and genetic improvement of eggplant. However, a more comprehensive and in-depth experimental investigation is required to elucidate the regulatory mechanisms of SmRBOHs under different stress conditions.

5. Conclusions

This study identified eight members of the eggplant RBOH gene family and analyzed their physicochemical properties, chromosomal localization, Motifs and conserved domains, phylogenetic evolutionary, cis-acting elements, and expression patterns. SmRBOHs exhibited diverse expression patterns in different tissues (the roots, stems, and leaves) and various treatments (salt, high-temperature, low-temperature, and V. dahliae). Among them, SmRBOHB was significantly upregulated under different stress conditions. Additionally, SmRBOHD, SmRBOHE1, and SmRBOHH2 also showed significant upregulation (>20-fold) under specific environments, making them potential candidate genes for stress tolerance. Subcellular localization analysis of these four genes revealed that SmRBOHs are localized on the plasma membrane. This study provides foundations for further exploring the stress resistance mechanisms of the SmRBOH genes in eggplant.