Next Article in Journal
Unraveling the Epigenetic Landscape of Mature B Cell Neoplasia: Mechanisms, Biomarkers, and Therapeutic Opportunities
Previous Article in Journal
Transcriptomic Identification of Immune-Related Hubs as Candidate Predictor Biomarkers of Therapeutic Response in Psoriasis
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 26
Issue 17
10.3390/ijms26178131
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genome-Wide Expression Profile of SOD Gene Family in Isatis indigotica and the Key Role of IiSOD2 and IiSOD7 in Alkaline Stress

by
Lengleng Ma
†,
Lingyang Kong
†,
Shan Jiang
,
Junbai Ma
,
Lianqing He
,
Jianhao Wu
,
Xiaozhuang Zhang
,
Wei Wu
,
Wei Ma
* and
Weichao Ren
*
College of Pharmacy, Heilongjiang University of Chinese Medicine, Harbin 150040, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2025, 26(17), 8131; https://doi.org/10.3390/ijms26178131
Submission received: 1 July 2025 / Revised: 15 August 2025 / Accepted: 20 August 2025 / Published: 22 August 2025
(This article belongs to the Section Molecular Plant Sciences)
Browse Figures
Versions Notes

Abstract

Superoxide dismutase (SOD) is a key enzyme in the plant antioxidant system. It plays an essential role in plant adversity stress by scavenging excess reactive oxygen species to protect cells from oxidative damage. Isatis indigotica, being a mildly saline-tolerant plant, can be grown in soils containing a certain amount of saline–alkaline content. In order to reveal the SOD gene family members and their potential roles under saline and alkaline stress, the present study used a bioinformatics approach to identify 9 potential IiSOD genes in the I. indigotica genome. It analyzed the expression patterns of SOD family genes (IiSODs) in response to alkaline stress. According to the results of quantitative real-time PCR (qRT-PCR), the expression levels of the IiSOD7 gene significantly increased within 120 h of alkaline stress treatment, while the expression level of the IiSOD8 gene was the highest among all detected genes at 120 h of alkaline stress. The rest of the genes showed different degrees of expression. Alkaline stress showed significant and dynamic changes in the content of indigo and indirubin in leaves of I. indigotica. Finally, the yeast one-hybrid assay confirmed that IiWRKY54 was able to activate the expression of IiSOD2 and IiSOD7. Combined with qRT-PCR analysis, it was further hypothesized that IiWRKY54 might enhance the alkaline tolerance of I. indigotica by regulating the expression of IiSOD2 and IiSOD7. Taken together, this study lays the foundation for elucidating the function of the IiSOD gene in salinity stress tolerance of I. indigotica as well as promoting the genetic breeding of alkaline-tolerant varieties of I. indigotica.

1. Introduction

Isatis indigotica Fortune is a herbaceous biennial belonging to the genus Isatis of the Brassicaceae. It has a long medicinal history in Asia and Europe. The whole plant of I. indigotica can be used as medicine. The root of I. indigotica is Isatidis radix, which has antiviral and anti-inflammatory pharmacological effects. It is clinically used to treat influenza, hepatitis, mumps, and other diseases [1]. Isatidis folium has antibacterial, antiviral, antioxidant, and other pharmacological effects [2]. In addition to being medicinal, I. indigotica leaves or stems, through the processing of dry powder for indigo, can be used as a medicinal and a plant dye [3,4]. I. Indigotica is widely grown in China as a medicinal cash crop. It usually faces the threat of biotic and abiotic stress during growth and development, resulting in financial losses. Abiotic stresses mainly include saline–alkaline, drought, low temperature, and high temperature, which humans cannot control. Soil salinization is one of the key factors affecting the yield and quality of I. indigotica. The harmful salts in soil mainly include NaCl, Na2SO4, Na2CO3, and NaHCO3 [5,6].
The alkaline salt stress caused by NaHCO3 and Na2CO3 usually leads to the increase in pH value in the rhizosphere environment of plants, which not only destroys the physiological function of roots but also leads to the disintegration of root cells and even the destruction of root structure, thus affecting the growth and development of plants, the quality of medicinal materials and physiological and biochemical indexes [7]. The most common stress symptoms of plants under alkaline stress are chlorotic leaves and reduced plant growth to a certain extent. For example, the chlorophyll content index (CCI) of blueberry leaves in alkaline soil showed a downward trend, and the concentration of some trace elements in leaves was unbalanced, indicating that the veins of leaves were chlorotic in alkaline soil [8]. Under alkaline stress, rice causes severe damage to the growth of old leaves but has little effect on the development of young leaves [9]. Studies have shown that three genes, GmFSD3, GmFSD4, and GmFSD5, in soybean (Glycine max (L.) Merr.) roots were significantly induced under alkaline stress [10]. Under low temperature, high temperature, and salt stress, the two genes HbCSD2 and HbCSD4 in mature leaves of Hevea brasiliensis were significantly up-regulated to enhance stress resistance [11].
When plants are subjected to abiotic stress, they show increased respiration, decreased photosynthesis, and a large amount of ROS [12]. Excessive ROS accumulation can lead to toxic by-products of oxygen metabolism and severe cell death [13,14]. SOD is present in peroxisomes, mitochondria, chloroplasts, and cytoplasm and is the first line of defense against reactive oxygen species (ROS)-mediated damage in cells [15]. SOD protects plants from biotic and abiotic stresses by scavenging excessive reactive oxygen species produced by organisms. According to the different binding modes with metal cofactors, SOD genes in plants can be divided into three subfamilies: Cu/Zn-SOD, Mn-SOD, and Fe-SOD [16]. The three subfamilies of SOD mainly exist in higher plants, and different subtypes have different functions [17]. The Mn-SOD gene encodes a synthetic manganese superoxide dismutase to increase SOD activity in plants and directly scavenge reactive oxygen species to resist oxidative stress [18]. Previous studies have shown that the three subfamilies of the SOD gene family are present in different organelles. For example, Cu/Zn-SOD is mainly distributed in chloroplasts, cytoplasm and peroxisomes [19]. Mn-SOD is distributed in mitochondria and peroxisomes [20,21]. In contrast, Fe-SOD was mainly detected in mitochondria and chloroplasts.
In this study, we systematically analyzed the evolutionary relationships, conserved motifs, and promoter elements characterizing the IiSOD gene family based on the chromosome-level genome of I. indigotica (Size: 293.88 MB, annotated 30,323 genes encoding proteins with high confidence, diploid chromosome number 2n = 14) [22]. Based on the inhibitory effect of alkaline stress on plant growth [23], the molecular mechanism by which the IiSOD gene enhances plant resilience by scavenging reactive oxygen species (ROS) accumulated in excess under adverse conditions was investigated for the first time in a NaHCO3 alkaline stress experiment. This study, a comprehensive analysis of the IiSOD gene family was carried out, which provided a theoretical basis for improving the saline–alkali tolerance of I. indigotica cultivars.

2. Results

2.1. Identification and Physicochemical Propertiesof SOD Gene Family in I. indigotica

In this study, 8 A. thaliana (AtSODs) protein sequences were queried, and 9 IiSOD genes were identified in the I. indigotica genome. Based on the structural domain analysis, 3 proteins were found to have Cu/Zn-SOD domains (Pfam: 00080), 6 proteins had Fe/Mn-SOD (PF02777 and PF00081) domains. This study analyzed the protein sequence and physicochemical properties of the IiSOD family members. The IiSOD gene CDSs ranged from 462 bp to 972 bp, and the encoded IiSOD protein was 153–323 amino acids. The molecular weight of IiSOD protein ranged from 15,163.77 Da to 34,597.74 Da, and the theoretical isoelectric point (pI) ranged from 4.85 to 9.46, indicating that IiSOD protein has both acidic and alkaline proteins (Table 1).
In addition, the results of the predicted subcellular localization showed that the IiSOD protein was mainly localized in the two organelles of mitochondria and chloroplasts (Table 1). The SOD proteins of the Cu/Zn-SOD subfamily were located in the chloroplast, and the SOD proteins of the Mn-SOD subfamily were located in the mitochondria. The proteins of the same subfamily may be located in different organelles. Except that IiSOD3 and IiSOD7 are located in chloroplasts, other members of Fe-SOD are located in mitochondria.
According to the signal peptide prediction results of IiSOD proteins shown in Supplementary Figure S1, the SP/CS/OTHER probabilities of the IiSOD protein family are all close to 0, indicating that these proteins do not depend on the classical secretory pathway and are non-secretory proteins. The predicted transmembrane domains of the IiSOD proteins are shown in Supplementary Figure S2, and the transmembrane domains of the IiSOD protein family (Transmembrane probability ≈ 0) indicate that the IiSOD protein family does not contain transmembrane domains and does not function in a membrane-bound mode. Of these, the IiSOD1, IiSOD3, IiSOD8, and IiSOD9 proteins all exhibited significant external localization characteristics (Outside probability ≈ 1.0), with a very low internal localization probability (Inside probability ≈ 0), suggesting that these proteins are extracellular.

2.2. The Phylogenetic Relationship of IiSODs

Phylogenetic trees are widely used to show the evolutionary relationships of gene families. Phylogenetic analysis of SOD protein sequences of I. indigotic (nine), A. thaliana (eight), Brassica oleracea (fourteen), and Vitis vinifera (nine) was used (Supplementary Table S1). Based on domain analysis and the phylogenetic tree, the SOD genes of all species were clustered into three major clades: Cu/Zn-SODs (I), Fe-SODs (II), and Mn-SODs (III), represented by light blue, pink, and light green, respectively (Figure 1). SOD protein sequences clustered in the same branch are more likely to exhibit functional similarity. The Cu/Zn-SOD group contained 17 SOD members (3 liSODs, 3 AtSODs, 6 BoSODs, and 5 VvSODs), the Fe-SOD included 14 members (4 liSODs, 3 AtSODs, 5 BoSODs, and 2 VvSODs), and the Mn-SOD group had 9 members (2 liSODs, 2 AtSODs, 3 BoSODs, and 2 VvSODs). The I. indigotica, A. thaliana, and B. oleracea belong to the cruciferous family, and the SOD family members are closest to the AtSOD and BoSOD gene family members in the phylogenetic tree. The results showed that the IiSOD, AtSOD, and BoSOD families were most closely related during evolution. Interestingly, the Cu/Zn-SOD and Fe-SOD groups of each species contained more SOD compared to the Mn-SOD group. In the Fe-SOD group, IiSOD8’s similarity to A. thaliana Fe-SOD (50%) was significantly higher than its similarity to A. thaliana Mn-SOD (20%).

2.3. Chromosomal and Collinearity Analysis of IiSODs Gene

According to the genome annotation file of I. indigotica, we analyzed the relative position of 9 IiSODs on chromosomes. The results confirmed that 9 IiSOD genes were distributed on seven different chromosomes. According to the chromosome distribution of A. thaliana and the widely accepted naming system, the IiSOD candidate genes were named IiSOD1-9. Among them, Chromosomes 2, 4, and 7 each have two genes distributed, and strangely, there is no gene distribution on chromosome 5. The results showed that the positional distribution of IiSOD genes was irregular and, in addition, no tandem duplication events were found (Figure 2B). Collinearity analysis revealed that a segmental duplication occurred in one gene pair (IiSOD3 and IiSOD7) in the IiSOD genes (Figure 2A), and they both belonged to the Fe-SOD subfamily. IiSOD3 and IiSOD7 may have undergone subfunctionalization, neofunctionalization or functional redundancy.

2.4. Conserved Motifs and Domains of IiSOD Proteins

The nine liSODs were divided into two groups (Figure 3A): one group consisted of three Cu/Zn-SODs, while the other group included six proteins: four Fe-SODs and two Mn-SODs. We further analyzed the conserved motifs of IiSOD, and showed that each subfamily of IiSOD has a unique motif composition (Figure 3B). These conserved motifs may have specific roles in their respective subgroups. Among them, motif 5 is related to the Cu/Zn-SOD domain (PF00080) and is only present in members of the Cu/Zn-SOD subfamily. Conserved motifs 1 and 2 are present in both Fe-SOD and Mn-SOD families, and motifs 5, 6, and 10 are contained in the Cu/Zn-SOD family. Each subfamily contains the same conserved motif. Analysis of the conserved domains showed that both IiFe-SODs and IiMn-SODs contained SOD_Fe_N (PF00081) and SOD_Fe_C (PF02777) domains among the nine liSODs, while IiCu/Zn-SODs contained only Sod_Cu (PF00080) domains (Figure 3C).

2.5. Cis-Regulatory Elements in the Promoter Region of IiSODs Gene

To predict the functions and regulatory roles of IiSOD genes, a region of 2000 bp upstream of the transcriptional activation sites of IiSOD genes was extracted using the plantCARE database, and cis-regulatory elements in the promoter regions of IiSODs were examined (Figure 4). The results showed that the promoter region of IiSODs contained abundant cis-regulatory elements, including the binding sites of MYB, and MYC, (Figure 4). The hormone-responsive cis-elements were mainly abscisic acid (ABRE), methyl jasmonate (MeJA), salicylic acid (SA), growth hormone, and gibberellin (GA). The promoters of IiSODs commonly contain MYC binding sites (MYC) and anaerobic-inducible elements (ARE), suggesting that the IiSOD gene may be directly regulated by bHLH-like MYC transcription factors, which can respond rapidly to jasmonic acid, saline drought, and oxidative stresses, and that the IiSOD gene can be activated by hypoxic signals to maintain ROS homeostasis, and that it has a combination of both antioxidant and hypoxia tolerance functions. In summary, IiSOD genes have different expression levels and functions under different environmental conditions and are involved in responding to various stresses.

2.6. IiSOD Gene Expression Profile Under Alkaline Stress

Under alkaline stress, the nine IiSOD genes showed significant differences (Figure 5). The expression of IiSOD7 was low in the CK group. It significantly increased in the treatment group, suggesting its expression was time-dependent under alkaline stress. The expression levels of IiSOD1, IiSOD2, IiSOD3, and IiSOD4 remained almost unchanged at 24 h of stress, reaching their peak after 72 h. Subsequently, the expression level decreased at 120 h of stress, showing a typical stress response pattern of “delay-peak-attenuation”. IiSOD6 was up-regulated after 24 h of stress and reached a peak at 120 h of stress. It is worth noting that IiSOD8 exhibits a dynamic change in increase-decrease-increase under alkaline stress, with expression reaching a peak at 24 h, rapidly declining at 72 h, and rising slightly again at 120 h. Furthermore, its expression level is the highest among all detected genes, indicating that IiSOD8 exhibits temporal fluctuations in response to alkaline stress. On the contrary, the IiSOD5 gene was completely silenced at 24 h under alkaline stress, indicating that it did not respond to early stress or was specifically inhibited. Although IiSOD9 showed a slow upward trend with stress time, the overall expression level was always the lowest.

2.7. Determination of Phenotype and SOD Activity of I. indigotica Under Alkaline Stress

After alkaline stress treatment, the phenotype of I. indigotica showed significant differences in leaf color and growth status, such as leaf chlorosis, yellowing and decreased growth. With the extension of alkaline stress treatment time, the effect of alkaline stress on plant phenotype is becoming increasingly apparent. Especially in the final sampling stage, the phenotypic differences are pronounced. The CK group showed higher plant height and healthier leaves. In contrast, the plant height of the 120 h alkaline stress treatment group decreased, the leaves wilted and the growth amount was seriously reduced (Figure 6A). Under the same concentration of NaCl salt stress, the effect on plant phenotype was relatively small. The leaves remained evergreen under stress for 72 h, but the leaves showed severe chlorosis and wilting, and yellowing at 120 h [24].
With the prolongation of alkaline stress time, the activity of SOD decreased first and then increased, indicating that the oxidative stress induced by alkaline stress activated the antioxidant defense system. Our previous studies have found that the level of reactive oxygen species in I. indigotica increased sharply under stress, which led to oxidative damage of cell structure in plants and usually started the antioxidant enzyme system to resist the increase in reactive oxygen species content [24]. As the first line of defense of the antioxidant enzyme system, SOD directly scavenges oxygen free radicals in plants and reduces the accumulation of reactive oxygen species. Therefore, the activity of the SOD enzyme in I. indigotica tissue samples is an important indicator to observe the antioxidant capacity of I. indigotica. The SOD activity of I. indigotica showed an increasing trend under alkaline stress treatment by the superoxide dismutase kit (Figure 6B). The SOD activity of I.indigotica plants decreased first and then increased under alkaline therapy at different times. Compared with the control group, the enzyme activity of SOD decreased under alkaline treatment for 24 h and 72 h, and the enzyme activity of SOD increased to the maximum value with the extension of alkaline treatment to 120 h. Therefore, it can be seen that in a specific concentration range, I. indigotica can resist the damage caused by reactive oxygen species under alkaline stress by increasing the activity of the SOD enzyme in the antioxidant system. The SOD activity was calculated using the following formula:
S O D   a c t i v i t y     ( U / g ) = A 1 × V 1 × V 2 × D ( 1 − A 1 ) × W × V 3
A1: inhibition percentage; V1: total volume of the reaction system; V2: total volume of crude enzyme solution; D: dilution factor of crude enzyme solution; W: sample mass; V3: volume of crude enzyme solution added to the reaction system.

2.8. Content of Indole Alkaloids in Leaves of I. indigotica Under Alkaline Stress

The content of indigo and indirubin in leaves of I. indigotica under alkaline stress showed a tendency of decreasing and then increasing with the prolongation of stress time (Figure 7). From the figure, it can be seen that the indigo content in I. indigotica leaves continued to decrease at 24 h and 72 h of alkaline stress, and the indigo content slightly recovered at 120 h, but it was still lower than that of CK (Figure 7A). The trend of indirubin was similar to that of indigo, which also showed a decrease and then an increase, but the decrease was more significant: at 24 h of alkaline stress, the content of indirubin decreased by about 50%, indicating that its sensitivity to alkaline stress was higher than that of indigo (Figure 7B). Supplementary Figure S3 shows the HPLC of indigo and indirubin under alkaline stress. The peak area is shown in Supplementary Table S4.

2.9. IiWRKY54 Binds to IiSOD2 and IiSOD7 Promoter Genes

According to the transcription factor of the alkaline-tolerant gene AtWRKY45, the phylogenetic tree was constructed with IiWRKY to screen IiWRKY54 for a close genetic relationship. Through yeast one-hybrid experiments, the vector pJG4-5-IiWRKY54 transcription factor was co-transformed into the Saccharomyces cerevisiae strain with the vector pLacZi-IiSOD2 or pLacZi-IiSOD7 promoter. Under the appropriate temperature conditions, the colony color changed to blue (Figure 8). This indicates that the IiWRKY54 transcription factor can activate the transcription of the IiSOD2 or IiSOD7 gene. IiWRKY54 can regulate the transcription of the downstream IiSOD2 or IiSOD7 gene promoter, thereby improving the stress response of I. indigotica to alkaline stress.

3. Discussion

Excessive accumulation of ROS under adversity stress in I. indigotica, an important medicinal and economic crop, significantly reduces biomass and affects the synthesis of secondary metabolites. SOD, as the first line of defense against oxidative stress in plants, plays a key role in maintaining ROS homeostasis by catalyzing the disproportionation reaction of superoxide anion (O2−). By systematically analyzing the functional regulatory mechanism of the IiSOD gene family under adversity stress, we can provide a theoretical basis for cultivating high-quality and stress-resistant varieties. The SOD gene family has been comprehensively studied in a variety of plants, such as Liriodendron chinense [25], Triticum turgidum [26], and Cassava [27]. This paper is based on the genome file of I. indigotica; 9 IiSOD genes were successfully identified in I. indigotica by bioinformatics methods, which was similar to the number of SOD genes reported in most plants. For example, there are 8 SOD genes in A. thaliana, 8 SOD genes in Liriodendron chinense [25], 14 SOD genes in Triticum turgidum [26], 8 SOD genes in Cassava [27], and 22 SOD genes in Ophiopogon japonicus [28]. Nine SOD genes were identified in Rosa roxburghii Tratt [29].
Physicochemical properties analysis showed that the IiSOD family was mainly in mitochondria and chloroplasts. In the Fe-SOD subfamily, except for IiSOD3 and IiSOD7, which are located in chloroplasts, the remaining members, IiSOD1 and IiSOD8, are distributed in mitochondria (Table 1) [30]. A phylogenetic tree was constructed using the SOD sequences of and I. indigotica, A. thaliana, B. oleracea, and V. vinifera. IiSOD (9), A. thaliana (8), B. oleracea (14), and V. vinifera (9) proteins were divided into three subfamilies. The I. indigotica, A. thaliana, and B. oleracea belong to the cruciferous family, and the SOD family members are closest to the AtSOD and BoSOD gene family members in the phylogenetic tree. Phylogenetic analysis showed that the members of the IiSOD gene family could be divided into three subfamilies: Fe-SOD, Mn-SOD, and Cu/Zn-SOD according to their sequence characteristics, and the members of the same subfamily were usually distributed in the same cell compartment, which was consistent with the report of SOD gene clustering and subcellular localization [31].
The conserved motifs of IiSOD are consistent across subfamilies, suggesting that gene functions may be similar within the same subfamily. In addition, conserved motif analysis further revealed significant differences between different subfamilies, such as the differentiation of Fe-SOD and Cu/Zn-SOD subfamilies in functional domains (Figure 3B) [32]. IiCu/Zn-SODs proteins contain the conserved SOD_Cu domain (Pfam: 00080), both IiFe-SOD and IiMn-SOD possess the Sod_Fe_N (Pfam: 00081) and Sod_Fe_C (Pfam: 02777) domains. These proteins are classified into three subfamilies, consistent with previous studies [33]. Fe-SODs and Mn-SODs from different plants come together and are separated by high guide values, suggesting that they may have originated from a common ancestral gene [34].
PlantCARE analysis showed that there were a large number of cis-acting elements in the promoter region of the IiSOD gene family in response to plant hormones (Figure 4), which was consistent with the fact that the SOD gene was regulated by ABA and MeJA in plants [35,36]. The promoter region of the IiSOD gene has a large number of MYB binding sites. Analysis of cis-acting elements in I. indigotica promoters showed different regulatory patterns of indigo growth and response to stress [37].
The qRT-PCR results showed that the IiSOD7 gene responded positively to alkaline stress, whereas the IiSOD8 gene had the highest expression levels under alkaline stress. This suggests that both the IiSOD7 and IiSOD8 genes play key roles in the response of I. indigotica to alkaline stress. The results of qRT-PCR combined with phylogenetic analysis showed that the IiSOD gene in response to alkaline stress was a member of the Cu/Zn-SOD and Fe-SOD subfamily. It could be inferred that the SOD protein types that played a role in scavenging reactive oxygen species in the leaves of I. indigotica under alkaline stress were Cu/Zn-SODs and Fe-SODs. In summary, qRT-PCR results showed that the expression patterns of IiSOD genes in I. indigotica under alkaline stress treatment were differentially expressed, indicating that these genes were specific in response to environmental stress under alkaline stress treatment.
Indigo and indirubin are secondary metabolites of I. indigotica formed by oxidative polymerization of indole precursor compounds (e.g., indoxyl, isatin) under adverse conditions [38]. HPLC analyses showed that the contents of indigo and indirubin showed a dynamic trend of decreasing and then increasing under alkaline stress, indicating that the response of I. indigotica to alkaline stress has a stage-regulated characteristic. In the early stage of stress, the osmotic and ionic stress triggered by the high pH environment prompted the plants to preferentially allocate carbon–nitrogen resources to the synthesis of osmotic-regulating substances and the maintenance of ionic homeostasis, resulting in the inhibition of secondary metabolic pathways and a decrease in the content of indigo and indirubin [39,40]. As the stress persisted, cells initiated secondary defense responses by activating the Ca2+-ROS-MAPK signaling cascade [41], at which time indigo and indirubin were synthesized in large quantities and their contents were significantly reduced due to their ROS scavenging ability [42,43].
It is worth noting that the indigo content of CK group leaves was as high as 20 mg/g at 0 h; after 72 h of alkaline stress, this value decreased to a minimum of 12 mg/g, a decrease of 40%. The content of indirubin also showed a synchronous decline, with both showing similar trends. The above results indicate that an alkaline environment significantly inhibits the accumulation of indigo and indirubin in I. indigotica leaves. This result is consistent with the drought and salt stress data detected by UPLC: in this study, the content of indigo, indirubin, tryptanthrin, and syringic acid in I. indigotica was significantly reduced [44].
According to the results of the Y1H experiment, the yeast colonies co-transformed with pLacZi-IiSOD2 or pLacZi-IiSOD7 and pJG4-5-IiWRKY54 showed blue, indicating that IiWRKY54 can specifically bind to the promoters of IiSOD2 and IiSOD7 and significantly activate their transcription. Because IiSOD2 and IiSOD7 are key genes in plant response to alkaline stress, and the overexpression of IiSOD7 can enhance the resistance of I. indigotica, IiWRKY54 may positively regulate the expression of IiSOD2 or IiSOD7, thereby improving the tolerance of I. indigotica to alkaline stress.

4. Materials and Methods

4.1. Plant Material and Treatment Sampling

The seeds of I. indigotica are from the Medicinal Botanical Garden of Heilongjiang University of Chinese Medicine. Firstly, the seeds of I. indigotica are screened, and the seeds with whole seeds and bright seed coats are selected for sowing. The seeds of I. indigotica were sown in a seedling tray containing vermiculite and nutrient soil (1:4), and one seed was sown in each hole. When the plant grows to 3–4 leaflets, it is transferred to a pot (15 cm high, 15.3 cm in diameter), and one plant is planted in each pot. When I. indigotica grows for about 70 days and reaches 5–6 leaves, it is ready for treatment.
Based on the fact that soils in parts of Heilongjiang, China, are characterized by typical soda salinity, with a salt composition dominated by sodium carbonate (Na2CO3) and sodium bicarbonate (NaHCO3), and with measured HCO3− concentrations ranging from 100 to 250 mmol/L (pH 8.0–9.2) [45,46,47]. To simulate the effect of Heilongjiang saline soil on the growth status of I. indigotica. A 200 mmol/L NaHCO3 alkaline solution (pH ≈ 8.2) was prepared to stress I. indigotica [48]. The experiment was set up for four stress treatment times, and three plants with uniform growth were selected as biological replicates for each stress group. The root soil of uniformly growing I. indigotica plants was watered with 200 mL of NaHCO3 alkaline solution. The 0 h (CK) group was watered with the same volume of sterile water for the same duration of stress. All plants were sampled at 24 h, 72 h, and 120 h after the start of treatment under the same cultivation and management conditions. The samples were frozen in liquid nitrogen immediately after collection and stored in a refrigerator at −80 °C.

4.2. Identification and Physicochemical Properties of IiSOD Gene in I. indigotica

The I. indigotica genome was characterized by two methods, protein blast (BlastP) and Hidden Markov Model (HMM) [49]. I. indigotica genome and annotation files were obtained from the Figshare database (Available online: https://figshare.com/) (accessed on 4 January 2025) [2,22]. The amino acid sequences of the eight AtSODs were used as the query sequences, and the E-value was set to 1 × 10−5 to search for potential IiSOD candidate genes. The amino acid sequences of the AtSODs were obtained from the A. thaliana genome database TAIR (Available online: http://www.arabidopsis.org/ (accessed on 10 January 2025)). The HMM spectra of Cu/Zn-SOD (PF00080) and Fe/Mn-SOD (PF02777 and PF00081) were downloaded from the InterPro database. Then, the IiSOD protein database was scanned using TBtools v2.210 software’s HMM search [50].
The MEME website (Available online: https://meme-suite.org/meme/db/motifs (accessed on 16 January 2025)) parameter is set to 12, and the rest of the default parameter values are selected to identify the conserved motifs in the IiSOD protein [51]. TBtools v2.210 software was used to visualize the conserved motifs of the IiSOD gene. The physical and chemical properties of IiSOD protein, such as molecular weight and isoelectric point (pI), were calculated using the ExPASy online website (Available online: https://web.expasy.org/compute_pi/ (accessed on 20 January 2025)) [52]. In addition, WoLF PSORT (Available online: https://www.genscript.com/tools/wolf-psort (accessed on 2 March 2025)) was used to predict the subcellular localization of IiSOD protein [53]. Signal peptides of IiSODs protein were predicted by the SignalP 5.0 website (Available online: https://services.healthtech.dtu.dk/services/SignalP-5.0/ (accessed on 8 March 2025)). The transmembrane structural domains of IiSOD proteins were predicted by the online TMHMM-2.0 website (Available online: https://services.healthtech.dtu.dk/services/TMHMM-2.0/ (accessed on 16 March 2025)).

4.3. Phylogenetic and Collinearity Analysis of IiSOD Protein

To observe the evolutionary relationship of the IiSOD gene family, the phylogenetic tree of I. indigotica and A. thaliana protein sequences was constructed. MEGA 11 software was used for sequence alignment, and IiSOD protein sequences were selected by the neighbor-joining (NJ) algorithm to build a rootless phylogenetic tree. The phylogenetic relationship between I. indigotica and A. thaliana SOD genes was studied. Parameter settings include the model being p-distance, the missing data method being partial deletion, the proportion being 50%, Bootstrap is set to 1000, and other parameters being default parameters [54]. The phylogenetic tree was beautified using the Evolview website (Available online: https://www.evolgenius.info/evolview/ (accessed on 22 March 2025)) online tool. The IiSOD protein position was extracted from the GFF3 annotation file of the genome of I. indigotica, and then the intraspecific collinear visualization was performed using the Circos of TBtools v2.210 v2.0 software.

4.4. Analysis of Cis-Acting Elements in IiSOD Promoter

Analysis of cis-elements in the IiSOD promoter helps understand information on gene expression regulation. TBtools v2.210 software extracted the gene structure information from the GFF3 gene annotation file of I. indigotica, and the sequence 2000 bp upstream of the IiSOD gene was further extracted [55]. The PlantCARE website (Available online: http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 28 March 2025)) was used to predict cis-acting elements, and the expected results were screened and simplified [56]. Finally, the results are visualized by TBtools v2.210 software.

4.5. RNA Extraction and qRT-PCR Analysis of I. Indigotica

Total RNA was extracted from I. indigotica plants under different temporal alkaline stress treatments using a total RNA extraction kit (DP441; Xinjing, Hangzhou, China). To minimize individual differences, only one fully expanded leaf (counting from top to bottom, the 3rd–4th leaves) was taken from each I. indigotica plant in each treatment group and mixed in equal amounts for RNA extraction. The concentration of RNA was then determined using an ultra-micro UV spectrophotometer (Eppendorf, Hamburg, Germany). Subsequently, the extracted I. indigotica RNA was reversely transcribed into I. indigotica cDNA using the Vazyme reverse transcriptase kit (Novozan, Nanjing, China).
The qRT-PCR detected the expression pattern of the IiSOD gene under alkaline stress. The qPCR primers were designed using the Primer 3 website [57], and the EF1-α gene was used as a reference gene. The gene sequences and primer sequences used are detailed in Supplementary Table S2. The qRT-PCR analysis was performed using a fluorescence quantitative PCR instrument (Agilent Technologies, Santa Clara, CA, USA). The specific procedures were as follows: 95 °C 3 min, followed by 40 cycles of primer annealing/extension, 95 °C 30 s, 60 °C 10 s and 95 °C 30 s, and finally one cycle of 95 °C 30 s, 65 °C 30 s and 95 °C 30 s. Three biological replicates were performed for each sample, and three technical replicates were used for each replicate. The relative expression of IiSOD genes was calculated using the 2−ΔΔCt method [58]. Expression levels of IiSOD genes were plotted using GraphPad. Prism. 8 software.

4.6. Phenotypes and Superoxide Dismutase Activity in Alkaline Stress

I.indigotica was stressed using an alkaline solution of 200 mmol/L NaHCO3. In each treatment group, 200 mL of NaHCO3 solution was poured on the I. indigotica root soil, while the same volume of sterile water was used for the blank group. Changes in phenology of I. indigotica plants regarding plant height, color, volume, degree of wilting, and fresh weight were recorded under the same cultivation and management conditions at 0 h (CK), 24 h, 72 h, and 120 h alkaline treatments. Photographs were taken in each treatment group at the time of sampling. The I. indigotica were frozen in liquid nitrogen and stored at −80 °C.
SOD enzyme activity assay kit (G0101W, Suzhou Greus Biotechnology Co., Ltd., Suzhou, China) was used to determine the activity of SOD enzyme in I. indigotica tissue samples by the WST-8 method. Weighed 0.1 g of I. indigotica tissue under different alkaline stress treatments, homogenized with the extract provided by the 1 mL kit under ice bath conditions, homogenized and centrifuged at 4 °C × 12,000 rpm for 10 min, and the supernatant was taken as the test solution. According to the kit’s instructions, the required reagent was added to a 96-well detachable microtiter plate (12 wells, eight strips) in proportion. After incubating for 30 min at room temperature (25 °C) under light-protected conditions, the absorbance was measured at a wavelength of 405 nm using a light-absorbing enzyme-labeled instrument (VersaMax, Beijing Yuechangxing Technology Co., Ltd., Beijing, China). Three repeated samples were measured for each treatment after calculating the SOD activity in the tissue samples of I. indigotica, using GraphPad.Prism 8 software was used for visualization. SOD is the first line of defense in the plant antioxidant system. It relies on metal cofactors (Cu2+/Zn2+, Mn3+, or Fe3+) to catalyze the dismutation reaction of superoxide anions (O2−). The reaction can be expressed as [59]:
2 O 2 · − + 2 H +   →   H 2 O 2 + O 2
Through this reaction, SOD rapidly converts highly toxic O2− into H2O2 and releases O2, thereby effectively alleviating the oxidative stress caused by ROS in cells.

4.7. Effects of Alkaline Stress on the Accumulation of Indole Alkaloids in I. indigotica

Indole alkaloids are secondary metabolites produced during plant growth and have essential ecological functions in adapting to environmental stress. Indole alkaloids are mainly enriched in the leaves of I. indigotica, including indigo, indirubin, tryptanthrin, etc. [42]. According to the 2020 edition of the Chinese pharmacopeia, indigo and indirubin are the quality control components of I. indigotica Fort. Indigo and indirubin can be natural antioxidants in the food or pharmaceutical industry [60]. Changes in the content of indigo and indirubin in I. indigotica under alkaline stress treatments (0 h (CK), 24 h, 72 h, 120 h) were detected using HPLC. The chromatograph was a Thermo U3000 series, and the column was an Agilent C18 (250 mm × 4.6 mm, five μm particle size). Indigo and indirubin control products were purchased from Sichuan Vicki Biotechnology Co., Ltd. (Chengdu, China), and about 0.5 g of powdered I. indigotica leaves from the alkaline stress treatment groups were precisely weighed and dissolved in 10 mL of N, N-dimethylformamide solution. Vortex shaking for about 5 min, ultrasonic extraction for 45 min (power 300 W, frequency 80 Hz), cooled to room temperature, and N, N-dimethylformamide was added to the scale. After shaking well, the test solution was filtered through a 0.45 μm organic microporous filter membrane to determine the test solution. The elution procedure was based on a methanol (A)-ultrapure water (B) gradient as the mobile phase [61]. Isocratic elution (0–20 min, 75:25); Flow rate: 1.0 mL/min; Detection wavelength: 290 nm; Column temperature: 25 °C; Injection volume: 10 μL [62].

4.8. Yeast One-Hybrid Assay

Yeast One-Hybrid (Y1H) is a typical method for detecting protein–DNA interactions [63]. WRKY transcription factors (TFs) widely regulate plant biotic and abiotic stress responses, growth, and development [64,65]. WRKY transcription factors specifically bind to the cis-acting element W-box (TTGACT/C) in the promoter region of downstream target genes to activate or inhibit the expression of downstream target genes [66,67]. AtWRKY45 plays a vital role in response to saline–alkaline and osmotic stress [68]. When constructing a phylogenetic tree, IiWRKY54 with a strong genetic relationship with AtWRKY45 was selected [69,70], which indicated that IiWRKY54 may be involved in the regulation of saline–alkaline stress response of I. indigotica. According to qRT-PCR analysis, IiSOD genes with high expression under alkaline stress were screened, and the PlantCARE website screened IiSOD2 and IiSOD7 genes with W-box cis-acting elements. It was found that IiSOD2 and IiSOD7 have both W-box cis-acting elements and high expression under alkaline stress. Therefore, IiSOD2, IiSOD7, and IiWRKY54 were selected for yeast experimental verification.
The recombinant plasmid was constructed using homologous recombination, and homology arm primers were designed using the Tiangen Seamless Cloning Primer Online Tool (Available online: http://yw.tiangen.com/) based on the CDS sequence of the IiWRKY54 transcription factor and the pJG4-5 vector sequence 20 bp above and below the EcoRI restriction site. The W-box fragment sequences of the promoter regions of the two IiSOD genes and the pLacZi vector sequence of 20 bp up and down the EcoRI restriction site were used to design homologous arm primers. The primer sequences for this experiment are detailed in Supplementary Table S3. After obtaining the target gene, the recombinant vector was constructed by the constant temperature one-step method, and the two IiSOD target genes were ligated with the linearized pLacZi vector. The IiWRKY54 gene was ligated to the linearized pJG4-5 vector (pLacZi and JG4-5 had Amp resistance from Coolaber, Beijing, China). The bait vector constructed by pLacZi-IiSOD2 and pLacZi-IiSOD7, and the prey vector constructed by pJG4-5-IiWRKY54 were co-transformed into the EGY48 yeast strain (purchased from Shanghai Weidi Company (Shanghai, China)). No-load pLacZi-no-load pJG4-5, no-load pJG4-5-pLacZ-IiSOD2, no-load pJG4-5-pLacZ-IiSOD7, and pJG4-5-IiWRKY54-no-load pLacZi were transformed into the EGY48 yeast strain as negative controls to exclude false negative results. The transformed yeast was plated on SD/-Trp-Ura-Broth medium and cultured at 30 °C for 3-5 days. The positive bacteria were selected and cultured in SD/-Trp-Ura-Broth medium, and the plate was coated with chromogenic medium and cultured at 30 °C for 1–3 days. The interaction results were determined by observing the color change in the colony.

4.9. Statistical Analysis

One-way ANOVA (Tukey’s test) was used to determine significant differences between the groups in qRT-PCR, superoxide dismutase activity, and indigo and indirubin content. Three replications were performed for both control and treatment groups.

5. Conclusions

In this study, the SOD gene family of I. indigotica was systematically analyzed, and 9 IiSOD members were identified. Based on the conserved domains, these genes were classified into three subfamilies: Cu/Zn-SOD, Fe-SOD, and Mn-SOD, which were unevenly distributed across seven chromosomes. Under alkaline stress, the SOD enzyme activity of I. indigotica was significantly increased, indicating that SOD plays a vital role in antioxidant defense. The combination of qRT-PCR and phylogenetic analyses revealed that members of the Fe-SOD and Mn-SOD subfamilies responded particularly well to alkaline stress. Alkaline stress showed significant and dynamic changes in the content of indigo and indirubin in leaves of I. indigotica, with the accumulation levels decreasing and increasing with stress duration.
The transcription factor IiWRKY54 regulates the transcriptional activation of IiSOD2 and IiSOD7; among them, the expression level of IiSOD7 was significantly up-regulated under alkaline stress. In this study, the biological information of the IiSOD gene family was systematically identified, which laid a foundation for an in-depth analysis of the molecular mechanism of the response of the IiSOD gene to saline–alkaline conditions. Studies have shown that in addition to improving the stress resistance of plants, the regulation of SOD activity also plays a positive role in the active ingredients of plants. In the future, the use of homologous or heterologous expression systems combined with gene editing methods to further verify the function of these genes will provide direction for plant breeding and stress resistance.

Supplementary Materials

The supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms26178131/s1.

Author Contributions

Conceptualization, Data curation, Writing—original draft, L.M.; Conceptualization, Data curation, Investigation, L.K.; Data curation, Investigation, S.J.; Data curation, Investigation, Software, J.M.; Data curation, Investigation, L.H.; Data curation, Methodology, Validation, J.W.; Data curation, Investigation, Validation, X.Z.; Data curation, Investigation, Validation, W.W.; Conceptualization, Funding acquisition, W.M.; Investigation, Methodology, Validation, Visualization, W.R. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Project name: National Key Research and development Project, research and demonstration of collection, screening and breeding technology of ginseng and other genuine medicinal materials, Project No. 2021YFD1600901, Heilongjiang Touyan Innovation Team Program (Grant Number: [2019] No. 5), Heilongjiang Province “double first-class” discipline collaborative innovation achievement project: Quality optimization and deep processing of characteristic Chinese medicinal materials in the lower cold region of great health industry, Project No. LJGXCG2023-058.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Wong, L.W.; Goh, C.B.S.; Tan, J.B.L. A Systemic Review for Ethnopharmacological Studies on Isatis indigotica Fortune: Bioactive Compounds and their Therapeutic Insights. Am. J. Chin. Med. 2022, 50, 161–207. [Google Scholar] [CrossRef]
  2. Jiang, M.; Wang, Z.; Ren, W.; Yan, S.; Xing, N.; Zhang, Z.; Li, H.; Ma, W. Identification of the bZIP gene family and regulation of metabolites under salt stress in isatis indigotica. Front. Plant Sci. 2022, 13, 1011616. [Google Scholar] [CrossRef] [PubMed]
  3. Feng, J.; Huang, D.; Yang, Y.; Chen, J.; Qiu, S.; Lv, Z.; Ma, X.; Li, Y.; Li, R.; Xiao, Y.; et al. Isatis indigotica: From (ethno) botany, biochemistry to synthetic biology. Mol. Hortic. 2021, 1, 17. [Google Scholar] [CrossRef] [PubMed]
  4. Wang, Y.H.; Jin, Y.; Chen, Z.Q.; Chen, S.B.; Zhan, Z.L. Textual research on medicinal blue herbs. China J. Chin. Mater. Medica 2020, 45, 5819–5828. [Google Scholar]
  5. Miller, A.F. Superoxide dismutases: Ancient enzymes and new insights. FEBS Lett. 2012, 586, 585–595. [Google Scholar] [CrossRef]
  6. Shi, D.; Sheng, Y. Effect of various salt–alkaline mixed stress conditions on sunflower seedlings and analysis of their stress factors. Environ. Exp. Bot. 2005, 54, 8–21. [Google Scholar] [CrossRef]
  7. Gong, R.; XI, X.; Zhang, C.-y. Alkali stress tolerance analysis of four Rhododendron cultivars. J. Nanjing. For. Univ. (Nat. Sci. Ed.) 2024, 48, 113–120. [Google Scholar]
  8. Jiang, Y.; Zeng, Q.; Wei, J.; Jiang, J.; Li, Y.; Chen, J.; Yu, H. Growth, Fruit Yield, Photosynthetic Characteristics, and Leaf Microelement Concentration of Two Blueberry Cultivars under Different Long-Term Soil pH Treatments. Agronomy 2019, 9, 357. [Google Scholar] [CrossRef]
  9. Wang, H.; Wu, Z.; Han, J.; Zheng, W.; Yang, C. Comparison of ion balance and nitrogen metabolism in old and young leaves of alkali-stressed rice plants. PLoS ONE 2012, 7, e37817. [Google Scholar] [CrossRef]
  10. Lu, W.; Duanmu, H.; Qiao, Y.; Jin, X.; Yu, Y.; Yu, L.; Chen, C. Genome-wide identification and characterization of the soybean SOD family during alkaline stress. PeerJ 2020, 8, e8457. [Google Scholar] [CrossRef]
  11. Yu, W.; Kong, G.; Chao, J.; Yin, T.; Tian, H.; Ya, H.; He, L.; Zhang, H. Genome-wide identification of the rubber tree superoxide dismutase (SOD) gene family and analysis of its expression under abiotic stress. PeerJ 2022, 10, e14251. [Google Scholar] [CrossRef] [PubMed]
  12. Nadarajah, K.K. ROS Homeostasis in Abiotic Stress Tolerance in Plants. Int. J. Mol. Sci. 2020, 21, 5208. [Google Scholar] [CrossRef] [PubMed]
  13. Knipfer, T.; Besse, M.; Verdeil, J.L.; Fricke, W. Aquaporin-facilitated water uptake in barley (Hordeum vulgare L.) roots. J. Exp. Bot. 2011, 62, 4115–4126. [Google Scholar] [CrossRef] [PubMed]
  14. Zorov, D.B.; Juhaszova, M.; Sollott, S.J. Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiol. Rev. 2014, 94, 909–950. [Google Scholar] [CrossRef]
  15. Zelko, I.N.; Mariani, T.J.; Folz, R.J. Superoxide dismutase multigene family: A comparison of the CuZn-SOD (SOD1), Mn-SOD (SOD2), and EC-SOD (SOD3) gene structures, evolution, and expression. Free Radic. Biol. Med. 2002, 33, 337–349. [Google Scholar] [CrossRef]
  16. Dupont, C.L.; Neupane, K.; Shearer, J.; Palenik, B. Diversity, function and evolution of genes coding for putative Ni-containing superoxide dismutases. Environ. Microbiol. 2008, 10, 1831–1843. [Google Scholar] [CrossRef]
  17. Gill, S.S.; Tuteja, N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant. Physiol. Biochem. 2010, 48, 909–930. [Google Scholar] [CrossRef]
  18. Wang, X.; Liu, X.; Zhou, Y.; Zhang, G. Molecular mechanism of expression and regulation of SOD gene in plant. Chin. J. Oil Crop Sci. 2014, 36, 275–280. [Google Scholar]
  19. Su, W.; Raza, A.; Gao, A.; Jia, Z.; Zhang, Y.; Hussain, M.A.; Mehmood, S.S.; Cheng, Y.; Lv, Y.; Zou, X. Genome-Wide Analysis and Expression Profile of Superoxide Dismutase (SOD) Gene Family in Rapeseed (Brassica napus L.) under Different Hormones and Abiotic Stress Conditions. Antioxidants 2021, 10, 1182. [Google Scholar] [CrossRef]
  20. Song, J.; Zeng, L.; Chen, R.; Wang, Y.; Zhou, Y. In silico identification and expression analysis of superoxide dismutase (SOD) gene family in Medicago truncatula. 3 Biotech 2018, 8, 348. [Google Scholar] [CrossRef]
  21. Perry, J.J.; Shin, D.S.; Getzoff, E.D.; Tainer, J.A. The structural biochemistry of the superoxide dismutases. Biochim. Biophys. Acta 2010, 1804, 245–262. [Google Scholar] [CrossRef]
  22. Kang, M.; Wu, H.; Yang, Q.; Huang, L.; Hu, Q.; Ma, T.; Li, Z.; Liu, J. A chromosome-scale genome assembly of Isatis indigotica, an important medicinal plant used in traditional Chinese medicine. Hortic. Res. 2020, 7, 18. [Google Scholar] [CrossRef]
  23. Wang, Z.; Ma, W. Genome-wide identification of the Trihelix gene family and abiotic expression pattern analysis in Isatis indigotica Fort. of traditional Chinese medicine satidis radix and isatidis folium. China J. Tradit. Chin. Med. Pharm. 2023, 38, 2109–2115. [Google Scholar]
  24. Ma, J.; Ren, W.; Jiang, S.; Kong, L.; Ma, L.; He, J.; Wang, D.; Liu, W.; Ma, W.; Liu, X. Identification and expression analysis of the RBOH gene family of Isatis indigotica Fort. and the potential regulation mechanism of RBOH gene on H2O2 under salt stress. Plant Cell Rep. 2025, 44, 52. [Google Scholar] [CrossRef]
  25. Chen, Y.; Wu, H.; Hao, Z.; Zhu, L.; Lu, L.; Shi, J.; Chen, J. The Identification and Expression Analysis of the Liriodendron chinense (Hemsl.) Sarg. SOD Gene Family. Forests 2023, 14, 628. [Google Scholar] [CrossRef]
  26. Tounsi, S.; Jemli, S.; Feki, K.; Brini, F.; Najib Saidi, M. Superoxide dismutase (SOD) family in durum wheat: Promising candidates for improving crop resilience. Protoplasma. 2023, 260, 145–158. [Google Scholar] [CrossRef] [PubMed]
  27. Zheng, L.; Assane Hamidou, A.; Zhao, X.; Ouyang, Z.; Lin, H.; Li, J.; Zhang, X.; Luo, K.; Chen, Y. Superoxide dismutase gene family in cassava revealed their involvement in environmental stress via genome-wide analysis. iScience 2023, 26, 107801. [Google Scholar] [CrossRef]
  28. Hou, R.; Wang, Z.; Zhu, Q.; Wang, J.; Zhou, Y.; Li, Y.; Liu, H.; Zhao, Q.; Huang, J. Identification and characterization of the critical genes encoding Cd-induced enhancement of SOD isozymes activities in Zhe-Maidong (Ophiopogon japonicus). Front. Plant Sci. 2024, 15, 1355849. [Google Scholar] [CrossRef]
  29. Jin, W.; Yuanxin, D.; Anbo, W. Identification and Expression Pattern Analysis of Rosa roxburghii SOD Gene Family. Biotechnol. Bull. 2024, 153–166. [Google Scholar] [CrossRef]
  30. Duan, W.; Xue, B.; He, Y.; Liao, S.; Li, X.; Li, X.; Liang, Y.K. Genome-Wide Identification and Expression Pattern Analysis of Dirigent Members in the Genus Oryza. Int. J. Mol. Sci. 2023, 24, 7189. [Google Scholar] [CrossRef]
  31. Han, L.M.; Hua, W.P.; Cao, X.Y.; Yan, J.A.; Chen, C.; Wang, Z.Z. Genome-wide identification and expression analysis of the superoxide dismutase (SOD) gene family in Salvia miltiorrhiza. Gene 2020, 742, 144603. [Google Scholar] [CrossRef] [PubMed]
  32. Wang, W.; Zhang, X.; Deng, F.; Yuan, R.; Shen, F. Genome-wide characterization and expression analyses of superoxide dismutase (SOD) genes in Gossypium hirsutum. BMC Genom. 2017, 18, 376. [Google Scholar] [CrossRef]
  33. Liu, Z.; Guo, Y.; Li, H.; Zhang, J.; Dong, Y.; Hu, C.; Long, J.; Chen, Y. The superoxide dismutase (SOD) gene family in perennial ryegrass: Characterization and roles in heat stress tolerance. Plant Physiol. Biochem. 2025, 226, 110061. [Google Scholar] [CrossRef] [PubMed]
  34. Chen, X.; Li, D.; Guo, J.; Wang, Q.; Zhang, K.; Wang, X.; Shao, L.; Luo, C.; Xia, Y.; Zhang, J. Identification and Analysis of the Superoxide Dismutase (SOD) Gene Family and Potential Roles in High-Temperature Stress Response of Herbaceous Peony (Paeonia lactiflora Pall.). Antioxidants 2024, 13, 1128. [Google Scholar] [CrossRef]
  35. Cui, Y.; Cao, Q.; Li, Y.; He, M.; Liu, X. Advances in cis-element- and natural variation-mediated transcriptional regulation and applications in gene editing of major crops. J. Exp. Bot. 2023, 74, 5441–5457. [Google Scholar] [CrossRef]
  36. Ezzat, A.; Szabo, S.; Szabo, Z.; Hegedus, A.; Berenyi, D.; Holb, I.J. Temporal Patterns and Inter-Correlations among Physical and Antioxidant Attributes and Enzyme Activities of Apricot Fruit Inoculated with Monilinia laxa under Salicylic Acid and Methyl Jasmonate Treatments under Shelf-Life Conditions. J. Fungi 2021, 7, 341. [Google Scholar] [CrossRef] [PubMed]
  37. Li, C.; Yu, W.; Xu, J.; Lu, X.; Liu, Y. Anthocyanin Biosynthesis Induced by MYB Transcription Factors in Plants. Int. J. Mol. Sci. 2022, 23, 11701. [Google Scholar] [CrossRef]
  38. Warzecha, H.; Frank, A.; Peer, M.; Gillam, E.M.J.; Guengerich, F.P.; Unger, M. Formation of the indigo precursor indican in genetically engineered tobacco plants and cell cultures. Plant Biotechnol. J. 2006, 5, 185–191. [Google Scholar] [CrossRef]
  39. Fu, H.; Liu, X.; Song, S.; Lv, W.; Yang, Y. Mechanisms of secondary metabolites regulating plant resistance to salinity and alkali stress. Plant. Physiol. J. 2023, 59, 727–740. [Google Scholar] [CrossRef]
  40. Yang, Y.; Guo, Y. Elucidating the molecular mechanisms mediating plant salt-stress responses. New. Phytol. 2017, 217, 523–539. [Google Scholar] [CrossRef]
  41. Fang, S.; Hou, X.; Liang, X. Response Mechanisms of Plants Under Saline-Alkali Stress. Front. Plant. Sci. 2021, 12, 667458. [Google Scholar] [CrossRef] [PubMed]
  42. Li, Q.; Chen, W.; Wang, Y.; Chen, J.; Zhang, L.; Zhang, Q. Comprehensive transcriptomic profiling of <italic>Isatis indigotica</italic> leaves reveals biosynthesis of indole active ingredients. Sci. Sin. Vitae 2018, 48, 412–422. [Google Scholar] [CrossRef]
  43. Jia, H.L.; Liu, L.Y.; Liu, H.X. Effect of NaHS on the expression of related genes in isatis indigotica alkaoid synthesis pathway. J. Shaanxi. Univ. Sci. Technol. 2020, 5, 52–57. [Google Scholar] [CrossRef]
  44. Wang, Z.; Cao, B.; Du, J.; Deng, T.; Wang, R.; Chen, Y.; Li, X.; Fu, J.; Pang, J.; Yang, M.; et al. Transcriptomic and proteomic-based analysis of the mechanisms by which drought and salt stresses affect the quality of Isatidis Folium. BMC Plant. Biol. 2025, 25, 332. [Google Scholar] [CrossRef]
  45. Li, J.H.; Liu, Y.; Han, M.; Wang, Z.; Lu, X.; Yang, C. Soil properties and enzyme activities varied with seasonal precipitation during vegetation succession in saline-alkali grassland in songnen plain. Chin. J. Soil Sci. 2025, 56, 201–208. [Google Scholar] [CrossRef]
  46. Wang, Z. Study on the Distribution and Spatial Change of SALINE Alkali Land in Songnen Plain. Master’s Thesis, Harbin Normal University, Harbin, China, 2020. [Google Scholar]
  47. Yu, H.; Wang, Z.; Mao, D.; Jia, M.; Chang, S.; Li, X. Spatiotemporal variations of soil salinization in China’s West Songnen Plain. Land Degrad. Dev. 2023, 34, 2366–2378. [Google Scholar] [CrossRef]
  48. He, J.; Kong, L.; Wang, P.; He, L.; Cao, H.; Qin, C.; Ren, W.; Liu, X.; Ma, W. Identification and expression analysis of NAC family genes in Eleutherococcus senticosus under salt stress. Mol. Biol. Rep. 2025, 52, 729. [Google Scholar] [CrossRef]
  49. Raza, A.; Su, W.; Gao, A.; Mehmood, S.S.; Hussain, M.A.; Nie, W.; Lv, Y.; Zou, X.; Zhang, X. Catalase (CAT) Gene Family in Rapeseed (Brassica napus L.): Genome-Wide Analysis, Identification, and Expression Pattern in Response to Multiple Hormones and Abiotic Stress Conditions. Int. J. Mol. Sci. 2021, 22, 4281. [Google Scholar] [CrossRef]
  50. Potter, S.C.; Luciani, A.; Eddy, S.R.; Park, Y.; Lopez, R.; Finn, R.D. HMMER web server: 2018 update. Nucleic Acids Res. 2018, 46, W200–W204. [Google Scholar] [CrossRef]
  51. Bailey, T.L.; Boden, M.; Buske, F.A.; Frith, M.; Grant, C.E.; Clementi, L.; Ren, J.; Li, W.W.; Noble, W.S. MEME SUITE: Tools for motif discovery and searching. Nucleic Acids Res. 2009, 37, W202–W208. [Google Scholar] [CrossRef]
  52. Hu, B.; Jin, J.; Guo, A.Y.; Zhang, H.; Luo, J.; Gao, G. GSDS 2.0: An upgraded gene feature visualization server. Bioinformatics 2015, 31, 1296–1297. [Google Scholar] [CrossRef]
  53. Chou, K.C.; Shen, H.B. Cell-PLoc: A package of Web servers for predicting subcellular localization of proteins in various organisms. Nat. Protoc. 2008, 3, 153–162. [Google Scholar] [CrossRef]
  54. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [Google Scholar] [CrossRef]
  55. Chen, C.; Wu, Y.; Li, J.; Wang, X.; Zeng, Z.; Xu, J.; Liu, Y.; Feng, J.; Chen, H.; He, Y.; et al. TBtools-II: A “one for all, all for one” bioinformatics platform for biological big-data mining. Mol. Plant 2023, 16, 1733–1742. [Google Scholar] [CrossRef] [PubMed]
  56. Lescot, M.; Dehais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouze, P.; Rombauts, S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002, 30, 325–327. [Google Scholar] [CrossRef] [PubMed]
  57. Ye, J.; Coulouris, G.; Zaretskaya, I.; Cutcutache, I.; Rozen, S.; Madden, T.L. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinform. 2012, 13, 134. [Google Scholar] [CrossRef]
  58. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
  59. Qu, K.; Wang, J.; Cheng, Y.; Bai, B.; Xia, X.; Geng, H. Identification of quantitative trait loci and candidate genes for grain superoxide dismutase activity in wheat. BMC Plant Biol. 2024, 24, 716. [Google Scholar] [CrossRef]
  60. Zhao, G.; Li, T.; Qu, X.; Zhang, N.; Lu, M.; Wang, J. Optimization of ultrasound-assisted extraction of indigo and indirubin from Isatis indigotica Fort. and their antioxidant capacities. Food Sci. Biotechnol. 2017, 26, 1313–1323. [Google Scholar] [CrossRef]
  61. Xiao, Y.; Xia, Y.; Cheng, P.; Fang, Y.; Da, G.; Huang, J.; Zhang, X. Reversed-phase high-performance liquid chromatography was used to simultaneously determine the contents of indigo and indigo red in large green leaves. Her. Med. 2015, 34, 1506–1508. [Google Scholar] [CrossRef]
  62. Liu, D.; Wang, C. Research progress on determination methods of plant indigo extract. BiomassChem. Eng. 2023, 57, 66–72. [Google Scholar]
  63. Li, Y.; Zhang, J.; Wang, S.; Liu, Y.; Yang, M.; Huang, Y. Genome-wide identification of the Pyrus R2R3-MYB gene family and PhMYB62 regulation analysis in Pyrus hopeiensis flowers at low temperature. Int. J. Biol. Macromol. 2024, 257, 128611. [Google Scholar] [CrossRef]
  64. Bjornson, M.; Pimprikar, P.; Nurnberger, T.; Zipfel, C. The transcriptional landscape of Arabidopsis thaliana pattern-triggered immunity. Nat. Plants 2021, 7, 579–586. [Google Scholar] [CrossRef]
  65. Jiang, J.; Ma, S.; Ye, N.; Jiang, M.; Cao, J.; Zhang, J. WRKY transcription factors in plant responses to stresses. J. Integr. Plant Biol. 2017, 59, 86–101. [Google Scholar] [CrossRef]
  66. Dai, L.; Xu, Y.; Du, Z.; Su, X.D.; Yu, J. Revealing atomic-scale molecular diffusion of a plant-transcription factor WRKY domain protein along DNA. Proc. Natl. Acad. Sci. USA 2021, 118, e2102621118. [Google Scholar] [CrossRef]
  67. Cheng, Z.; Luan, Y.; Meng, J.; Sun, J.; Tao, J.; Zhao, D. WRKY Transcription Factor Response to High-Temperature Stress. Plants 2021, 10, 2211. [Google Scholar] [CrossRef] [PubMed]
  68. Zhou, X.; Sun, Z.; Huang, Y.; He, D.; Lu, L.; Wei, M.; Lin, S.; Luo, W.; Liao, X.; Jin, S.; et al. WRKY45 positively regulates salinity and osmotic stress responses in Arabidopsis. Plant Physiol. Biochem. 2025, 219, 109408. [Google Scholar] [CrossRef] [PubMed]
  69. Qu, R.; Cao, Y.; Tang, X.; Sun, L.; Wei, L.; Wang, K. Identification and expression analysis of the WRKY gene family in Isatis indigotica. Gene 2021, 783, 145561. [Google Scholar] [CrossRef]
  70. Zhang, L.; Wu, D.; Zhang, W.; Shu, H.; Sun, P.; Huang, C.; Deng, Q.; Wang, Z.; Cheng, S. Genome-Wide Identification of WRKY Gene Family and Functional Characterization of CcWRKY25 in Capsicum chinense. Int. J. Mol. Sci. 2023, 24, 11389. [Google Scholar] [CrossRef] [PubMed]
Ijms 26 08131 g001
Figure 1. SODs phylogenetic tree of I. indigotica, A. thaliana, B. oleracea, and V. vinifera, 40 SOD proteins were constructed using the neighbor-joining (NJ) method. The SOD protein is divided into I Cu/Zn-SODs, II Fe-SODs, and III Mn-SODs. The proteins of the I. indigotica are labeled with red, and various colors and shapes distinguish different varieties of SODs.
Figure 1. SODs phylogenetic tree of I. indigotica, A. thaliana, B. oleracea, and V. vinifera, 40 SOD proteins were constructed using the neighbor-joining (NJ) method. The SOD protein is divided into I Cu/Zn-SODs, II Fe-SODs, and III Mn-SODs. The proteins of the I. indigotica are labeled with red, and various colors and shapes distinguish different varieties of SODs.
Ijms 26 08131 g001
Ijms 26 08131 g002
Figure 2. Collinearity analysis and chromosomal localization of IiSODs gene. (A) Interspecific collinearity analysis of the IiSODs gene. The gray background shows all the same linear segments in the genome of I. indigotica. The blue line shows the identical linear IiSOD gene pairs. (B) The number of chromosomes of the IiSODs gene are shown on the left side of each chromosome. The respective genes were marked on the right side of the chromosome.
Figure 2. Collinearity analysis and chromosomal localization of IiSODs gene. (A) Interspecific collinearity analysis of the IiSODs gene. The gray background shows all the same linear segments in the genome of I. indigotica. The blue line shows the identical linear IiSOD gene pairs. (B) The number of chromosomes of the IiSODs gene are shown on the left side of each chromosome. The respective genes were marked on the right side of the chromosome.
Ijms 26 08131 g002
Ijms 26 08131 g003
Figure 3. Phylogenetic tree, conserved motifs, and domain analysis of IiSOD. (A) Neighbor-joining tree of IiSODs protein. (B) IiSOD conserved motif analysis. (C) Conserved structure domains of IiSOD. The blue, yellow, and green rectangles indicate the SOD_Cu structural domain (PF00080), SOD_Fe_N structural domain (PF00081), and SOD_Fe_C structural domain (PF02777), respectively.
Figure 3. Phylogenetic tree, conserved motifs, and domain analysis of IiSOD. (A) Neighbor-joining tree of IiSODs protein. (B) IiSOD conserved motif analysis. (C) Conserved structure domains of IiSOD. The blue, yellow, and green rectangles indicate the SOD_Cu structural domain (PF00080), SOD_Fe_N structural domain (PF00081), and SOD_Fe_C structural domain (PF02777), respectively.
Ijms 26 08131 g003
Ijms 26 08131 g004
Figure 4. The cis-regulatory elements in the promoter region of the IiSOD gene family. Different colors in the IiSODs promoter region indicate cis-elements with various functions.
Figure 4. The cis-regulatory elements in the promoter region of the IiSOD gene family. Different colors in the IiSODs promoter region indicate cis-elements with various functions.
Ijms 26 08131 g004
Ijms 26 08131 g005
Figure 5. Expression level of the IiSOD gene under alkaline stress. Data are means (mean ± SD) of three replicates. The significance of IiSOD gene expression level was analyzed by one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test (* p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.0001).
Figure 5. Expression level of the IiSOD gene under alkaline stress. Data are means (mean ± SD) of three replicates. The significance of IiSOD gene expression level was analyzed by one-way analysis of variance (ANOVA) and Tukey’s multiple comparison test (* p < 0.05, ** p < 0.01, and *** p < 0.001, **** p < 0.0001).
Ijms 26 08131 g005
Ijms 26 08131 g006
Figure 6. Effects of alkaline stress on the phenotype and SOD enzyme activity of I. indigotica. (A) Phenotype of I. indigotica. The plant height of I. indigotica decreased, leaves wilted, and growth was reduced with increasing treatment time. (B) SOD activity. Data are means (mean ± SD) of three replicates. Significance of SOD enzyme activity was analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test (*: p < 0.05, ****: p < 0.0001).
Figure 6. Effects of alkaline stress on the phenotype and SOD enzyme activity of I. indigotica. (A) Phenotype of I. indigotica. The plant height of I. indigotica decreased, leaves wilted, and growth was reduced with increasing treatment time. (B) SOD activity. Data are means (mean ± SD) of three replicates. Significance of SOD enzyme activity was analyzed using one-way ANOVA followed by Tukey’s multiple comparisons test (*: p < 0.05, ****: p < 0.0001).
Ijms 26 08131 g006
Ijms 26 08131 g007
Figure 7. Effect of alkaline stress on indigo and indirubin content in leaves of I. indigotica. (A) Indigo content; (B) indirubin content. Data are means (mean ± SD) of three replicates. One-way ANOVA followed by Tukey’s multiple comparisons test was used for the significance of chemical content in leaves of I. indigotica under alkaline stress (****: p < 0.0001).
Figure 7. Effect of alkaline stress on indigo and indirubin content in leaves of I. indigotica. (A) Indigo content; (B) indirubin content. Data are means (mean ± SD) of three replicates. One-way ANOVA followed by Tukey’s multiple comparisons test was used for the significance of chemical content in leaves of I. indigotica under alkaline stress (****: p < 0.0001).
Ijms 26 08131 g007
Ijms 26 08131 g008
Figure 8. Yeast one-hybrid screening of target genes that bind to IiWRKY54. The colony color of pLacZi-IiSOD2pro or pLacZi-IiSOD7pro and pJG4-5-IiWRKY54 became blue, indicating that the IiWRKY54 transcription factor interacts with IiSOD2 or IiSOD7 genes.
Figure 8. Yeast one-hybrid screening of target genes that bind to IiWRKY54. The colony color of pLacZi-IiSOD2pro or pLacZi-IiSOD7pro and pJG4-5-IiWRKY54 became blue, indicating that the IiWRKY54 transcription factor interacts with IiSOD2 or IiSOD7 genes.
Ijms 26 08131 g008
Table 1. Identification data of IiSOD genes in I. indigotica genome.
Table 1. Identification data of IiSOD genes in I. indigotica genome.
Gene NameGene IDAmino AcidsChromosome
Location		pIMolecular
Weight (Da)		Subcellular LocationPfam
Domain
IiSOD1Iin00748.t1260Chr17.0229,539.48mitochondrionIMA, IMC
IiSOD2Iin05388.t1167Chr26.4817,057.09chloroplastCZ
IiSOD3Iin06861.t1307Chr24.8534,597.74chloroplastIMA, IMC
IiSOD4Iin08997.t1153Chr35.4515,163.77chloroplastCZ
IiSOD5Iin09368.t1323Chr37.6434,325.95chloroplastCZ
IiSOD6Iin15648.t1232Chr48.7425,463.07mitochondrionIMA, IMC
IiSOD7Iin22527.t1257Chr67.7428,582.51chloroplastIMA, IMC
IiSOD8Iin24760.t1182Chr79.4620,410.29mitochondrionIMA, IMC
IiSOD9Iin27319.t1187Chr76.7920,318.94mitochondrionIMA, IMC
pI: isoelectric points; CZ: Copper/zinc superoxide dismutase; IMA: Iron/manganese superoxide dismutases, alpha-hairpin domain; IMC: Iron/manganese superoxide dismutases, C-terminal domain.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Ma, L.; Kong, L.; Jiang, S.; Ma, J.; He, L.; Wu, J.; Zhang, X.; Wu, W.; Ma, W.; Ren, W. Genome-Wide Expression Profile of SOD Gene Family in Isatis indigotica and the Key Role of IiSOD2 and IiSOD7 in Alkaline Stress. Int. J. Mol. Sci. 2025, 26, 8131. https://doi.org/10.3390/ijms26178131

AMA Style

Ma L, Kong L, Jiang S, Ma J, He L, Wu J, Zhang X, Wu W, Ma W, Ren W. Genome-Wide Expression Profile of SOD Gene Family in Isatis indigotica and the Key Role of IiSOD2 and IiSOD7 in Alkaline Stress. International Journal of Molecular Sciences. 2025; 26(17):8131. https://doi.org/10.3390/ijms26178131

Chicago/Turabian Style

Ma, Lengleng, Lingyang Kong, Shan Jiang, Junbai Ma, Lianqing He, Jianhao Wu, Xiaozhuang Zhang, Wei Wu, Wei Ma, and Weichao Ren. 2025. "Genome-Wide Expression Profile of SOD Gene Family in Isatis indigotica and the Key Role of IiSOD2 and IiSOD7 in Alkaline Stress" International Journal of Molecular Sciences 26, no. 17: 8131. https://doi.org/10.3390/ijms26178131

APA Style

Ma, L., Kong, L., Jiang, S., Ma, J., He, L., Wu, J., Zhang, X., Wu, W., Ma, W., & Ren, W. (2025). Genome-Wide Expression Profile of SOD Gene Family in Isatis indigotica and the Key Role of IiSOD2 and IiSOD7 in Alkaline Stress. International Journal of Molecular Sciences, 26(17), 8131. https://doi.org/10.3390/ijms26178131

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop