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Article

Molecular Responses of the NAC Gene Family in Acanthus ebracteatus: Involvement in Abiotic Stress Responses and Biosynthesis of Verproside and Verbascoside

by
Jun Yao
†,
Huiting Fan
†,
Canming Xing
and
Ying Zhang
*
Zhanjiang Key Laboratory of Mangrove Ecosystem Protection and Restoration, Engineering and Technological Research Center in Protection and Utilization of Mangrove Rare and Endangered Species, Lingnan Normal University, Zhanjiang 524048, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2025, 16(8), 1354; https://doi.org/10.3390/f16081354
Submission received: 16 July 2025 / Revised: 18 August 2025 / Accepted: 19 August 2025 / Published: 20 August 2025
(This article belongs to the Special Issue Forests That Heal: Importance of Medicinal Trees and Forest Understory Plants Conservation and Use)
Browse Figures
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Abstract

The NAC gene family, as a group of plant-specific transcription factors, plays crucial roles in plant growth, development, abiotic stress regulation, and biosynthesis of medicinal components. However, research on this family in the medicinal true mangrove Acanthus ebracteatus remains unreported. In this study, 56 NAC genes (AeNAC01-AeNAC56) were identified from A. ebracteatus transcriptome data, all encoding proteins with the NAM domain. Phylogenetic analysis classified them into two groups, with 51 in Group I and 5 in Group II. Comparative transcriptome analysis of roots, leaves, and flowers, validated by qRT-PCR, revealed lower AeNAC genes expression in leaves, with AeNAC10, AeNAC31, and AeNAC48 showing the lowest levels. Under salt, cold, and waterlogging, AeNAC03/44, /48/56 exhibited differential expression, suggesting their key roles in stress responses. Metabolome analysis further demonstrated that AeNAC14 and AeNAC48 significantly correlated with the biosynthesis of verproside and verbascoside, major bioactive phenythanoid glycodides in A. ebracteatus leaves with anti-inflammatory and antioxidant properties. This study provides insights into the A. ebracteatus NAC gene family, identifying candidate targets for understanding the synergistic regulation of abiotic stress responses and medicinal component biosynthesis, which is significant for optimizing the plant’s growth and medicinal value via genetic engineering.

1. Introduction

Medicinal plants are increasingly exposed to abiotic stresses like salt stress, low temperature, and flooding due to global climate change, which severely hinder their growth and development and disrupt ecosystem stability [1]. Delving into the molecular mechanisms that control non-biological stress response is highly important for uncovering plant adaptation strategies, breeding stress-resistant new varieties, and preserving endangered species. Transcription factors are essential for regulating plant stress response. Transcription factors are essential for regulating plant stress response, with the NAC (No apical meristem (NAM), Arabidopsis thaliana transcription activation factor (ATAF), cup-shaped cotyledon (CUC)) gene families being particularly noteworthy due to their versatile roles in plant growth, development, and stress response [2]. These plant-specific transcription factors have been extensively studied in various economic and ecological plants [3]. For example, the identification and expression analysis of NAC family genes in Limonium bicolor elucidated the key role of NAC transcription factors in its response to abiotic stress [4]. Comparative transcriptome analysis revealed that the NAC genes are highly expressed in the inflorescence of Trachyspermum ammi, implicating their involvement in terpenoid biosynthesis [5]. Transgenic validation shows that overexpression of the NAC2 gene in Salvia miltiorrhiza inhibits the synthesis of tanshinone I and tanshinone IIA, but Sm-NAC2 RNAi-mediated silencing significantly promotes the biosynthesis of four tanshinones (tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I) [6]. In Andrographis paniculata, ApNAC1 is predominantly expressed in leaves, and its expression is induced by methyl jasmonate (MeJA), suggesting a role in andrographis biosynthesis [7].
The NAC gene family is widely distributed across plants but varies significantly in plants. Up to now, a large number of NAC genes have been identified from various plants using transcriptome and genome data. For example, the most numerous (410) NAC genes were found in Brassica napus, while only 9 NAC genes were found in Marchantia polymorpha [8]. A total of 68 NAC genes were identified in the genome of Kandelia obovata, while 142 NAC genes were found in the genome of Avicennia marina. Both are true mangroves and are also utilized as medicinal plants. This discovery highlights the significance of NAC family genes in aiding plants in adapting to intertidal environments and surviving low temperatures [9,10]. NAC transcription factors also exhibit organ-specific expression patterns [1]. In the model plant A. thaliana, transcriptome data show that different NAC genes have unique expression profiles in organs such as roots, leaves, and flowers [11]. In Houttuynia cordata, qRT-PCR showed that ApNAC1 was mainly distributed in the leaves, and much higher than in the roots and stems [7]. The transcriptome and qRT-PCR of Liriodendron chinense revealed differential expression of LcNAC genes in different tissues, with 6 genes highly expressed in floral organs, suggesting their roles in reproductive development [12]. Under abiotic stress conditions, NAC genes regulate plant stress adaptation through differential expression. For example, RNA-Seq analysis of soybean leaves revealed significant changes in the expression levels of multiple NAC family members under waterlogging stress [13]. The gene OsNAC3 is induced to express in rice under salt stress, regulating the accumulation of Na+ and related gene expression, thereby affecting the sensitivity of rice to salt stress [14]. In the cold resistance regulation of apples, MdNAC104 not only directly activates MdCBF1 and MdCBF3 in the CBF (C-repeat Binding Factor) dependent pathway, but also activates anthocyanin synthesis and the expression of antioxidant enzyme-related genes through CBF independent pathway [15]. Despite the extensive studies on NAC transcription factors in various plant species, including medicinal plants and mangroves as mentioned above, our knowledge regarding the NAC gene family in A. ebractetus remains notably scarce.
The medicinal mangrove plant A. ebractetus is a true mangrove species belonging to the genus Acanthus L. in the family Acanthaceae, which is widely distributed in Southeast Asia, South Asia, and Australia. In China, it is mainly distributed in mangrove wetlands in Hainan, Guangdong, and Guangxi Provinces [16]. Widely used in traditional Thai medicine to treat inflammation, wound healing, and skin diseases [17,18]. This plant is rich in polyphenolic compounds, alkaloids, and terpenoids [19]. Among them, verbascoside has antioxidant and anti-inflammatory effects [20]. The 2-hydroxy-4 H-[1, 4] benzoxazin-3-one (HBOA) from A. ebractetus has hepatoprotective, anti-inflammatory, analgesic, anti-tumor, and other effects [21]. The latest research has found that HBOA has the functions of promoting wound healing, providing photoprotection, and enhancing fibroblast proliferation and migration; it can also inhibit the expression of MMP-9 induced by ultraviolet radiation [18]. Extracts from plants of A. ebractetus also have anti-aging, anti-inflammatory, and neuroprotective effects [22]. It has an inhibitory effect on tumor cells such as cervical cancer [23]. However, its restricted habitat of A. ebractetus in China [24], coupled with its endangered status [25], is hindering its medicinal development. The systematic identification, classification, or characterization of NAC genes in A. ebractetus at the transcriptome-wide level are expected to be discovered. Further, investigating the NAC gene family’s role in stress response and medicinal component biosynthesis in A. ebractetus is of great scientific importance.
This study identified NAC family genes in A. ebractetus using transcriptome data. Bioinformatics analysis revealed evolutionary and structural characteristics of AeNACs. Expression profiles of AeNACs in roots, leaves, and flowers were analyzed, examining correlations with verproside and verbascoside contents in leaves. qRT-PCR analysis under abiotic stress conditions elucidated expression characteristics and regulatory roles of AeNACs. This provides a basis for molecular breeding to optimize the growth performance of A. ebractetus in the future.

2. Materials and Methods

2.1. RNA Extraction and cDNA Library Preparation

The plant materials (roots, leaves, and flowers) were collected from three healthy and pest-free A. ebractetus plants living in Danxian Port, Lianjiang City, Guangdong Province (21°46′ N, 109°98′ E) for subsequent RNA extraction. The above three healthy plants were identified by marking their branches when collecting them for propagation.
Total RNA of each material of A. ebractetus weighed 1 g each and was extracted using the FastPure Plant Total RNA Isolation Kit (Vazyme, Nanjing, China). The concentration and purity of the total RNA were assessed using Nanodrop2000 (Thermo Scientific, Shanghai, China), and the RNA integrity was evaluated through 1.0% agarose gel electrophoresis.
mRNA was enriched from total RNA using Oligo (dT) magnetic beads (Thermo Scientific, Shanghai, China). The enriched mRNA was then fragmented into ~200–300 bp fragments using fragmentation buffer (Thermo Scientific, Shanghai, China) under thermal conditions (94 °C for 8 min) to facilitate subsequent cDNA synthesis. The first cDNA strand was synthesized with reverse transcriptase (Thermos Scientific RevertAid premix, Shanghai, China), followed by second-strand synthesis using End Repair Mix (Thermos Scientific Rapid DNA end repair kit, Shanghai, China) to create blunt ends. An adenine base was added to the ends for adapter sequence attachment. The products were purified, sorted, and PCR amplified to purify the library. Transcriptome sequencing was conducted at Shanghai Majorbio Company (Shanghai, China).

2.2. Plant Materials Under Abiotic Stress

The fresh woody branches from the above three A. ebractetus with markings were collected and planted as cuttings in the nursery of the Mangrove Research Institute of Lingnan Normal University. When 6–8 new leaves grew from the cuttings of each tree, leaves with consistent growth and no pests or diseases were picked and propagated into seedlings through hydroponics [26]. Stress experiments were conducted only after the seedling height reached 30–40 cm.
For every treatment, the seedlings chosen were from the above three different plants and were in identical growth conditions. The seedlings were cultured in an artificial climate incubator (RLD-260D-2DW, Ningbo Ledian, Ningbo, China) for 10 days under the following conditions: temperature 25 °C, light cycle of 16 h/8 h, light intensity of 20,000 LX, and relative humidity of 70%.
For abiotic stress treatments, all experiments were initiated simultaneously under controlled environmental conditions identical to those described above to ensure consistency. Specific treatments were designed as follows: the temperature was set to 4 °C for cold treatment and 35 °C for heat treatment. Under salt stress, seedlings were irrigated with 2 L of 300 mM NaCl Hogland solution. For waterlogging stress, potted seedlings were immersed in Hogland solution so that the liquid level covered all root systems. Each stress treatment was conducted for 0 h, 24 h, and 48 h, with 3 biological replicates, and 0 h served as the control group [27]. Fresh leaves of each seedling at each time point under different treatments were collected for subsequent RNA extraction and qRT-PCR analysis.

2.3. Bioinformatic Analysis of the AeNACs Gene Family

The raw paired-end reads from the second-generation transcriptome analysis were trimmed and quality controlled by fastp [28] with default parameters. The clean reads were then separately aligned to Avicennia marina as the reference genome in orientation mode using HISAT2 v2.2.1 [29] software. StringTie [30] was used to assemble the mapped reads of each sample.
The hidden Markov model of the NAC domain (PF02365) was downloaded from the Pfam database (http://pfam.xfam.org/). BlastP detection was performed on the transcriptome data using the HMMER online tool (https://www.ebi.ac.uk/Tools/hmmer/ (accessed on 21 May 2025)), and domain prediction was conducted on CCD (https://www.ncbi.nlm.nih.gov/cdd/wrpsb.cgi (accessed on 21 May 2025)) to remove duplicate sequences. The physical and chemical properties of the AeNAC protein were analyzed using Expasy (https://web.expasy.org/protparam/ (accessed on 16 July 2025)) and Deeploc 2.1 software (http://www.cbs.dtu.dk/services/DeepLoc-2.1 (accessed on 16 July 2025)) for subcellular localization prediction [31]. Conservative motif analysis was performed using the Meme software (https://memesuite.org/meme/tools/meme (accessed on 16 July 2025)) and visualized with TBtools v 2.225 (https://github.com/CJ-Chen/TBtools) (accessed on 16 July 2025). Systematic evolutionary analysis of NAC proteins in A. ebractetus, A. thaliana, and A. marina, was conducted using MEGA11 v 11.0.11, employing the Maximum Likelihood (ML) method with a bootstrap value of 1000 times [32]. GO analysis of AeNAC genes was conducted using the goatools (v 1.4.4, https://pypi.org/project/goatools/ (accessed on 16 July 2025)) software.

2.4. Expression Analysis of the AeNAC Genes in Different Tissues of A. ebractetus

Transcriptome data of leaf, root, and flower of A. ebractetus were employed to investigate the role of AeNACs genes. Differential expression analysis was performed in three materials using the DESeq2 package (v 1.46.0), where genes with log2 (fold-change) > 1 and adjusted p-value < 0.05 were upregulated compared to leaves, while those with log2 (fold-change) < −1 and adjusted p-value < 0.05 were downregulated. Weighted Gene Co-expression Network Analysis (WGCNA) was conducted to investigate the correlation of differentially expressed AeNAC genes in three materials using the R package WGCNA (v 1.72.1).
Utilize qRT-PCR to analyze the expression profiles of AeNAC genes in various plant tissues such as leaf, root, stem, and flower. Extract total RNA from 0.2 g of plant material using the FastPure Plant Total RNA Isolation Kit (Vazyme, Nanjing, China). Synthesize cDNA from 2 μg of total RNA using the RTC101 from reverse transcription reagent form Vazyme (Vazyme, Nanjing, China) and store the cDNA at −20 °C. Design primers with Primer 6.0 software based on AeNAC gene sequences, with the primer sequences listed in Table S1. The Actin gene served as the internal reference gene [27]. qRT-PCR was performed using PerfectStart Green qPCR SuperMix (Transgen, Beijing, China) on the Heal Force CG-05 real-time fluorescence quantitative PCR instrument (Heal Force, Shanghai, China). The reaction system includes forward and reverse primers, diluted template cDNA, PerfectStart Green qPCR SuperMix, universal passive reference dye, and nuclease-free water, with a total reaction volume of 20 μL. The reaction conditions were as follows: 94 °C for 30 s; followed by 30 cycles of 94 °C for 5 s, 60 °C for 15 s, 72 °C for 10 s. The relative gene expression levels were calculated using the 2−ΔΔCt method, with 3 biological replicates per reaction. The consistency between the results of qRT-PCR and RNA-seq was measured using the Pearson correlation coefficient (PPC).

2.5. Analysis of Verproside and Verbascoside Contents

Leaf tissues, the same as the material used for transcriptome sequencing, were collected from A. ebractetus. Approximately 1 g of leaf material was obtained per sample. The collected samples were promptly frozen in liquid nitrogen, dried, and then ground into powder. Metabolite extraction was carried out, and the samples were analyzed by Shanghai Majorbio Biotechnology Co., Ltd., Shanghai, China. Pearson’s correlation analysis was used to examine the relationship between the AeNACs gene and the expression levels of Verproside and Verbascoside in the leaves of A. ebracteatus.

3. Results

3.1. Analysis of Physical and Chemical Properties, Conservative Sequences, and Evolutionary Tree of AeNACs Gene Family

A total of 56 AeNAC family genes were identified from A. ebractetus and named AeNAC01~AeNAC56 (Figure 1A, Table 1) after removing sequences with incomplete structural domains. The genes encode proteins ranging from 116–687 amino acids, with molecular weights of 13.48–76.40 kDa and isoelectric points of 4.03–10.21. 26.32% of the proteins have an isoelectric point greater than 7, falling within the basic range. Only two proteins, AeNAC05 and AeNAC09, are classified as hydrophobic based on their average hydrophilicity index. The rest of the AeNAC proteins are considered hydrophilic, with average hydrophilicity coefficients less than 0. However, there are variations in hydrophilicity among the different proteins. The AeNAC family proteins exhibit differences in hydrophilicity, with an instability index ranging from 4.03 to 10.21, indicating high stability. The aliphatic indices range from 15.0 to 68.98. Subcellular localization analysis showed that seven proteins (AeNAC06/18/32/34/35/37/46) were localized to the endoplasmic reticulum, AeNAC15 was localized to both the endoplasmic reticulum and nucleus, while the others were localized to the nucleus, suggesting their role in transcriptional regulation [33]. Conservative motif analysis identified 10 motifs (Figure 1B), with some proteins containing specific motifs. All 56 AeNAC proteins contained the NAM domain (Figure 1C). Comparison with Arabidopsis NAC family proteins showed two groups (Figure 2): Group II had fewer AeNAC proteins, only in branches ANAC063 and ONAC003, compared to Group I, which included proteins from different branches like ONAC022, NAP, ATAF, OsNAC8, TIP, and NAC2. The supplementary clustering analysis of the NAC family proteins from A. marina, A. ebractetus, and Arabidopsis was illustrated in Figure S1. In this polytree, the AmNAC proteins of A. marina are distributed across all branches.

3.2. Enrichment Analysis of AeNAC Gene Family GO in A. ebractetus

The GO enrichment analysis results (Figure 3) show that the 56 AeNAC transcription factors are primarily associated with molecular function and biological processes. Biological processes consist mainly of molecular processes and metabolic processes, while molecular functions mainly involve binding and catalytic activity processes.

3.3. Tissue-Specific Expression Analysis of AeNAC Genes

Based on transcriptome sequencing results, the expression data of 56 AeNAC genes in the leaf, root, and flower tissues in A. ebracteatus were analyzed. The data were then subjected to heatmap clustering analysis, as depicted in Figure 4A. The 56 AeNAC genes in A. ebracteatus showed varying expression levels in different tissues, with some genes having low expression in leaves but high expression in roots and flowers, while others had high expression in roots but low expression in both roots and flowers. Overall, expression levels in leaves were generally low. Nine AeNAC genes were chosen, with the Actin gene serving as the internal reference gene. qRT-PCR technology was utilized to examine the expression of NAC genes in various tissue parts of roots, leaves, and flowers of A. ebracteatus, as depicted in Figure 4B. For validating the reliability of the transcriptome data, one gene was selected for each subfamily cluster based on the AeNAC genes distributed in the polygenetic analysis (Figure 2). Additionally, the NAC2 subfamily cluster chose two genes, AeNAC03 and AeNAC10. Analysis of the tissue expression characteristics of candidate genes revealed that the relative expression levels of AeNAC03, AeNAC10, AeNAC31, and AeNAC48 in leaves were lower than those in flowers and roots. The consistency between the results of qRT-PCR and RNA-seq of these four genes was measured using the Pearson correlation coefficient (PPC) in Figure S2. Meanwhile, the expression levels of AeNAC27 and AeNAC55 in leaves were lower than those in flowers. These results are completely consistent with the transcriptome analysis findings.
Further studies showed that all eight candidate genes, except AeNAC27, were highly expressed in stems. Since stems are crucial organs for supporting the aboveground parts of plants and transporting water and nutrients, combined with the research conclusion in Arabidopsis that NAC transcription factors regulate plant secondary wall synthesis and vascular bundle development [34], it is showed that these AeNAC genes highly expressed in stems may be involved in important physiological processes such as mechanical strength formation, vascular tissue differentiation, and material transport [34]. In floral tissues, all seven candidate genes, except AeNAC10 and AeNAC47, showed high expression levels, suggesting that they may play a role in the floral organ development of A. ebracteatus [35]. In root tissues, the relative expression levels of other candidate genes were relatively low except for AeNAC03, AeNAC10, and AeNAC27. It is particularly noteworthy that AeNAC10, AeNAC31, and AeNAC48 had the lowest expression levels in leaves. This significant tissue-specific expression difference fully reflects the high functional differentiation of NAC family members.

3.4. Correlation Analysis Between the Expression Level of AeNAC Genes and the Contents of Verbascoside and Verprosid

The entire A. ebracteatus plant has medicinal value [36], but as an endangered mangrove species, the development and utilization of its medicinal components must avoid damaging its germplasm resources. In early work, our research team successfully achieved asexual reproduction of this plant through leaf cuttings [26]. Efforts should therefore focus on developing medicinal and edible applications using plant leaves, which would effectively support the ecological restoration of this endangered species. Correlation analysis was conducted between the expression level of the AeNAC gene and the content of verproside and verbascoside in the leaves of A. ebracteatus (Figure 5). AeNAC13 showed no correlation with either compound, while AeNAC14 and AeNAC48 were found to actively regulate these metabolites: AeNAC14 and AeNAC48 were positively correlated with verproside content but negatively correlated with verbascoside content.

3.5. Analysis of AeNAC Gene Expression in Young Leaves of A. ebracteatus Under Abiotic Stress

Under waterlogging stress (Figure 6A), AeNAC03/48/56 genes were upregulated after 48 h of treatment, with only AeNAC03 being upregulated after 24 h, and the other three genes were inhibited. Under cold stress (Figure 6B), AeNAC44 and AeNAC48 genes increased in expression with prolonged stress time, while AeNAC56 decreased. Under salt stress (Figure 6C), all four NAC genes were upregulated at 24 h and downregulated at 48 h, except for AeNAC44.

4. Discussion

Transcription factors are essential components that control gene expression and are vital for plant growth, development, and response to the environment. Studying transcription factor families systematically can offer valuable insights into how plants respond to environmental changes. NAC transcription factors play a key role in helping plants adapt to challenging ecological conditions by activating or repressing stress-related genes, regulating physiological and biochemical pathways, and enhancing stress tolerance in plants [37]. During plant organ development, certain NAC members control flower organ morphogenesis, leaf senescence, and cell organ differentiation [38]. Studies on medicinal plants such as Andrographis paniculata [39], Eleutherococcus senticosus [40], and Fagopyrum tataricum [41] have demonstrated the significant role of the NAC gene family in regulating secondary metabolism, thereby controlling the production of medicinal and edible components. The number of NAC genes identified from the genomes of medicinal true mangrove plants such as K. obovata and A. marina is higher than the 56 AeNAC genes identified from the medicinal mangrove plant A. ebracteatus based on the transcriptome [9,10]. The release of subsequent genomic data with high quality will inevitably increase the number of AeNAC genes [10]. There were 142 AmNAC genes distributed in each branch of the clustering analysis of the NAC family genes in A thaliana (Figure S2). The AmNAC genes belong to the OSNAC7 subclade with no AeNAC gene, which were proposed to act as a critical switch in the regulatory network related to cell call formation [10]. OsNAC300, a NAC TF in rice roots, was found to be involved in the response and tolerance to cadmium stress [42], and AeNAC52 is the sole gene in A. ebracteatus that falls under this category.
During drought stress, lipid-anchored NAC in Medicago falcata can be transported to the nucleus through deprotonation to stimulate glyoxylate I expression [43]. AeNAC proteins are mainly located in the nucleus, with 7 also found on the endoplasmic reticulum. These membrane-bound AeNAC proteins may have regulatory mechanisms similar to MfNAC in sensing external pressure [43], and 34 AeNAC proteins belong to the NAC2 subfamily [31]. Members of this subfamily are involved in responses to abiotic stress, closely related to physiological activities under stress, such as cold and salt stress [44]. There are tissue expression differences in plant NAC genes. Among the 68 KoNAC genes in K. obovata, 28 are highly expressed in roots, 13 in fruits, and only 18 in leaves [9]. Transcriptome analysis of AeNAC genes showed relatively low expression levels in leaf tissues. The NAC gene in certain stress-resistant plants can enhance leaf aging and stress response by upregulating, leading to nutrient redistribution and stress relief [45]. Arabidopsis NAC family genes are crucial in natural senescence and stress-induced leaf senescence [46]. The Arabidopsis mutant lacking NAC1 did not show significant leaf senescence, as AT5G39610 (ANAC092) is crucial in regulating leaf senescence-related genes and plays a key role in promoting senescence under salt stress [47]. The low expression level of AeNAC genes in A. ebracteatus leaves may suggest unique survival strategies for extending leaf lifespan and maintaining photosynthetic efficiency, and further validation is required. Most of the nine candidate AeNAC genes detected showed high expression in stem tissue, indicating potential roles in stem vascular bundle development, cell wall formation, and mechanical support [48]. Higher expression levels of AeNACs in flowers suggest involvement in flower organ development and metabolic regulation for plant reproductive processes [49]. The low expression of AeNAC10 and AeNAC47 in flowers may be due to functional differentiation or inactivation during molecular evolution [14]. Low expression levels of AeNAC10/3/48 in leaves may result from regulatory network dependence on synergistic regulation by other transcription factors or miRNAs [50].
The stress resistance of medicinal mangrove plants in intertidal habitats is regulated by the NAC family genes, impacting their growth and development [51]. The AeNACs gene shows significant temporal differential expression in response to various types and durations of abiotic stress. The KoNAC68 gene in K. obovata is activated under low temperature conditions, suggesting its involvement in low temperature stress [9]. By utilizing virus-induced gene silencing technology to suppress the expression of CaNAC2 in Capsicum annuum seedlings, the susceptibility of pepper seedlings to low temperature is heightened [45]. During cold stress treatment, AeNAC44 and AeNAC48 were continuously upregulated, similar to the regulatory effects of OsNAC5 and OsABI5 in Oryza sativa [51]. However, the expression of AeNAC56 decreased, which is similar to some functional features of GmNAC11 and GmNAC20 in Glycine max [13]. ANAC019/055/072 overexpression can improve A. thaliana’s resistance to drought, cold, and salt [52]. VaNAC26 [53] and StNAC05 [54] gene expression levels were increased in response to salt stress. DEG analysis of A. marina under flooding treatment revealed that five AmNAC genes showed differential expression in the leaves: AmNAC02/069/122/123 were upregulated, while AmNAC059 was downregulated [10]. After 48 h of waterlogging stress, AeNAC03/48/56 showed an increase in expression, while AeNAC44 exhibited a decrease in the leaves of A. ebracteatus. These NAC genes play a role in specific biological processes linked to adapting to intertidal environments [10].
The regulation of transcription levels is a critical aspect in the biosynthesis process of secondary metabolites in medicinal plants, with a multi-omics approach promoting in-depth analysis of transcription factor regulatory networks [55]. The accumulation of metabolites is closely linked to the expression level of key enzyme genes in the secondary metabolite synthesis pathway, and NAC transcription factors have the ability to regulate target genes by directly binding to their promoter region [56]. The levels of certain NAC transcription factors in Andrographis paniculata showed a strong positive correlation with the production of andrographon, indicating their potential regulatory role in its biosynthesis [39]. Camellia sinensis NAC transcription factors play a role in controlling the production of secondary metabolites like tea polyphenols and caffeine in tea plants [57]. Researchers have isolated various glycosylated cyclic terpenoid glycosides and phenolic glycosides through chromatographic and mass spectrometry techniques. Verproside and verbascoside have garnered attention for their antioxidant and cell protective effects, making them suitable for use in cosmetics and medicinal products [58]. A. ebracteatus, as a traditional medicinal plant, is known for its richness in verbascoside [20]. The AeNAC14 and AeNAC48 genes were discovered to correlate with the biosynthesis of verproside and verbascoside in the leaves of A. ebracteatus. Additionally, the AeNAC48 gene exhibited varying levels of expression in response to water, salt, and cold stress, indicating its potential involvement in regulating the production and storage of these compounds. This hypothesis can be confirmed through experiments manipulating gene expression in the future.

5. Conclusions

Figure 7 outlines the regulatory roles of AeNAC genes in A. ebracteatus abiotic stress responses and the biosynthesis of verproside and verbascoside. Our identification of 56 AeNAC genes, with diverse properties and nuclear localization, highlights the family’s functional diversity in this medicinal mangrove. Tissue-specific expression patterns and differential stress responses (especially AeNAC48) revealed specialized roles in coordinating developmental and environmental adaptations. Importantly, AeNAC14 and AeNAC48 exhibit distinct correlations with verproside (positive) and verbascoside (negative), identifying key regulators of these bioactive phenylethanoid glycosides critical to medicinal efficacy. These findings offer practical value: stress-responsive (AeNAC03, 44, 48, 56) and metabolite-regulating (AeNAC14, 48) candidates enable targeted breeding to enhance mangrove resilience and optimize medicinal compound production. This work advances understanding of NAC-mediated mechanisms while providing actionable targets for conserving and improving this ecologically and pharmaceutically significant species.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f16081354/s1, Table S1: RT- PCR primer sequences; Figure S1: Phylogenetic analysis of NAC family members from Avicennia marina, Acanthus ebractetus, and Arabidopsis thaliana; Figure S2: The consistency between the results of qRT-PCR and RNA-seq of AeNAC03, AeNAC10, AeNAC31, and AeNAC48 in leaf, root and flower.

Author Contributions

J.Y. and H.F.: experimental implementation, writing—original draft. C.X.: Experimental implementation. J.Y.: writing—review and editing, supervision. Y.Z.: review and editing, supervision. Funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Guangdong Province Science and Technology Innovation Strategic Project (2022A105).

Data Availability Statement

The transcriptome sequencing data were deposited in the China National Center for Bioinformation with the submission number: CRA027976.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Identification of conserved motifs and gene structure of AeNAC family members. (A) AeNACs, (B) motif analysis, (C) domain analysis.
Figure 1. Identification of conserved motifs and gene structure of AeNAC family members. (A) AeNACs, (B) motif analysis, (C) domain analysis.
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Figure 2. Phylogenetic analysis of AeNAC family members. (The red star indicates the AeNAC genes).
Figure 2. Phylogenetic analysis of AeNAC family members. (The red star indicates the AeNAC genes).
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Figure 3. GO enrichment analysis of NAC genes in Acanthus ebracteatus.
Figure 3. GO enrichment analysis of NAC genes in Acanthus ebracteatus.
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Figure 4. Tissue-specific expression of AeNAC genes family in Acanthus ebracteatus ((A) AeNACs transcriptome expression profile in root, leaf and flower tissues, Red indicates high expression, while blue indicates low expression; (B) Bar charts showing the qRT-PCR verification of nine candidate genes in leaf, root, stem and flower tissues; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001; Actin serves as the internal reference gene; Significance analysis was conducted using t-tests, n = 3).
Figure 4. Tissue-specific expression of AeNAC genes family in Acanthus ebracteatus ((A) AeNACs transcriptome expression profile in root, leaf and flower tissues, Red indicates high expression, while blue indicates low expression; (B) Bar charts showing the qRT-PCR verification of nine candidate genes in leaf, root, stem and flower tissues; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001; Actin serves as the internal reference gene; Significance analysis was conducted using t-tests, n = 3).
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Figure 5. Pearson correlation analysis between the expression level of AeNAC genes and the content of verbascoside and verproside in the leaves of Acanthus ebracteatus. (Purple means the correlation is negative; red means the correlation is positive; the size of the circle indicates the level of correlation; * indicates p < 0.05, ** indicates p < 0.01; Significance analysis was conducted using t-tests, n = 3).
Figure 5. Pearson correlation analysis between the expression level of AeNAC genes and the content of verbascoside and verproside in the leaves of Acanthus ebracteatus. (Purple means the correlation is negative; red means the correlation is positive; the size of the circle indicates the level of correlation; * indicates p < 0.05, ** indicates p < 0.01; Significance analysis was conducted using t-tests, n = 3).
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Figure 6. Expression of AeNAC03, AeNAC44, AeNAC48, and AeNAC56 in Acanthus ebracteatus seedling leaves by qRT-PCR under different abiotic stress. ((A) waterlogging stress; (B) cold stress; (C) salt stress; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001; Significance analysis was conducted using t-tests, n = 3).
Figure 6. Expression of AeNAC03, AeNAC44, AeNAC48, and AeNAC56 in Acanthus ebracteatus seedling leaves by qRT-PCR under different abiotic stress. ((A) waterlogging stress; (B) cold stress; (C) salt stress; * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001; Significance analysis was conducted using t-tests, n = 3).
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Figure 7. Conceptual model summarizing the molecular responses of AeNAC genes involved in abiotic stress response and the biosynthesis of verproside and verbascoside. The asterisk indicates high expression of the gene. The upward arrow signifies an upregulation of the gene, whereas the downward arrow signifies a downregulation of the gene.
Figure 7. Conceptual model summarizing the molecular responses of AeNAC genes involved in abiotic stress response and the biosynthesis of verproside and verbascoside. The asterisk indicates high expression of the gene. The upward arrow signifies an upregulation of the gene, whereas the downward arrow signifies a downregulation of the gene.
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Table 1. Physicochemical properties and subcellular localization of the AeNAC family.
Table 1. Physicochemical properties and subcellular localization of the AeNAC family.
NameGene IDAmino Acid NumberMolecular Weight/kDaPIInstability
Index		Aliphatic
Index		Grand Average of
Hydropathicity		Subcellular
Localization		Subfamily
S1_transcript_362.p2AeNAC0112414,365.5210.2129.7160.56−0.765nuclearTIP
S1_transcript_6038.p2AeNAC0219923,536.479.0849.5983.22−0.127nuclearNAC2
S1_transcript_12966.p2AeNAC0311913,913.918.4547.2573.7−0.451nuclearNAC2
S1_transcript_13336.p2AeNAC0419923,305.239.0551.985.68−0.129nuclearNAC2
S1_transcript_15934.p2AeNAC0515318,571.098.5633.47101.830.541nuclearNAC2
S1_transcript_22914.p1AeNAC0658365,328.655.0250.166.45−0.567endoplasmic reticulumNAC2
S1_transcript_23963.p2AeNAC0715818,199.829.6929.7669.11−0.744nuclearTIP
S1_transcript_24008.p2AeNAC0811613,612.578.4848.2172.24−0.483nuclearNAC2
S1_transcript_25936.p2AeNAC0915318,571.098.5633.47101.830.541 nuclearNAC2
S1_transcript_26270.p1AeNAC1039744,883.274.4445.8360.71−0.796nuclearNAC2
S1_transcript_26719.p2AeNAC1116218,530.129.5929.9967.41−0.757nuclearTIP
S1_transcript_29832.p1AeNAC1240745,615.434.5849.0468.08−0.614nuclearNAC2
S1_transcript_30968.p2AeNAC1317219,811.99.327.7383.84−0.44nuclearTIP
S1_transcript_34215.p1AeNAC1429733,731.024.1336.1278.45−0.516nuclearNAC2
S1_transcript_34259.p1AeNAC1552258,413.434.8347.2362.43−0.687Nuclear/endoplasmic reticulumNAC2
S1_transcript_34372.p1AeNAC1667375,870.845.343.0280.1−0.286nuclearNAC2
S1_transcript_35586.p1AeNAC1743748,643.94.3741.3681.46−0.378nuclearNAC2
S1_transcript_36673.p1AeNAC1852358,233.574.8849.6968.49−0.532endoplasmic reticulumNAC2
S1_transcript_37244.p1AeNAC1927230,569.364.0332.2477.46−0.563nuclearNAC2
S1_transcript_41915.p1AeNAC2044649,895.664.7635.0970.85−0.533nuclearNAC2
S1_transcript_42193.p1AeNAC2160567,929.595.0943.6978.15−0.342nuclearNAC2
S1_transcript_42738.p1AeNAC2244249,680.414.6938.3969.05−0.529nuclearNAC2
S1_transcript_44059.p1AeNAC2340845,744.554.5648.9467.92−0.622nuclearNAC2
S1_transcript_44264.p1AeNAC2454360,904.424.9648.5162.71−0.663nuclearNAC2
S1_transcript_44351.p1AeNAC2546752,632.464.6349.9769.76−0.599nuclearNAC2
S1_transcript_44403.p1AeNAC2668776,403.885.3248.8368.27−0.658nuclearTIP
S1_transcript_44485.p1AeNAC2760767,731.675.2652.4865.83−0.696nuclearTIP
S1_transcript_45348.p1AeNAC2866173,322.635.4751.3369.03−0.617nuclearTIP
S1_transcript_45359.p2AeNAC2917921,066.19.4720.969.72−0.558nuclearNAC2
S1_transcript_46629.p1AeNAC3046752,669.484.651.9369.14−0.613nuclearNAC2
S1_transcript_47372.p1AeNAC3139945,107.486.2952.267.19−0.666nuclearOsNAC8
S1_transcript_47382.p1AeNAC3263571,551.55.0347.465.28−0.625endoplasmic reticulumNAC2
S1_transcript_47723.p1AeNAC3358165,364.16.0543.8465.8−0.698nuclearTIP
S1_transcript_47969.p1AeNAC3458265,509.614.9649.1164.36−0.664endoplasmic reticulumNAC2
S1_transcript_48049.p1AeNAC3552358,184.54.8549.8867.93−0.539endoplasmic reticulumNAC2
S1_transcript_48109.p1AeNAC3660667,302.744.9553.4170.81−0.63nuclearTIP
S1_transcript_48112.p1AeNAC3758365,250.64.8950.3769.79−0.528endoplasmic reticulumNAC2
S1_transcript_48470.p1AeNAC3849455,797.224.8939.4271.6−0.595nuclearNAC2
S1_transcript_48616.p1AeNAC3941447,320.444.5431.7573.72−0.579nuclearNAC2
S1_transcript_48819.p1AeNAC4060366,371.635.3949.0769.2−0.637nuclearTIP
S1_transcript_48998.p1AeNAC4156062,848.864.6738.5177.86−0.461nuclearNAC2
S1_transcript_49250.p1AeNAC4255462,233.334.6836.9977.29−0.454nuclearNAC2
S1_transcript_49481.p1AeNAC4356863,491.694.6530.6781.58−0.43nuclearNAC2
S1_transcript_49861.p1AeNAC4411913,478.6510.171568.15−0.512nuclearATAF
S1_transcript_49863.p1AeNAC4553059,424.235.0639.8970.85−0.543nuclearNAC2
S1_transcript_50300.p1AeNAC4661068,749.754.7536.7572.05−0.427endoplasmic reticulumNAC2
S1_transcript_50937.p1AeNAC4716418,370.745.4448.4267.26−0.687nuclearANAC063
S1_transcript_51143.p1AeNAC4843148,594.145.1449.368.03−0.85nuclearONAC003
S1_transcript_52047.p1AeNAC4919022,305.589.5438.0958.47−0.674nuclearNAP
S1_transcript_52096.p1AeNAC5029934,360.726.2150.8662.64−0.688nuclearATAF
S1_transcript_52114.p1AeNAC5142547,446.075.5968.9868.38−0.724nuclearANAC063
S1_transcript_52170.p1AeNAC5237541,184.815.5950.665.28−0.682nuclearANAC063
S1_transcript_52233.p1AeNAC5342146,993.354.9961.9770.4−0.661nuclearANAC063
S1_transcript_52656.p1AeNAC5429433,181.46.0842.5464.46−0.659nuclearATAF
S1_transcript_52875.p1AeNAC5531235,189.818.1747.9367.47−0.538nuclearONAC022
S1_transcript_52913.p1AeNAC5630935,983.889.1440.6458.03−0.725nuclearNAP
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MDPI and ACS Style

Yao, J.; Fan, H.; Xing, C.; Zhang, Y. Molecular Responses of the NAC Gene Family in Acanthus ebracteatus: Involvement in Abiotic Stress Responses and Biosynthesis of Verproside and Verbascoside. Forests 2025, 16, 1354. https://doi.org/10.3390/f16081354

AMA Style

Yao J, Fan H, Xing C, Zhang Y. Molecular Responses of the NAC Gene Family in Acanthus ebracteatus: Involvement in Abiotic Stress Responses and Biosynthesis of Verproside and Verbascoside. Forests. 2025; 16(8):1354. https://doi.org/10.3390/f16081354

Chicago/Turabian Style

Yao, Jun, Huiting Fan, Canming Xing, and Ying Zhang. 2025. "Molecular Responses of the NAC Gene Family in Acanthus ebracteatus: Involvement in Abiotic Stress Responses and Biosynthesis of Verproside and Verbascoside" Forests 16, no. 8: 1354. https://doi.org/10.3390/f16081354

APA Style

Yao, J., Fan, H., Xing, C., & Zhang, Y. (2025). Molecular Responses of the NAC Gene Family in Acanthus ebracteatus: Involvement in Abiotic Stress Responses and Biosynthesis of Verproside and Verbascoside. Forests, 16(8), 1354. https://doi.org/10.3390/f16081354

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