Genome-Wide Identification and Expression Profiling of the NAC Transcription Factor Family in the Waterlogging-Tolerant Tree Magnolia sinostellata

Genome-Wide Identification and Expression Profiling of the NAC Transcription Factor Family in the Waterlogging-Tolerant Tree Magnolia sinostellata | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-Wide Identification and Expression Profiling of the NAC Transcription Factor Family in the Waterlogging-Tolerant Tree Magnolia sinostellata Xiaoai Fang, Lu Fan, Huijuan Zhou, Huiling Yan, Bin Xie, Fangbing Ding, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8390673/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The NAC transcription factors are master regulators of plant responses to abiotic stresses; however, their functions in woody ornamentals, particularly under waterlogging, remain poorly explored. Magnolia sinostellata , a rare and waterlogging-tolerant species, provides an ideal system to decipher these mechanisms. Results In this study, a genome-wide analysis identified 96 MsNAC genes in M. sinostellata , which were phylogenetically classified into 17 distinct subgroups. Gene structure and motif analyzes revealed considerable diversity, with several members harboring auxiliary functional domains beyond the conserved NAC domain, suggesting potential functional diversification. Promoter cis-acting element analysis uncovered an abundance of stress-responsive elements, among which abscisic acid (ABA, 19.3%) and methyl jasmonate (MeJA, 13.7%) responses were the most prominent, highlighting a complex regulatory landscape intertwined with hormonal and light signaling pathways. Collinearity and evolutionary pressure analyzes demonstrated that the expansion of the MsNAC family was primarily driven by gene duplication events under strong purifying selection (Ka/Ks < 1). Transcriptomic and RT-qPCR analyzes further revealed that MsNAC genes exhibit tissue-specific and temporally dynamic expression patterns under waterlogging stress. Notably, roots, as the primary sensing organ, mounted the most pronounced response. The key candidate MsNAC 66 was significantly and persistently upregulated, while others, including MsNAC 14, MsNAC 16, MsNAC 63, MsNAC 65, and MsNAC 70, displayed transient induction patterns. Conclusions Our study provides the first comprehensive genomic characterization of the NAC family in M. sinostellata and identifies several core candidate genes with putative roles in the root-centric adaptive response to waterlogging, as determined by their expression dynamics. These findings not only advance our understanding of NAC-mediated stress tolerance in woody plants but also offer valuable genetic resources for the molecular breeding of waterlogging-resistant magnolia cultivars. Magnolia sinostellata NAC transcription factor Genome-wide analysis Waterlogging stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Climate change has escalated the frequency of extreme weather events, particularly heavy rainfall and flooding, rendering waterlogging stress a critical abiotic constraint on plant growth, development, and geographical distribution [ 1 ]. In waterlogged or poorly drained soils, oxygen depletion (hypoxia) severely disrupts root respiration and energy metabolism. Prolonged waterlogging instigates a cascade of physiological disturbances, including reactive oxygen species (ROS) accumulation, membrane damage, and photosynthetic inhibition, ultimately culminating in growth arrest or plant death [ 2 ]. To mitigate waterlogging stress, plants have evolved sophisticated adaptive strategies involving both morphological modifications (e.g., aerenchyma formation, adventitious root development) and molecular reprogramming (e.g., induction of anaerobic metabolism-related genes) [ 3 ]. Within these adaptive frameworks, transcription factors (TFs) serve as central regulators by modulating the expression of stress-responsive genes. The NAC (NAM, ATAF1/2, CUC2) family represents a plant-specific class of TFs, typically phylogenetically classified into 18 subgroups [ 4 ]. NAC proteins are characterized by a conserved N-terminal DNA-binding NAC domain and a highly divergent C-terminal transcriptional regulation region [ 5 , 6 ]. Recent research has underscored their pleiotropic roles in various aspects of plant development (e.g., root architecture, leaf senescence) and stress responses (e.g., drought, waterlogging, cold) [ 7 ]. This functional versatility originates from their capacity to regulate gene expression through DNA binding, protein-protein interactions, and involvement in epigenetic modifications [ 5 , 8 ]. Consequently, the NAC family has emerged as a focal point in plant biology research. Genome-wide identifications of NAC genes have been extensively reported across diverse plant species, including Arabidopsis thaliana (117 genes) [ 9 ], rice (151 genes) [ 10 ], wheat (559 genes) [ 11 ], maize (157 genes) [ 12 ], tartary buckwheat (80 genes) [ 13 ], tomato (93 genes) [ 14 ], pear (183 genes) [ 15 ], bitter gourd (90 genes) [ 16 ], and papaya (66 genes) [ 17 ]. The regulatory mechanisms and functional roles of NAC s in different plants have been widely reported. For example, in A. thaliana , SOMBRERO (SMB) and Bearskin 1/2 (BRN1/2) regulate the maturation and shedding of root cap cells [ 18 ], while NAC1 promotes lateral root formation by activating auxin response genes [ 19 ]. Rice OsNAP and A. thaliana ANAC092 / ORE1 promote leaf senescence by activating senescence-associated genes (SAGs) and chlorophyll degradation genes such as NYC1 [ 20 ]. CUC1/2/3 regulate the separation of cotyledons and flower organs by inhibiting the proliferation of boundary cells or activating the LSH gene family [ 21 , 22 ]. In stress responses, rice SNAC1 enhances drought resistance by activating drought-responsive genes such as OsAP37 and inhibiting ROS accumulation [ 23 ]; A. thaliana ANAC096 interacts with the ABA signaling pathway to regulate the dehydration response [ 24 ]. A. thaliana SMB mediates root salt aversion by regulating auxin distribution [ 25 ], and soybean GmNAC20 improves salt tolerance by promoting proline accumulation [ 26 ]. Apple MdNAC104 enhances cold resistance through CBF-dependent and CBF-independent pathways [ 27 ], while banana MaNAC1 interacts with the ICE1-CBF pathway [ 28 ]. A. thaliana SHYG promotes rapid elongation of petioles through ethylene signaling to cope with flooding stress [ 29 ]. Furthermore, NAC s can coordinate development and stress response by integrating hormones (such as ABA, JA), ROS, and light signals. For instance, rice O sNAC120 balances GA-mediated growth and ABA-mediated drought response [ 30 ], while A. thaliana JUNGBRUNNEN1 ( JUB1 ) delays leaf senescence and enhances drought tolerance by inhibiting ROS accumulation [ 31 ]. Beyond model plants and crops, systematic studies on the NAC gene family have also been conducted in ornamental species. RcNAC091 was highly expressed in rose ( Rosa chinensis ) under drought and salt stress, enhancing tolerance by regulating ROS scavenging and cell wall modification pathways while playing a dual regulatory role in flower organ development [ 32 ]. Multiple PmNAC genes (such as PmNAC1 ) were differentially expressed in plum blossoms ( Prunus mume ), driving xylem development and activation of the ABA signaling pathway to improve frost damage tolerance [ 33 ]. The HhNAC genes (such as HhNAC25 ) in Hibiscus hamabo were significantly induced under various abiotic stresses such as salt, drought, and heavy metals, maintaining cell membrane integrity and photosynthetic efficiency by activating the MAPK signaling cascade and stress-response genes [ 34 ]. Under heat and waterlogging stress, the PsNAC gene family (including PsNAC72 ) was differentially expressed in peonies, participating in heat shock protein synthesis and hypoxia response to improve energy metabolism efficiency and root damage avoidance [ 35 ]. Collectively, these studies indicate that NAC transcription factors are core hubs for plants to adapt to the environment and regulate development, providing important targets for agricultural biotechnology. Magnolia species are economically important as ornamental and medicinal trees in China. However, most exhibit high sensitivity to waterlogging, which greatly limits their promotion and application in urban and wetland landscapes. Magnolia sinostellata is a rare and endemic species within the Magnoliaceae family in China, primarily distributed in regions such as Zhejiang and Fujian. It thrives in cool, humid, and water-sufficient environments, representing a rare example of waterlogging tolerance within the Magnoliaceae family and holding considerable value for scientific research and horticulture [ 36 , 37 ]. Our previous studies have indicated that M. sinostellata possesses significant waterlogging tolerance, positioning it as a key resource for mining superior genes and for the genetic improvement of magnolia plants [ 38 ]. However, the molecular mechanism underlying its waterlogging tolerance remains elusive, hindering the application of molecular breeding strategies in Magnolia. Given the central regulatory role of the NAC family in plant responses to abiotic stresses, including waterlogging, and the absence of systematic studies on NAC genes in Magnoliaceae, we embarked on this research. The recent availability of the M. sinostellata genome sequence enables a genome-wide investigation of stress-responsive gene families. The objectives of this study were to: (1) systematically identify and characterize the MsNAC gene family in M. sinostellata ; (2) analyze their phylogenetic relationships, gene structures, conserved motifs, and promoter cis-elements; (3) explore their tissue-specific expression profiles under waterlogging stress; and (4) identify core candidate genes putatively involved in waterlogging tolerance for future functional studies. Materials and methods Identification and characterization of the MsNAC gene family The whole-genome sequencing data for M. sinostellata in this study was deposited in NCBI under BioProject accession PRJNA1169943. The A. thaliana genome and protein sequences were downloaded from TAIR ( https://www.arabidopsis.org/ ). To comprehensively identify NAC family members, a combined strategy utilizing Hidden Markov Model (HMM)-based searches and BLASTp homology analysis was employed. First, the NAC domain HMM profile (PF02365) obtained from Pfam [ 39 ] was used to query the M. sinostellata proteome using HMMER v3.2.1 with an E-value cutoff of 1×10⁻⁵ (Supplementary Table S1 ) [ 40 ]. Simultaneously, known NAC protein sequences from various plant species (acquired from PlantTFDB; http://planttfdb.gao-lab.org ) were used as queries for BLASTp searches against the M. sinostellata proteome (E-value 50%) [ 4 ]. Non-redundant candidate sequences from both approaches were merged. The presence of the complete NAC domain in each candidate was rigorously verified using SMART [ 41 ], the Conserved Domain Database (CDD) [ 42 ], and Pfam. Final confirmation was performed using the NCBI Conserved Domain Search tool [ 43 ]. The resulting non-redundant set was designated as the MsNAC gene family. Physicochemical properties (theoretical isoelectric point pI, molecular weight, instability index) of the identified MsNAC proteins were predicted using ExPASy's ProtParam tool [ 44 ]. Subcellular localization was predicted using Cell-PLoc 2.0 [ 45 ]. Gene structure, conserved motif and phylogenetic analysis Gene structure diagrams (intron/exon organization) were generated based on the genome annotation GFF3 file using the Gene Structure Display Server (GSDS; http://gsds.cbi.pku.edu.cn/ ) [ 46 ]. To identify conserved motifs beyond the NAC domain, the protein sequences of the MsNAC members were analyzed using the MEME Suite ( http://meme-suite.org/ ) with the following parameters: maximum number of motifs set to 20, and the optimum motif width set between 6 and 50 amino acids [ 47 ]. For phylogenetic analysis, the full complement of NAC protein sequences from A. thaliana (AtNACs) was retrieved from TAIR. The MsNAC and AtNAC protein sequences were aligned using ClustalW. A phylogenetic tree was reconstructed using MEGA 7.0 software [ 48 ] with the maximum likelihood method. The reliability of the tree topology was assessed with 1000 bootstrap replicates. The final tree was visualized and annotated using EvolView ( https://www.evolgenius.info/evolview/ ) [ 49 ]. Promoter cis-acting element analysis The 2.0 kb genomic sequences upstream of the transcription start sites of all identified MsNAC genes were extracted as putative promoter regions. These sequences were subsequently submitted to the PlantCARE database ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) for the prediction of cis-acting regulatory elements [ 50 ]. The initial predictions were manually inspected to eliminate redundant and low-confidence annotations. The positions and types of the identified cis-elements were then visualized using GSDS 2.0 [ 46 ] to illustrate the promoter architecture. Chromosomal localization, collinearity and selective pressure analysis of MsNAC gene family The chromosomal locations of MsNAC genes were mapped using the genome annotation file (GFF3 format) through the Gene Location Visualization module in TBtools v2.084 [ 51 ]. To investigate gene duplication events, we performed synteny analysis using the Multiple Collinearity Scan toolkit (MCScanX) implemented in TBtools with default parameters. For evolutionary selection pressure analysis, we first aligned all amino acid sequences using MUSCLE in ParaAT v2.0 [ 52 ]. Subsequently, we calculated nonsynonymous (Ka) and synonymous (Ks) substitution rates using KaKs_Calculator v2.0 [ 53 ], interpreting the results as follows: Ka/Ks 1 representing positive selection. Expression analysis of MsNAC gene family RNA-seq raw reads for M. sinostellata roots, stems, and leaves under waterlogging stress and control conditions were retrieved from the NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA1274043. The raw reads were subjected to quality control and adapter trimming using Trimmomatic v0.33 [ 54 ] with parameters: ILLUMINACLIP:2:30:10, LEADING:20, TRAILING:20, SLIDINGWINDOW:4:20, MINLEN:50. The high-quality clean reads were then aligned to the M. sinostellata reference genome using HISAT2 (v2.2.1) [ 55 ] with default parameters. Read counts for each gene were generated using featureCounts [ 56 ] based on the genome annotation file. Gene expression levels were normalized and expressed as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Genes with an FPKM > 0.5 in at least one sample were retained for subsequent analysis. Differential gene expression analysis was conducted using the DESeq2 (v1.16.1) package in R [ 57 ], with significance thresholds set at |log2 (fold change)| > 1 and an adjusted p-value (padj) < 0.05 (Benjamini-Hochberg procedure). Three biological replicates were included for each sample. Annotation and enrichment analysis in Gene Ontology (GO) Databases To perform Gene Ontology (GO) enrichment analysis, the GO annotation file was first downloaded from the Gene Ontology database ( http://geneontology.org/ ) [ 58 ]. The analysis was then conducted using the clusterProfiler R package [ 59 ], supplemented with custom R scripts for specific analytical requirements. RT-qPCR Analysis Gene expression analysis was performed using a CFX96 Touch Deep Well real-time PCR system (Bio-Rad, Hercules, CA, USA). Reactions were carried out in accordance with the manufacturer's protocol for Hieff® qPCR SYBR Green Master Mix (No Rox) (11201ES, Yeasen, Shanghai, China). The housekeeping gene Actin was employed as normalization, and the relative gene expression levels were calculated using the 2^−ΔΔCt^ method [ 60 ]. Each assay included three biological replicates, and primer sequences are provided in Supplementary Table S7. Results Genome-wide identification and characterization of MsNAC genes A comprehensive analysis of the M. sinostellata genome led to the identification of 96 non-redundant NAC transcription factor genes, designated MsNAC1 to MsNAC96 (Supplementary Table S2 , S3). These genes were characterized by considerable diversity in their encoded proteins (Supplementary Table S4). The molecular weight (MW) ranged from 9,854.19 to 90,893.35 Da, the theoretical isoelectric point (pI) from 4.43 to 10.01, and protein length from 85 ( MsNAC 79) to 801 (MsNAC96) amino acids. Based on the instability index, 35 MsNAC proteins were classified as stable (index 40). The aliphatic index ranged from 54.71 to 89.53, and the grand average of hydropathicity (GRAVY) was negative for all proteins, indicating that MsNAC proteins are generally hydrophilic. Subcellular localization predictions suggested that most MsNAC proteins are localized to the nucleus, consistent with their role as transcription factors (Supplementary Table S4). The wide range of these indices indicates that MsNAC proteins possess diverse biochemical properties and likely function in different cellular contexts. Phylogenetic tree and NAC conserved domain analysis To elucidate the evolutionary relationships among the MsNAC proteins, a phylogenetic tree was constructed using the full-length sequences of the 96 MsNACs and 117 NAC proteins from A. thaliana (AtNACs) via the maximum likelihood method. The MsNAC proteins were classified into 17 distinct subgroups (Fig. 1 ), largely congruent with the established nomenclature for Arabidopsis, along with several unclassified members. The distribution of MsNAC members among these subgroups was uneven. The NAM, ANAC063, TREN, and OsNAC7 subgroups were the most populous, containing 10, 8, 8, and 8 members, respectively, whereas the OsNAC8 and AtNAC3 subgroups contained only a single member each. The widespread distribution of MsNAC and AtNAC proteins across shared clades indicates evolutionary conservation of the NAC family between M. sinostellata and A. thaliana. Notably, 14 MsNAC genes formed independent branches and could not be classified into any known subgroup, suggesting that they may represent lineage-specific evolutionary innovations that potentially contribute to the unique biology of M. sinostellata , including its waterlogging tolerance. Chromosomal distribution, gene structure, and protein motif analysis Analysis of gene structures revealed considerable diversity among MsNAC genes, with intron numbers ranging from 3 to 11 and exon numbers from 4 to 12 (Fig. 2 C). Conserved motif analysis using MEME identified 20 distinct motifs (Fig. 2 B). Motifs 1 to 10, which correspond to the core NAC domain (PF02365), were present in all MsNAC members. The motif composition was diversified across the family, with several motifs present in nearly all MsNAC s, likely reflecting conserved domains characteristic of the NAC family. Interestingly, MsNAC11 , MsNAC36 , and MsNAC96 were found to harbor additional domains (e.g., PF01126, PF02298, PF02798, PF13417) beyond the NAC domain, suggesting potential functional diversification (Fig. 2 B). Chromosomal mapping revealed that the 96 MsNAC genes are unevenly distributed across the 19 chromosomes of M. sinostellata (Fig. 3 ). Chromosomes 6 (13 genes), 14 (11 genes), 5 (10 genes), and 8 (9 genes) harbored the highest densities of MsNAC genes, while chromosomes 19 and 20 contained only one gene each. Several tandem gene clusters were identified, such as MsNAC14 - 16 on chromosome 12 and MsNAC36 - 38 on chromosome 2, suggesting that local gene duplication has contributed to the expansion of the family. A notable bias towards telomeric regions was observed in the distribution of MsNAC genes, which may have implications for chromatin accessibility and gene regulation. Cis-acting elements of MsNAC gene family Analysis of the 2,000 bp promoter regions upstream of the translation start sites of all MsNAC genes identified 2,352 cis-acting elements, which were grouped into nine major functional categories (Fig. 4 , Supplementary Table S5). The most abundant elements were associated with light responsiveness (1,100 elements, 46.7%), abscisic acid (ABA) response (454 elements, 19.3%), and methyl jasmonate (MeJA) response (322 elements, 13.7%). Additionally, promoters contained elements responsive to salicylic acid (SA, 62 elements) and MYBHv1 binding sites (MBS, 90 elements). The abundance of these regulatory motifs suggests that MsNAC genes are likely involved in diverse physiological processes through the integration of multiple phytohormone signaling pathways (ABA, MeJA, SA), modulation of light-responsive processes, and coordination of stress adaptation mechanisms, potentially via interactions with MYB transcription factors. Interestingly, MsNAC73 exhibited the highest density of cis-regulatory elements (71) (Fig. 4 ), implying its potential as a central hub for coordinating multiple stress responses. Collinearity and selective pressure analysis of MsNAC gene family To explore the evolutionary relationships of NAC genes across species, we constructed collinearity maps of M. sinostellata with A. thaliana, M. sinica, and M. biondii (Fig. 5 ). Comparative genomic analysis revealed limited homologous gene pairs: 4 between M. sinostellata and A. thaliana, and 1 between M. sinostellata and M. sinica (Table 1). This limited collinearity may reflect the large evolutionary distance between these species or incomplete genome assembly. MsNAC 16 showed a collinear relationship with M. sinica, suggesting strong evolutionary conservation predating ancestral divergence events. To assess evolutionary selection pressures, we calculated Ka/Ks ratios for 27 duplicated gene pairs. All pairs exhibited Ka/Ks ratios less than 1, ranging from 0.0039 to 0.4248, indicating strong purifying selection. The duplicated gene pair MsNAC 65- MsNAC 14 had a Ka/Ks value close to 0, indicating particularly strong purifying selection pressure and functional conservation during evolution (Supplementary Table S6). Expression profiling of genes under waterlogging stress Expression profiling of MsNAC genes under waterlogging stress To investigate the involvement of MsNAC genes in M. sinostellata 's response to waterlogging stress, we analyzed their expression profiles in roots, stems, and leaves at various treatment time points (0 h, 6 h, 72 h) using available transcriptomic data (Fig. 6 , Supplementary Figure S1 and S4). Hierarchical clustering based on FPKM values revealed distinct tissue-specific and stress-responsive expression patterns (Fig. 6 A). Our analysis unveiled unique tissue-specific expression profiles for MsNAC genes (Fig. 6 B, Supplementary Figure S1 and S4). Specifically, MsNAC 3, MsNAC 4, MsNAC 10, MsNAC 29, MsNAC 67, MsNAC 75, MsNAC 83, MsNAC 85, MsNAC 87, MsNAC 91, and MsNAC 92 exhibited a preference for root tissue, exhibiting high transcript levels in root controls (RCK_0 h) but minimal expression in stems and leaves. On the other hand, MsNAC 6, MsNAC 59, and MsNAC 69 displayed a preference for stem tissue, with abundant transcripts in stem controls (SCK_0 h) but limited expression in other tissues. Additionally, MsNAC 17, MsNAC 30, MsNAC 36, MsNAC 38, MsNAC 42, MsNAC 47 and MsNAC 53 exhibited a preference for leaf tissue, demonstrating moderate expression levels in leaf controls (LCK_0 h) while being nearly absent in roots and stems. Under waterlogging stress, MsNAC genes exhibited distinct expression dynamics across tissues. In leaves, MsNAC 41, MsNAC 60, and MsNAC 96 were consistently upregulated, whereas MsNAC 38 and MsNAC 79 showed an initial increase at 6 h followed by downregulation at 72 h. In roots, a strikingly distinct expression pattern was observed for MsNAC 66, which exhibited significant and sustained upregulation throughout the stress period, suggesting a potential role in maintaining long-term adaptation. In contrast, another group, including MsNAC14 , MsNAC16 , MsNAC63 , MsNAC65 , and MsNAC70 , displayed a transient induction pattern, characteristic of early-responsive regulators. In stems, MsNAC7 , MsNAC22 , and MsNAC46 were moderately induced at 6 h but declined by 72 h; in contrast, MsNAC 88 displayed delayed induction, being significantly activated only at 72 h. Transcriptomic analysis of M. sinostellata under waterlogging stress unveiled distinct spatial and temporal expression profiles among MsNAC genes, underscoring their involvement in tissue-specific development and stress response mechanisms. GO enrichment analysis GO enrichment analysis was performed to infer the potential biological functions of the MsNAC proteins (Fig. 7 ). The results revealed significant enrichment in terms related to meristem development and maintenance (e.g., shoot apical meristem specification, embryonic meristem initiation), regulation of transcription, root cap development, leaf senescence, and flavonoid biosynthetic processes. This enrichment profile underscores the potential dual roles of MsNAC genes in regulating both developmental programs and stress response mechanisms, the latter potentially through the modulation of protective compounds like flavonoids. Validation of RNA-seq Data by RT-qPCR Based on the transcriptomic data, which indicated more differentially expressed genes in root tissues, we selected several root-expressed genes for RT-qPCR validation. The RT-qPCR results confirmed that the expression levels of the selected MsNAC genes were consistent with the RNA-seq data (Fig. 8 ). These results strongly support the central role of these genes in mediating waterlogging stress responses in root tissues. Discussion This study presents the first genome-wide analysis of the NAC transcription factor family in the waterlogging-tolerant woody species M. sinostellata . While our findings provide strong correlative evidence linking specific MsNAC s to the stress response, we acknowledge that the functional characterization of these candidates remains to be experimentally validated. Evolutionary Expansion and Diversification of the MsNAC Family We identified 96 MsNAC genes in M. sinostellata , a number comparable to tomato (93) [ 14 ] and cassava (96) [ 61 ], but smaller than in species like poplar (270) [ 6 ] and oat (333) [ 62 ]. This variation is often attributed to differential whole-genome duplication (WGD) and segmental duplication events during plant evolution [ 6 ]. The presence of 27 duplicated gene pairs within the MsNAC family (Supplementary Table S6), coupled with Ka/Ks ratios all significantly less than 1 (purifying selection), indicates that small-scale duplication events, particularly tandem duplications, have been a significant force in its expansion, with strong selective pressure to maintain essential functions. The extreme conservation observed in the MsNAC65 - MsNAC14 pair (Ka/Ks ≈ 0) suggests these genes perform critical, non-redundant functions. Phylogenetic analysis classified most MsNAC s into 17 subgroups conserved with A. thaliana [ 9 ], indicating the early establishment of major NAC functional clades. However, the 14 unclassified MsNAC genes are of particular interest. Similar lineage-specific NAC members have been reported in other species [ 63 , 64 ], and we hypothesize that these unclassified MsNAC s may represent evolutionary innovations contributing to the unique waterlogging tolerance of M. sinostellata . Structural and Promoter Analysis Suggests Complex Regulatory Capacity The diversity in gene structure (intron number 3–11) and the identification of additional functional domains in proteins like MsNAC 11, MsNAC 36, and MsNAC 96 point to functional diversification. Promoter analysis revealed a complex regulatory landscape, dominated by light-, ABA-, and MeJA-responsive elements. The co-occurrence of these elements suggests the existence of a sophisticated light-hormone-MYB regulatory network governing MsNAC expression. This integration may be crucial for M. sinostellata to coordinate energy metabolism (via light signaling) with stress defense programs (via ABA/JA) in its natural shady, humid, and potentially waterlogged understory habitat. Tissue-Specific and Dynamic Expression Under Waterlogging Stress Expression profiling revealed a spatiotemporally complex response to waterlogging, with the most pronounced changes in roots, the primary organ sensing hypoxia. In roots, the sustained upregulation of MsNAC66 strongly suggests its potential as a key regulator of long-term tolerance, analogous to ANAC013 in Arabidopsis [ 65 ]. In contrast, genes like MsNAC 14, MsNAC 16, MsNAC 63, MsNAC 65, and MsNAC 70 showed transient induction, characteristic of early-response regulators that initiate defense programs. Notably, a set of root-preferential genes ( MsNAC 3, MsNAC 4, MsNAC 29, MsNAC 67, MsNAC 85, MsNAC 91, MsNAC 92) was persistently downregulated, likely representing an energy-saving strategy by repressing non-essential growth processes during stress, a strategy also observed in other species [ 66 ]. In leaves, sustained upregulation of MsNAC 41, MsNAC 60, and MsNAC96 may mediate systemic responses, while transient patterns in others (e.g., MsNAC 38, MsNAC 79) might relate to modulating senescence. In stems, the delayed strong activation of MsNAC 88 points to a potential role in longer-term acclimation, such as aerenchyma or adventitious root formation. A Proposed Model for MsNAC Function in Waterlogging Tolerance Synthesizing our findings, we propose a root-centric model for the role of MsNAC s in M. sinostellata 's waterlogging tolerance: (1) Immediate Alert & Defense: Early-responsive MsNAC s (e.g., MsNAC 14/16/63/65/70) are transiently induced to activate initial hypoxic responses (e.g., fermentation, ROS scavenging); (2) Sustained Core Defense: A subset (e.g., MsNAC 66) is stably upregulated to maintain core tolerance mechanisms for prolonged survival. (3) Energy Economy Management: Root-preferential developmental MsNAC s (e.g., MsNAC 3/4) are repressed to conserve energy. (4) Systemic Coordination & Acclimation: Specific MsNAC s in aerial parts adjust photosynthesis, and potentially promote aerenchyma formation (stems) and adventitious root development, improving overall aeration. This model highlights the functional diversification and specialization within the MsNAC family, facilitating a coordinated response from initial signaling to long-term anatomical adaptation. Limitations and Future Perspectives It is important to note that the roles proposed for the MsNAC candidates in our working model are primarily inferred from their expression patterns and promoter analyzes. While these correlational data are highly suggestive, direct functional evidence is required to confirm their biological roles. Future work should focus on functional validation using approaches such as heterologous expression in model plants (e.g., A. thaliana ) to assess their impact on waterlogging tolerance phenotypes, and/or the use of techniques like EMSA and dual-luciferase assays to identify their direct downstream targets. The candidate genes prioritized in this study, particularly the root-specific MsNAC66 and the transiently induced cluster, provide a high-value starting point for such in-depth mechanistic investigations. Conclusions In conclusion, this study provides the first genome-wide identification and characterization of the NAC transcription factor family in M. sinostellata . We identified 96 MsNAC genes that have evolved under strong purifying selection, with lineage-specific expansions potentially contributing to waterlogging adaptation. The complex cis-regulatory landscape of these genes suggests an advanced capacity to integrate light, hormone, and stress signals. Most importantly, the spatiotemporally dynamic expression profiles under waterlogging stress prioritize key candidate genes for future functional validation, such as the persistently upregulated MsNAC66 and the transiently induced MsNAC14 / 16/63/65 / 70 . The resources and insights generated here lay a solid foundation for unraveling the molecular mechanisms of waterlogging tolerance in woody perennials and for the molecular breeding of resilient Magnolia cultivars.This study fills a critical knowledge gap regarding stress-responsive transcription factors in the Magnoliaceae family and provides valuable genetic resources for the molecular breeding of resilient ornamental trees, which is increasingly important in an era of climate change marked by frequent flooding events. Abbreviations ABA Abscisic acid FPKM Fragments Per Kilobase of transcript per Million mapped reads GO Gene Ontology HMM Hidden Markov Model JA Jasmonic acid MeJA Methyl jasmonate NAC NAM, ATAF1/2, CUC2 ROS Reactive oxygen species RT-qPCR Reverse transcription quantitative polymerase chain reaction SA Salicylic acid TF Transcription factor WGD Whole-genome duplication Declarations Acknowledgements We thank all our colleagues for providing useful discussions and technical assistance. Authors contributions F.X. conceived and designed the study, conducted the bioinformatics analysis, prepared the experimental materials, performed the experiments and data analysis, wrote the manuscript, and prepared the figures and tables. F.L., Z.H., Y.H., and D.F. were involved in sample collection and data analysis. L.R., L.H., and X.B. participated in the study design and revised the manuscript. W.Y. conceived and designed the study, contributed to data analysis, wrote and revised the manuscript, and provided funding support. All authors read and approved the final manuscript. Funding This work was financially supported by the Youth Project of the Basic Research Program of Shaanxi Academy of Sciences (grant no. 2024k-20); Xi’an Agricultural Technology Research Project (grant no. 24NYGG0071); Applied Technology R&D Project of Shaanxi Academy of Sciences (grant no. 2025k-09); Shaanxi Provincial Innovation Capability Support Plan-Regional Science and Technology Innovation Demonstration Station (grant no. 2023XYSF-08); Platform Project of Shaanxi Academy of Sciences (grant no. 2024j-03); General Project of Shaanxi Key Research and Development Program, 2025SF-YBXM-502 (grant no. 2025SF-YBXM-502); Innovation Talent Promotion Program of Shaanxi Provincial Department of Science and Technology-Science and Technology Innovation Team Project (grant no. 2021TD-33); The Industrialization project of “One Institute, One Product” under the Science and Technology Plan of Shaanxi Academy of Sciences (grant no. 2019K-02). The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Availability of data and materials The raw RNA-seq data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1274043. All genome assembly data and raw transcriptome sequencing reads of M. sinostellata in this study have been deposited in NCBI under accession number PRJNA1169943. The genome annotation information of M. sinostellata has been uploaded to the figshare database (https://figshare.com/account/items/27184485/edit). The genome assembly data of M. sinostellata from this study have also been stored in Science DB under accession number CSTR 31253.11.sciencedb.31088, with a DOI of 10.57760/sciencedb.31088. All other data generated or analyzed during this study are included in this published article and its supplementary information files. Ethics approval and consent to participate We confirm that all experimental research on M. sinostellata , including the collection of plant material, complied with relevant institutional, national, and international guidelines and legislation. The M. sinostellata plants used in this experiment were authorized by Xi'an Botanical Garden of Shaanxi Province, Institute of Botany of Shaanxi Province. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details 1 Xi’an Botanical Garden of Shaanxi Province, Institute of Botany of Shaanxi Province, Xi’an, 710061, China 2 College of Tropical Agriculture and Forestry, Hainan University, Haikou, 570100, China References Voesenek LAC, Bailey-Serres J. Flood adaptive traits and processes: an overview. New Phytol. 2015;206(1):57–73. Fukao T, Barrera-Figueroa BE, Juntawong P, Peña-Castro JM. Submergence and waterlogging stress in plants: a review highlighting research opportunities and understudied aspects. Front Plant Sci. 2019;10:340. Gibbs DJ, Lee SC, Md Isa N, Gramuglia S, Fukao T, Bassel GW, et al. Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants. Nature. 2011;479(7373):415–8. Nuruzzaman M, Sharoni AM, Kikuchi S. Roles of NAC transcription factors in the regulation of biotic and abiotic stress responses in plants. Front Microbiol. 2013;4:248. Puranik S, Sahu PP, Srivastava PS, Prasad M. NAC proteins: regulation and role in stress tolerance. Trends Plant Sci. 2012;17(6):369–81. Han K, Zhao Y, Sun Y, Li Y. NACs, generalists in plant life. Plant Biotechnol J. 2023;21(12):2433–57. Xiong H, He H, Chang Y, Miao B, Liu Z, Wang Q, et al. Multiple roles of NAC transcription factors in plant development and stress responses. J Integr Plant Biol. 2025;67(1):1–22. Han K, Zhao Y, Liu J, Tian Y, El-Kassaby YA, Qi Y, et al. Genome-wide investigation and analysis of NAC transcription factor family in Populus tomentosa and expression analysis under salt stress. Plant Biol. 2024;26(5):764–76. Ooka H, Satoh K, Doi K, Nagata T, Otomo Y, Murakami K, et al. Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsis thaliana . DNA Res. 2003;10(6):239–47. Nuruzzaman M, Manimekalai R, Sharoni AM, Satoh K, Kondoh H, Ooka H, et al. Genome-wide analysis of NAC transcription factor family in rice. Gene. 2010;465(1–2):30–44. Guerin C, Roche J, Allard V, Ravel C, Mouzeyar S, Bouzidi MF. Genome-wide analysis, expansion and expression of the NAC family under drought and heat stresses in bread wheat ( T. aestivum L). PLoS ONE. 2019;14(3):e0213390. Lu M, Sun QP, Zhang DF, Wang TY, Pan JB. Identification of stress-related NAC transcription factor members in maize ( Zea mays L.) and characterization of the expression pattern of these genes. Biochem Biophys Res Commun. 2015;462(2):144–50. Liu M, Ma Z, Sun W, Huang L, Wu Q, Tang Z, et al. Genome-wide analysis of the NAC transcription factor family in Tartary buckwheat ( Fagopyrum tataricum ). BMC Genomics. 2019;20(1):113. Jin JF, Wang ZQ, He QY, Wang JY, Li PF, Xu JM, et al. Genome-wide identification and expression analysis of the NAC transcription factor family in tomato (Solanum lycopersicum) during aluminum stress. BMC Genomics. 2020;21(1):288. Gong X, Zhao L, Song X, Lin Z, Gu B, Yan J, et al. Genome-wide analyses and expression patterns under abiotic stress of NAC transcription factors in white pear ( Pyrus bretschneideri ). BMC Plant Biol. 2019;19(1):251. Ning Y, Liu J, Song B, Xu H, Liu Z, Chen L. Genome-wide analyses of the NAC transcription factor family to reveal the potential candidate genes responding to powdery mildew in balsam pear. Plant Biotechnol Rep. 2023;17(6):917–30. Arroyo-Álvarez E, Chan-León A, Girón-Ramírez A, Fuentes G, Estrella-Maldonado H, Santamaría JM. Genome-Wide analysis of WRKY and NAC transcription factors in Carica papaya L. and their possible role in the loss of drought tolerance by recent cultivars through the domestication of their wild ancestors. Plants. 2023;12(15):2775. Bennett T, van den Toorn A, Sanchez-Perez GF, Campilho A, Willemsen V, Snel B, et al. SOMBRERO, BEARSKIN1, and BEARSKIN2 regulate root cap maturation in Arabidopsis. Plant Cell. 2010;22(3):640–54. Xie Q, Frugis G, Colgan D, Chua NH. Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development. Genes Dev. 2000;14(23):3024–36. Liang C, Wang Y, Zhu Y, Tang J, Hu B, Liu L, et al. OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice. Proc Natl Acad Sci U S A. 2014;111(28):10013–8. Aida M, Ishida T, Fukaki H, Fujisawa H, Tasaka M. Genes involved in organ separation in Arabidopsis: An analysis of the cup-shaped cotyledon mutant. Plant Cell. 1997;9(6):841–57. Takeda S, Hanano K, Kariya A, Shimizu S, Zhao L, Matsui M, et al. CUP-SHAPED COTYLEDON1 transcription factor activates the expression of LSH4 and LSH3, two members of the ALOG gene family, in shoot organ boundary cells. Plant J. 2011;66(6):1066–77. Hu H, Dai M, Yao J, Xiao B, Li X, Zhang Q, et al. Overexpressing a NAC transcription factor enhances drought resistance and salt tolerance in rice. Plant Physiol. 2006;141(3):1159–75. Xu ZY, Kim SY, Hyeon DY, Kim DH, Dong T, Park Y, et al. The Arabidopsis NAC transcription factor ANAC096 cooperates with bZIP-type transcription factors in dehydration and osmotic stress responses. Plant Cell. 2013;25(11):4708–24. Zheng L, Hu Y, Yang T, Wang Z, Wang D, Jia L, et al. A root cap-localized NAC transcription factor controls root halotropic response to salt stress in Arabidopsis. Nat Commun. 2024;15(1):2061. Yarra R, Wei W. The NAC-type transcription factor GmNAC20 improves cold, salinity tolerance, and lateral root formation in transgenic rice plants. Funct Integr Genomics. 2021;21(3–4):473–87. Mei C, Yang J, Mei Q, Jia D, Yan P, Feng B, et al. MtNAC104 positively regulates apple cold tolerance via CBF-dependent and CBF-independent pathways. Plant Biotechnol J. 2023;21(10):2057–73. Shan W, Kuang JF, Lu WJ, Chen JY. Banana fruit NAC transcription factor MaNAC1 is a direct target of MaICE1 and involved in cold stress through interacting with MaCBF1 . Plant Cell Environ. 2014;37(9):2116–27. Rauf M, Arif M, Fisahn J, Xue GP, Balazadeh S, Mueller-Roeber B. NAC transcription factor speedy hyponastic growth regulates flooding-induced leaf movement in Arabidopsis. Plant Cell. 2013;25(12):4941–51. Xie Z, Jin L, Sun Y, Zhan C, Tang S, Qin T, et al. OsNAC120 balances plant growth and drought tolerance by integrating GA and ABA signaling in rice. Plant Commun. 2024;5(1):100782. Wu A, Allu AD, Garapati P, Siddiqui H, Dortay H, Zanor MI, et al. JUNGBRUNNEN1, a reactive oxygen species-responsive NAC transcription factor, regulates longevity in Arabidopsis. Plant Cell. 2012;24(2):482–506. Geng LF, Su L, Fu LF, Lin S, Zhang JM, Liu QH, et al. Genome-wide analysis of the rose ( Rosa chinensis ) NAC family and characterization of RcNAC091 . Plant Mol Biol. 2022;108(4–5):605–19. Zhuo X, Zheng T, Zhang Z, Zhang Y, Jiang L, Ahmad S, et al. Genome-wide analysis of the NAC transcription factor gene family reveals differential expression patterns and cold-stress responses in the woody plant Prunus mume . Genes. 2018;9(10):494. Wang ZQ, Ni LJ, Liu DN, Fu ZK, Hua JF, Lu ZG, et al. Genome-wide identification and characterization of NAC family in Hibiscus hamabo Sieb. et Zucc. under various abiotic stresses. Int J Mol Sci. 2022;23(6):3055. Wang Q, Zhou L, Yuan M, Peng F, Zhu X, Wang Y. Genome-wide identification of NAC gene family members of tree peony ( Paeonia suffruticosa Andrews) and their expression under heat and waterlogging stress. Int J Mol Sci. 2024;25(17):9312. Wang CT, Ye CL. The reasons for the endangerment of Endemic wild Precious plants in Zhejiang Province and the protection countermeasures. Fujian Forestry Sci Technol. 2007;34(2):202–4. Wu YF, Xu JN, Shen YM, Shi CG, Ren MJ, Qiu JX, et al. Analysis of the distribution characteristics of the endangered plant Magnolia sinostellata and its association with the properties of Habitat Soil. For Resour Manage. 2023;1:62–9. Wang J, Wang YL, Wu YF. Establishment and application of evaluation system of morphological waterlogging tolerance of magnoliaceae. Landsc Archit Plants. 2022;5(44):210. El-Gebali S, Mistry J, Bateman A, Eddy SR, Luciani A, Potter SC, et al. The Pfam protein families database in 2019. Nucleic Acids Res. 2019;47(D1):D427–32. Finn RD, Clements J, Eddy SR. HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 2011;39(suppl2):W29–37. Letunic I, Khedkar S, Bork P. SMART: recent updates, new developments and status in 2020. Nucleic Acids Res. 2021;49(D1):D458–60. Lu S, Wang J, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, et al. CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Res. 2020;48(D1):D265–8. Marchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, et al. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res. 2017;45(D1):D200–3. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, et al. Protein Identification and Analysis Tools on the ExPASy Server. In: Walker JM, editor. The Proteomics Protocols Handbook. Totowa, NJ: Humana; 2005. pp. 571–607. Chou KC, Shen HB. Cell-PLoc 2.0: an improved package of web-servers for predicting subcellular localization of proteins in various organisms. Nat Sci. 2010;2(10):1090–103. Hu B, Jin J, Guo AY, Zhang H, Luo J, Gao G. GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics. 2015;31(8):1296–7. Bailey TL, Johnson J, Grant CE, Noble WS. The MEME Suite. Nucleic Acids Res. 2015;43(W1):W39–49. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33(7):1870–4. Subramanian B, Gao S, Lercher MJ, Hu S, Chen WH. Evolview v3: a webserver for visualization, annotation, and management of phylogenetic trees. Nucleic Acids Res. 2019;47(W1):W270–5. Lescot M, Déhais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, et al. 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(1):325–7. Chen C, Wu Y, Li J, Wang X, Zeng Z, Xu J, et al. TBtools-II: A one for all, all for one bioinformatics platform for biological big-data mining. Mol Plant. 2023;16(11):1733–42. Zhang Z, Xiao J, Wu J, Zhang H, Liu G, Wang X, et al. ParaAT: A parallel tool for constructing multiple protein-coding DNA alignments. Biochem Biophys Res Commun. 2012;419(4):779–81. Wang D, Zhang Y, Zhang Z, Zhu J, Yu J. KaKs_Calculator 2.0: a toolkit incorporating gamma-series methods and sliding window strategies. Genomics Proteom Bioinf. 2010;8(1):77–80. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114–20. Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019;37(8):907–15. Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30(7):923–30. Love MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550. The Gene Ontology Consortium. The Gene Ontology resource: enriching a GOld mine. Nucleic Acids Res. 2021;49(D1):D325–34. Wu T, Hu E, Xu S, Chen M, Guo P, Dai Z, et al. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. Innov (Camb). 2021;2(3):100141. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 – ∆∆CT method. Methods. 2001;25(4):402–8. Hu W, Wei Y, Xia Z, Yan Y, Hou X, Zou M, et al. Genome-Wide Identification and Expression Analysis of the NAC Transcription Factor Family in Cassava. PLoS ONE. 2015;10(8):e0136993. Xu Y, Cheng J, Hu H, Yan L, Jia J, Wu B. Genome-Wide Identification of NAC Family Genes in Oat and Functional Characterization of AsNAC109 in Abiotic Stress Tolerance. Plants. 2024;13(7):1017. Singh AK, Sharma V, Pal AK, Acharya V, Ahuja PS. Genome-wide organization and expression profiling of the NAC transcription factor family in potato ( Solanum tuberosum L). DNA Res. 2013;20(4):403–23. Xie WL, Wang YQ. Genome-wide identification of the NAC gene family in Apocynum pictum and its expression analysis under drought stress. Agric Res Arid Areas. 2025;43(2):54–65. Eysholdt-Derzso E, Renziehausen T, Frings S, Frohn S, von Bongartz K, Igisch CP, et al. Endoplasmic reticulum-bound ANAC013 factor is cleaved by RHOMBOID-LIKE 2 during the initial response to hypoxia in Arabidopsis thaliana . Proc Natl Acad Sci U S A. 2023;120(19):e2221308120. Ding Q, Ran J, Chen X, Gao Z, Qian X, Zhang C, et al. Identification of the NAC Family and a Functional Analysis of NoNAC36a Under Flooding Stress in Watercress ( Nasturtium officinale R. Br). Horticulturae. 2024;10(11):1219. Additional Declarations No competing interests reported. Supplementary Files MagnoliasinostellataNAC.xlsx Supportinginformation.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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09:58:02","extension":"xml","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154861,"visible":true,"origin":"","legend":"","description":"","filename":"580f16939ece45c4ab4a0011ca0ebf341structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8390673/v1/1424267d01f163289407481c.xml"},{"id":98990025,"identity":"e3eaa79b-41e6-4ba0-8dee-d26bb7335fe0","added_by":"auto","created_at":"2025-12-25 09:58:02","extension":"html","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":171730,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8390673/v1/e91eba2db2fcb4aa3eed7372.html"},{"id":98990000,"identity":"d01ce38d-67e0-4bfc-a978-06eb7eac2693","added_by":"auto","created_at":"2025-12-25 09:58:01","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":269388,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree of\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. sinostellata\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e NAC proteins.\u003c/strong\u003e The dendrogram was constructed using MEGA7 with the maximum likelihood method. Proteins were clustered into 17 subgroups indicated by different background colors. Different shapes represent different species. Numbers on branches represent bootstrap values (1000 repetitions), with only values ≥ 70% shown.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8390673/v1/e49d5583f8785c3b28458ae6.jpeg"},{"id":98990003,"identity":"448cc19b-975d-49c3-8548-594ea9f3d484","added_by":"auto","created_at":"2025-12-25 09:58:01","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":311148,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic tree, conserved motifs and gene structures of the MsNAC gene family. \u003c/strong\u003e(A) Phylogenetic relationship of MsNAC proteins. (B) Conserved protein motifs identified by MEME analysis. The colorful boxes represent different motifs. (C) Gene structure analysis showing exon-intron organization. Yellow bars represent exons, black lines represent introns. Clustering is based on phylogenetic analysis results.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8390673/v1/0b66aea92d11cbf37a403545.jpeg"},{"id":98990009,"identity":"44e4c796-44ed-4429-93a1-d155b5804de0","added_by":"auto","created_at":"2025-12-25 09:58:02","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":59115,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromosomal distribution of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMsNAC\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes. \u003c/strong\u003eThe 96 \u003cem\u003eMsNAC\u003c/em\u003e genes are unevenly distributed across 19 chromosomes of \u003cem\u003eM. sinostellata\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8390673/v1/925e2b9e35ded0d4008e1492.jpeg"},{"id":99312275,"identity":"c41e3672-5f23-4a7b-978b-74dbab4a17b2","added_by":"auto","created_at":"2025-12-31 16:18:33","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":268921,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePredicted cis-elements in the promoters of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMsNAC\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genes. \u003c/strong\u003eThe distribution of cis-acting elements in the promoter regions is illustrated, with different colors representing various types of elements. The number of cis-acting elements is shown in the colored boxes. Elements are annotated for abscisic acid response, auxin response, drought-inducibility, gibberellin response, light response, low-temperature response, MeJA response, MYB binding site, and salicylic acid response.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8390673/v1/f974578a1a30b17e33eab16d.jpeg"},{"id":99312885,"identity":"6ba634b5-2660-444b-aca6-b21216dd1ae8","added_by":"auto","created_at":"2025-12-31 16:19:33","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":101886,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGenome-wide collinearity analysis of NAC gene families among \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. sinostellata\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. thaliana\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. sinica\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eRed lines indicate orthologous gene pairs. The limited number of collinear gene pairs may reflect evolutionary distance or genome assembly completeness.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8390673/v1/ebf58cd78ea7768a20e4f0b2.jpeg"},{"id":98990010,"identity":"b9310333-beb9-4899-aa4b-bc5f49600edc","added_by":"auto","created_at":"2025-12-25 09:58:02","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":188839,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression patterns analysis of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMsNAC\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene family in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. sinostellata\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e under waterlogging stress. \u003c/strong\u003e(A) Box plots showing the expression of \u003cem\u003eMsNAC\u003c/em\u003e genes in different tissues (root, stem, leaf) at different waterlogging treatment times (0 h, 6 h, 72 h), reflecting the distribution of gene expression levels (log₁₀ (FPKM + 1)); (B) Heatmap of \u003cem\u003eMsNAC\u003c/em\u003e gene expression under different tissues and waterlogging treatment times. Hierarchical clustering is used to show the similarity of gene expression patterns, with color intensity representing the level of expression (red for high, blue for low). (C) Expression profile of \u003cem\u003eMsNAC\u003c/em\u003egenes under waterlogging stress. Heatmap depicting the expression patterns (log-transformed FPKM values) of \u003cem\u003eMsNAC\u003c/em\u003e genes in roots, stems, and leaves at 0 h, 6 h, and 72 h of waterlogging treatment.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8390673/v1/22c9d97f14a718f804f5c0c7.jpeg"},{"id":98990016,"identity":"ca9d4b5f-3f24-4bdd-8444-b44115c9e832","added_by":"auto","created_at":"2025-12-25 09:58:02","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":142627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChord diagram and bubble plot of GO functional enrichment for \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMsNAC\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenes.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8390673/v1/856abf0cd2f142865a3f9d5c.jpeg"},{"id":98990013,"identity":"35001dad-06dc-46a6-8c7c-0b3a2b40c26f","added_by":"auto","created_at":"2025-12-25 09:58:02","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":152982,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression verification of six candidate \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMsNAC\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e hub genes under waterlogging stress.\u003c/strong\u003e Differential expression was visualized through colored bars (RT-qPCR) and connected scatter points (RNA-Seq). All data are the means±SE of three biological replicates, with the significance of intergroup differences indicated by asterisks above: *** (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001), ** (\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01), and * (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8390673/v1/e6c5f8486bd79af8ac3fa035.jpeg"},{"id":100629744,"identity":"946135c1-481d-4ff9-a774-08be08d33ffa","added_by":"auto","created_at":"2026-01-19 21:12:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2890461,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8390673/v1/44440425-52ea-4ede-bd90-d65611c626ee.pdf"},{"id":99312705,"identity":"c00fb73e-959a-4e36-a585-64d43de49e7a","added_by":"auto","created_at":"2025-12-31 16:19:21","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":209940,"visible":true,"origin":"","legend":"","description":"","filename":"MagnoliasinostellataNAC.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8390673/v1/736d781b2b705248d6919d06.xlsx"},{"id":98990005,"identity":"9f559d22-4c40-4ad0-a6c6-1e46adedb151","added_by":"auto","created_at":"2025-12-25 09:58:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":568379,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8390673/v1/909a4a0fca92c012a26f7c84.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-Wide Identification and Expression Profiling of the NAC Transcription Factor Family in the Waterlogging-Tolerant Tree Magnolia sinostellata","fulltext":[{"header":"Introduction","content":"\u003cp\u003eClimate change has escalated the frequency of extreme weather events, particularly heavy rainfall and flooding, rendering waterlogging stress a critical abiotic constraint on plant growth, development, and geographical distribution [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In waterlogged or poorly drained soils, oxygen depletion (hypoxia) severely disrupts root respiration and energy metabolism. Prolonged waterlogging instigates a cascade of physiological disturbances, including reactive oxygen species (ROS) accumulation, membrane damage, and photosynthetic inhibition, ultimately culminating in growth arrest or plant death [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. To mitigate waterlogging stress, plants have evolved sophisticated adaptive strategies involving both morphological modifications (e.g., aerenchyma formation, adventitious root development) and molecular reprogramming (e.g., induction of anaerobic metabolism-related genes) [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Within these adaptive frameworks, transcription factors (TFs) serve as central regulators by modulating the expression of stress-responsive genes.\u003c/p\u003e \u003cp\u003eThe NAC (NAM, ATAF1/2, CUC2) family represents a plant-specific class of TFs, typically phylogenetically classified into 18 subgroups [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. NAC proteins are characterized by a conserved N-terminal DNA-binding NAC domain and a highly divergent C-terminal transcriptional regulation region [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Recent research has underscored their pleiotropic roles in various aspects of plant development (e.g., root architecture, leaf senescence) and stress responses (e.g., drought, waterlogging, cold) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. This functional versatility originates from their capacity to regulate gene expression through DNA binding, protein-protein interactions, and involvement in epigenetic modifications [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Consequently, the NAC family has emerged as a focal point in plant biology research. Genome-wide identifications of NAC genes have been extensively reported across diverse plant species, including \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (117 genes) [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], rice (151 genes) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], wheat (559 genes) [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], maize (157 genes) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], tartary buckwheat (80 genes) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], tomato (93 genes) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], pear (183 genes) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], bitter gourd (90 genes) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and papaya (66 genes) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe regulatory mechanisms and functional roles of \u003cem\u003eNAC\u003c/em\u003es in different plants have been widely reported. For example, in \u003cem\u003eA. thaliana\u003c/em\u003e, SOMBRERO (SMB) and Bearskin 1/2 (BRN1/2) regulate the maturation and shedding of root cap cells [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], while \u003cem\u003eNAC1\u003c/em\u003e promotes lateral root formation by activating auxin response genes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Rice \u003cem\u003eOsNAP\u003c/em\u003e and \u003cem\u003eA. thaliana ANAC092\u003c/em\u003e/\u003cem\u003eORE1\u003c/em\u003e promote leaf senescence by activating senescence-associated genes (SAGs) and chlorophyll degradation genes such as NYC1 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. CUC1/2/3 regulate the separation of cotyledons and flower organs by inhibiting the proliferation of boundary cells or activating the LSH gene family [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In stress responses, rice \u003cem\u003eSNAC1\u003c/em\u003e enhances drought resistance by activating drought-responsive genes such as \u003cem\u003eOsAP37\u003c/em\u003e and inhibiting ROS accumulation [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]; \u003cem\u003eA. thaliana ANAC096\u003c/em\u003e interacts with the ABA signaling pathway to regulate the dehydration response [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. \u003cem\u003eA. thaliana\u003c/em\u003e SMB mediates root salt aversion by regulating auxin distribution [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and soybean \u003cem\u003eGmNAC20\u003c/em\u003e improves salt tolerance by promoting proline accumulation [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Apple \u003cem\u003eMdNAC104\u003c/em\u003e enhances cold resistance through CBF-dependent and CBF-independent pathways [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], while banana \u003cem\u003eMaNAC1\u003c/em\u003e interacts with the ICE1-CBF pathway [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. \u003cem\u003eA. thaliana SHYG\u003c/em\u003e promotes rapid elongation of petioles through ethylene signaling to cope with flooding stress [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Furthermore, \u003cem\u003eNAC\u003c/em\u003es can coordinate development and stress response by integrating hormones (such as ABA, JA), ROS, and light signals. For instance, rice O\u003cem\u003esNAC120\u003c/em\u003e balances GA-mediated growth and ABA-mediated drought response [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], while \u003cem\u003eA. thaliana\u003c/em\u003e JUNGBRUNNEN1 (\u003cem\u003eJUB1\u003c/em\u003e) delays leaf senescence and enhances drought tolerance by inhibiting ROS accumulation [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBeyond model plants and crops, systematic studies on the NAC gene family have also been conducted in ornamental species. \u003cem\u003eRcNAC091\u003c/em\u003e was highly expressed in rose (\u003cem\u003eRosa chinensis\u003c/em\u003e) under drought and salt stress, enhancing tolerance by regulating ROS scavenging and cell wall modification pathways while playing a dual regulatory role in flower organ development [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Multiple \u003cem\u003ePmNAC\u003c/em\u003e genes (such as \u003cem\u003ePmNAC1\u003c/em\u003e) were differentially expressed in plum blossoms (\u003cem\u003ePrunus mume\u003c/em\u003e), driving xylem development and activation of the ABA signaling pathway to improve frost damage tolerance [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The \u003cem\u003eHhNAC\u003c/em\u003e genes (such as \u003cem\u003eHhNAC25\u003c/em\u003e) in Hibiscus hamabo were significantly induced under various abiotic stresses such as salt, drought, and heavy metals, maintaining cell membrane integrity and photosynthetic efficiency by activating the MAPK signaling cascade and stress-response genes [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Under heat and waterlogging stress, the \u003cem\u003ePsNAC\u003c/em\u003e gene family (including \u003cem\u003ePsNAC72\u003c/em\u003e) was differentially expressed in peonies, participating in heat shock protein synthesis and hypoxia response to improve energy metabolism efficiency and root damage avoidance [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Collectively, these studies indicate that NAC transcription factors are core hubs for plants to adapt to the environment and regulate development, providing important targets for agricultural biotechnology.\u003c/p\u003e \u003cp\u003e \u003cem\u003eMagnolia\u003c/em\u003e species are economically important as ornamental and medicinal trees in China. However, most exhibit high sensitivity to waterlogging, which greatly limits their promotion and application in urban and wetland landscapes. \u003cem\u003eMagnolia sinostellata\u003c/em\u003e is a rare and endemic species within the Magnoliaceae family in China, primarily distributed in regions such as Zhejiang and Fujian. It thrives in cool, humid, and water-sufficient environments, representing a rare example of waterlogging tolerance within the Magnoliaceae family and holding considerable value for scientific research and horticulture [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Our previous studies have indicated that \u003cem\u003eM. sinostellata\u003c/em\u003e possesses significant waterlogging tolerance, positioning it as a key resource for mining superior genes and for the genetic improvement of magnolia plants [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. However, the molecular mechanism underlying its waterlogging tolerance remains elusive, hindering the application of molecular breeding strategies in Magnolia.\u003c/p\u003e \u003cp\u003eGiven the central regulatory role of the NAC family in plant responses to abiotic stresses, including waterlogging, and the absence of systematic studies on \u003cem\u003eNAC\u003c/em\u003e genes in Magnoliaceae, we embarked on this research. The recent availability of the \u003cem\u003eM. sinostellata\u003c/em\u003e genome sequence enables a genome-wide investigation of stress-responsive gene families. The objectives of this study were to: (1) systematically identify and characterize the \u003cem\u003eMsNAC\u003c/em\u003e gene family in \u003cem\u003eM. sinostellata\u003c/em\u003e; (2) analyze their phylogenetic relationships, gene structures, conserved motifs, and promoter cis-elements; (3) explore their tissue-specific expression profiles under waterlogging stress; and (4) identify core candidate genes putatively involved in waterlogging tolerance for future functional studies.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003eIdentification and characterization of the\u003c/b\u003e \u003cb\u003eMsNAC\u003c/b\u003e \u003cb\u003egene family\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe whole-genome sequencing data for \u003cem\u003eM. sinostellata\u003c/em\u003e in this study was deposited in NCBI under BioProject accession PRJNA1169943. The \u003cem\u003eA. thaliana\u003c/em\u003e genome and protein sequences were downloaded from TAIR (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). To comprehensively identify NAC family members, a combined strategy utilizing Hidden Markov Model (HMM)-based searches and BLASTp homology analysis was employed. First, the NAC domain HMM profile (PF02365) obtained from Pfam [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] was used to query the \u003cem\u003eM. sinostellata\u003c/em\u003e proteome using HMMER v3.2.1 with an E-value cutoff of 1\u0026times;10⁻⁵ (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Simultaneously, known NAC protein sequences from various plant species (acquired from PlantTFDB; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://planttfdb.gao-lab.org\u003c/span\u003e\u003cspan address=\"http://planttfdb.gao-lab.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) were used as queries for BLASTp searches against the \u003cem\u003eM. sinostellata\u003c/em\u003e proteome (E-value\u0026thinsp;\u0026lt;\u0026thinsp;1\u0026times;10⁻⁵, identity\u0026thinsp;\u0026gt;\u0026thinsp;50%) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Non-redundant candidate sequences from both approaches were merged. The presence of the complete NAC domain in each candidate was rigorously verified using SMART [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], the Conserved Domain Database (CDD) [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and Pfam. Final confirmation was performed using the NCBI Conserved Domain Search tool [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The resulting non-redundant set was designated as the \u003cem\u003eMsNAC\u003c/em\u003e gene family. Physicochemical properties (theoretical isoelectric point pI, molecular weight, instability index) of the identified MsNAC proteins were predicted using ExPASy's ProtParam tool [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Subcellular localization was predicted using Cell-PLoc 2.0 [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGene structure, conserved motif and phylogenetic analysis\u003c/h2\u003e \u003cp\u003eGene structure diagrams (intron/exon organization) were generated based on the genome annotation GFF3 file using the Gene Structure Display Server (GSDS; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.cbi.pku.edu.cn/\u003c/span\u003e\u003cspan address=\"http://gsds.cbi.pku.edu.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. To identify conserved motifs beyond the NAC domain, the protein sequences of the MsNAC members were analyzed using the MEME Suite (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme-suite.org/\u003c/span\u003e\u003cspan address=\"http://meme-suite.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with the following parameters: maximum number of motifs set to 20, and the optimum motif width set between 6 and 50 amino acids [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. For phylogenetic analysis, the full complement of NAC protein sequences from \u003cem\u003eA. thaliana\u003c/em\u003e (AtNACs) was retrieved from TAIR. The MsNAC and AtNAC protein sequences were aligned using ClustalW. A phylogenetic tree was reconstructed using MEGA 7.0 software [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e] with the maximum likelihood method. The reliability of the tree topology was assessed with 1000 bootstrap replicates. The final tree was visualized and annotated using EvolView (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.evolgenius.info/evolview/\u003c/span\u003e\u003cspan address=\"https://www.evolgenius.info/evolview/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePromoter cis-acting element analysis\u003c/h3\u003e\n\u003cp\u003eThe 2.0 kb genomic sequences upstream of the transcription start sites of all identified \u003cem\u003eMsNAC\u003c/em\u003e genes were extracted as putative promoter regions. These sequences were subsequently submitted to the PlantCARE database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for the prediction of cis-acting regulatory elements [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The initial predictions were manually inspected to eliminate redundant and low-confidence annotations. The positions and types of the identified cis-elements were then visualized using GSDS 2.0 [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] to illustrate the promoter architecture.\u003c/p\u003e \u003cp\u003e \u003cb\u003eChromosomal localization, collinearity and selective pressure analysis of\u003c/b\u003e \u003cb\u003eMsNAC\u003c/b\u003e \u003cb\u003egene family\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe chromosomal locations of \u003cem\u003eMsNAC\u003c/em\u003e genes were mapped using the genome annotation file (GFF3 format) through the Gene Location Visualization module in TBtools v2.084 [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. To investigate gene duplication events, we performed synteny analysis using the Multiple Collinearity Scan toolkit (MCScanX) implemented in TBtools with default parameters. For evolutionary selection pressure analysis, we first aligned all amino acid sequences using MUSCLE in ParaAT v2.0 [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Subsequently, we calculated nonsynonymous (Ka) and synonymous (Ks) substitution rates using KaKs_Calculator v2.0 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], interpreting the results as follows: Ka/Ks\u0026thinsp;\u0026lt;\u0026thinsp;1, indicating purifying selection, Ka/Ks\u0026thinsp;=\u0026thinsp;1 suggesting neutral evolution, and Ka/Ks\u0026thinsp;\u0026gt;\u0026thinsp;1 representing positive selection.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression analysis of\u003c/b\u003e \u003cb\u003eMsNAC\u003c/b\u003e \u003cb\u003egene family\u003c/b\u003e\u003c/p\u003e \u003cp\u003eRNA-seq raw reads for \u003cem\u003eM. sinostellata\u003c/em\u003e roots, stems, and leaves under waterlogging stress and control conditions were retrieved from the NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA1274043. The raw reads were subjected to quality control and adapter trimming using Trimmomatic v0.33 [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] with parameters: ILLUMINACLIP:2:30:10, LEADING:20, TRAILING:20, SLIDINGWINDOW:4:20, MINLEN:50. The high-quality clean reads were then aligned to the \u003cem\u003eM. sinostellata\u003c/em\u003e reference genome using HISAT2 (v2.2.1) [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] with default parameters. Read counts for each gene were generated using featureCounts [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] based on the genome annotation file. Gene expression levels were normalized and expressed as Fragments Per Kilobase of transcript per Million mapped reads (FPKM). Genes with an FPKM\u0026thinsp;\u0026gt;\u0026thinsp;0.5 in at least one sample were retained for subsequent analysis. Differential gene expression analysis was conducted using the DESeq2 (v1.16.1) package in R [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], with significance thresholds set at |log2 (fold change)| \u0026gt; 1 and an adjusted p-value (padj)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (Benjamini-Hochberg procedure). Three biological replicates were included for each sample.\u003c/p\u003e\n\u003ch3\u003eAnnotation and enrichment analysis in Gene Ontology (GO) Databases\u003c/h3\u003e\n\u003cp\u003eTo perform Gene Ontology (GO) enrichment analysis, the GO annotation file was first downloaded from the Gene Ontology database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://geneontology.org/\u003c/span\u003e\u003cspan address=\"http://geneontology.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. The analysis was then conducted using the clusterProfiler R package [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], supplemented with custom R scripts for specific analytical requirements.\u003c/p\u003e\n\u003ch3\u003eRT-qPCR Analysis\u003c/h3\u003e\n\u003cp\u003eGene expression analysis was performed using a CFX96 Touch Deep Well real-time PCR system (Bio-Rad, Hercules, CA, USA). Reactions were carried out in accordance with the manufacturer's protocol for Hieff\u0026reg; qPCR SYBR Green Master Mix (No Rox) (11201ES, Yeasen, Shanghai, China). The housekeeping gene Actin was employed as normalization, and the relative gene expression levels were calculated using the 2^\u0026minus;ΔΔCt^ method [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Each assay included three biological replicates, and primer sequences are provided in Supplementary Table S7.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eGenome-wide identification and characterization of\u003c/b\u003e \u003cb\u003eMsNAC\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eA comprehensive analysis of the \u003cem\u003eM. sinostellata\u003c/em\u003e genome led to the identification of 96 non-redundant NAC transcription factor genes, designated MsNAC1 to MsNAC96 (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, S3). These genes were characterized by considerable diversity in their encoded proteins (Supplementary Table S4). The molecular weight (MW) ranged from 9,854.19 to 90,893.35 Da, the theoretical isoelectric point (pI) from 4.43 to 10.01, and protein length from 85 (\u003cem\u003eMsNAC\u003c/em\u003e79) to 801 (MsNAC96) amino acids. Based on the instability index, 35 MsNAC proteins were classified as stable (index\u0026thinsp;\u0026lt;\u0026thinsp;40) and 61 as unstable (index\u0026thinsp;\u0026gt;\u0026thinsp;40). The aliphatic index ranged from 54.71 to 89.53, and the grand average of hydropathicity (GRAVY) was negative for all proteins, indicating that MsNAC proteins are generally hydrophilic. Subcellular localization predictions suggested that most MsNAC proteins are localized to the nucleus, consistent with their role as transcription factors (Supplementary Table S4). The wide range of these indices indicates that MsNAC proteins possess diverse biochemical properties and likely function in different cellular contexts.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePhylogenetic tree and NAC conserved domain analysis\u003c/h2\u003e \u003cp\u003eTo elucidate the evolutionary relationships among the MsNAC proteins, a phylogenetic tree was constructed using the full-length sequences of the 96 MsNACs and 117 NAC proteins from A. thaliana (AtNACs) via the maximum likelihood method. The MsNAC proteins were classified into 17 distinct subgroups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), largely congruent with the established nomenclature for Arabidopsis, along with several unclassified members. The distribution of \u003cem\u003eMsNAC\u003c/em\u003e members among these subgroups was uneven. The NAM, ANAC063, TREN, and OsNAC7 subgroups were the most populous, containing 10, 8, 8, and 8 members, respectively, whereas the OsNAC8 and AtNAC3 subgroups contained only a single member each. The widespread distribution of MsNAC and AtNAC proteins across shared clades indicates evolutionary conservation of the NAC family between \u003cem\u003eM. sinostellata\u003c/em\u003e and A. thaliana. Notably, 14 \u003cem\u003eMsNAC\u003c/em\u003e genes formed independent branches and could not be classified into any known subgroup, suggesting that they may represent lineage-specific evolutionary innovations that potentially contribute to the unique biology of \u003cem\u003eM. sinostellata\u003c/em\u003e, including its waterlogging tolerance.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChromosomal distribution, gene structure, and protein motif analysis\u003c/h3\u003e\n\u003cp\u003eAnalysis of gene structures revealed considerable diversity among \u003cem\u003eMsNAC\u003c/em\u003e genes, with intron numbers ranging from 3 to 11 and exon numbers from 4 to 12 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Conserved motif analysis using MEME identified 20 distinct motifs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Motifs 1 to 10, which correspond to the core NAC domain (PF02365), were present in all \u003cem\u003eMsNAC\u003c/em\u003e members. The motif composition was diversified across the family, with several motifs present in nearly all \u003cem\u003eMsNAC\u003c/em\u003es, likely reflecting conserved domains characteristic of the NAC family. Interestingly, \u003cem\u003eMsNAC11\u003c/em\u003e, \u003cem\u003eMsNAC36\u003c/em\u003e, and \u003cem\u003eMsNAC96\u003c/em\u003e were found to harbor additional domains (e.g., PF01126, PF02298, PF02798, PF13417) beyond the NAC domain, suggesting potential functional diversification (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eChromosomal mapping revealed that the 96 \u003cem\u003eMsNAC\u003c/em\u003e genes are unevenly distributed across the 19 chromosomes of \u003cem\u003eM. sinostellata\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Chromosomes 6 (13 genes), 14 (11 genes), 5 (10 genes), and 8 (9 genes) harbored the highest densities of \u003cem\u003eMsNAC\u003c/em\u003e genes, while chromosomes 19 and 20 contained only one gene each. Several tandem gene clusters were identified, such as \u003cem\u003eMsNAC14\u003c/em\u003e-\u003cem\u003e16\u003c/em\u003e on chromosome 12 and \u003cem\u003eMsNAC36\u003c/em\u003e-\u003cem\u003e38\u003c/em\u003e on chromosome 2, suggesting that local gene duplication has contributed to the expansion of the family. A notable bias towards telomeric regions was observed in the distribution of \u003cem\u003eMsNAC\u003c/em\u003e genes, which may have implications for chromatin accessibility and gene regulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCis-acting elements of\u003c/b\u003e \u003cb\u003eMsNAC\u003c/b\u003e \u003cb\u003egene family\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAnalysis of the 2,000 bp promoter regions upstream of the translation start sites of all \u003cem\u003eMsNAC\u003c/em\u003e genes identified 2,352 cis-acting elements, which were grouped into nine major functional categories (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, Supplementary Table S5). The most abundant elements were associated with light responsiveness (1,100 elements, 46.7%), abscisic acid (ABA) response (454 elements, 19.3%), and methyl jasmonate (MeJA) response (322 elements, 13.7%). Additionally, promoters contained elements responsive to salicylic acid (SA, 62 elements) and MYBHv1 binding sites (MBS, 90 elements). The abundance of these regulatory motifs suggests that \u003cem\u003eMsNAC\u003c/em\u003e genes are likely involved in diverse physiological processes through the integration of multiple phytohormone signaling pathways (ABA, MeJA, SA), modulation of light-responsive processes, and coordination of stress adaptation mechanisms, potentially via interactions with MYB transcription factors. Interestingly, \u003cem\u003eMsNAC73\u003c/em\u003e exhibited the highest density of cis-regulatory elements (71) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), implying its potential as a central hub for coordinating multiple stress responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCollinearity and selective pressure analysis of\u003c/b\u003e \u003cb\u003eMsNAC\u003c/b\u003e \u003cb\u003egene family\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore the evolutionary relationships of NAC genes across species, we constructed collinearity maps of \u003cem\u003eM. sinostellata\u003c/em\u003e with A. thaliana, M. sinica, and M. biondii (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Comparative genomic analysis revealed limited homologous gene pairs: 4 between \u003cem\u003eM. sinostellata\u003c/em\u003e and A. thaliana, and 1 between \u003cem\u003eM. sinostellata\u003c/em\u003e and M. sinica (Table\u0026nbsp;1). This limited collinearity may reflect the large evolutionary distance between these species or incomplete genome assembly. \u003cem\u003eMsNAC\u003c/em\u003e16 showed a collinear relationship with M. sinica, suggesting strong evolutionary conservation predating ancestral divergence events. To assess evolutionary selection pressures, we calculated Ka/Ks ratios for 27 duplicated gene pairs. All pairs exhibited Ka/Ks ratios less than 1, ranging from 0.0039 to 0.4248, indicating strong purifying selection. The duplicated gene pair \u003cem\u003eMsNAC\u003c/em\u003e65-\u003cem\u003eMsNAC\u003c/em\u003e14 had a Ka/Ks value close to 0, indicating particularly strong purifying selection pressure and functional conservation during evolution (Supplementary Table S6).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eExpression profiling of genes under waterlogging stress\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eExpression profiling of \u003cem\u003eMsNAC\u003c/em\u003e genes under waterlogging stress\u003c/div\u003e \u003cp\u003eTo investigate the involvement of \u003cem\u003eMsNAC\u003c/em\u003e genes in \u003cem\u003eM. sinostellata\u003c/em\u003e's response to waterlogging stress, we analyzed their expression profiles in roots, stems, and leaves at various treatment time points (0 h, 6 h, 72 h) using available transcriptomic data (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S4). Hierarchical clustering based on FPKM values revealed distinct tissue-specific and stress-responsive expression patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Our analysis unveiled unique tissue-specific expression profiles for \u003cem\u003eMsNAC\u003c/em\u003e genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S4). Specifically, \u003cem\u003eMsNAC\u003c/em\u003e3, \u003cem\u003eMsNAC\u003c/em\u003e4, \u003cem\u003eMsNAC\u003c/em\u003e10, \u003cem\u003eMsNAC\u003c/em\u003e29, \u003cem\u003eMsNAC\u003c/em\u003e67, \u003cem\u003eMsNAC\u003c/em\u003e75, \u003cem\u003eMsNAC\u003c/em\u003e83, \u003cem\u003eMsNAC\u003c/em\u003e85, \u003cem\u003eMsNAC\u003c/em\u003e87, \u003cem\u003eMsNAC\u003c/em\u003e91, and \u003cem\u003eMsNAC\u003c/em\u003e92 exhibited a preference for root tissue, exhibiting high transcript levels in root controls (RCK_0 h) but minimal expression in stems and leaves. On the other hand, \u003cem\u003eMsNAC\u003c/em\u003e6, \u003cem\u003eMsNAC\u003c/em\u003e59, and \u003cem\u003eMsNAC\u003c/em\u003e69 displayed a preference for stem tissue, with abundant transcripts in stem controls (SCK_0 h) but limited expression in other tissues. Additionally, \u003cem\u003eMsNAC\u003c/em\u003e17, \u003cem\u003eMsNAC\u003c/em\u003e30, \u003cem\u003eMsNAC\u003c/em\u003e36, \u003cem\u003eMsNAC\u003c/em\u003e38, \u003cem\u003eMsNAC\u003c/em\u003e42, \u003cem\u003eMsNAC\u003c/em\u003e47 and \u003cem\u003eMsNAC\u003c/em\u003e53 exhibited a preference for leaf tissue, demonstrating moderate expression levels in leaf controls (LCK_0 h) while being nearly absent in roots and stems.\u003c/p\u003e \u003cp\u003eUnder waterlogging stress, \u003cem\u003eMsNAC\u003c/em\u003e genes exhibited distinct expression dynamics across tissues. In leaves, \u003cem\u003eMsNAC\u003c/em\u003e41, \u003cem\u003eMsNAC\u003c/em\u003e60, and \u003cem\u003eMsNAC\u003c/em\u003e96 were consistently upregulated, whereas \u003cem\u003eMsNAC\u003c/em\u003e38 and \u003cem\u003eMsNAC\u003c/em\u003e79 showed an initial increase at 6 h followed by downregulation at 72 h. In roots, a strikingly distinct expression pattern was observed for \u003cem\u003eMsNAC\u003c/em\u003e66, which exhibited significant and sustained upregulation throughout the stress period, suggesting a potential role in maintaining long-term adaptation. In contrast, another group, including \u003cem\u003eMsNAC14\u003c/em\u003e, \u003cem\u003eMsNAC16\u003c/em\u003e, \u003cem\u003eMsNAC63\u003c/em\u003e, \u003cem\u003eMsNAC65\u003c/em\u003e, and \u003cem\u003eMsNAC70\u003c/em\u003e, displayed a transient induction pattern, characteristic of early-responsive regulators. In stems, \u003cem\u003eMsNAC7\u003c/em\u003e, \u003cem\u003eMsNAC22\u003c/em\u003e, and \u003cem\u003eMsNAC46\u003c/em\u003e were moderately induced at 6 h but declined by 72 h; in contrast, \u003cem\u003eMsNAC\u003c/em\u003e88 displayed delayed induction, being significantly activated only at 72 h. Transcriptomic analysis of \u003cem\u003eM. sinostellata\u003c/em\u003e under waterlogging stress unveiled distinct spatial and temporal expression profiles among \u003cem\u003eMsNAC\u003c/em\u003e genes, underscoring their involvement in tissue-specific development and stress response mechanisms.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGO enrichment analysis\u003c/h2\u003e \u003cp\u003eGO enrichment analysis was performed to infer the potential biological functions of the \u003cem\u003eMsNAC\u003c/em\u003e proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The results revealed significant enrichment in terms related to meristem development and maintenance (e.g., shoot apical meristem specification, embryonic meristem initiation), regulation of transcription, root cap development, leaf senescence, and flavonoid biosynthetic processes. This enrichment profile underscores the potential dual roles of \u003cem\u003eMsNAC\u003c/em\u003e genes in regulating both developmental programs and stress response mechanisms, the latter potentially through the modulation of protective compounds like flavonoids.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eValidation of RNA-seq Data by RT-qPCR\u003c/h2\u003e \u003cp\u003eBased on the transcriptomic data, which indicated more differentially expressed genes in root tissues, we selected several root-expressed genes for RT-qPCR validation. The RT-qPCR results confirmed that the expression levels of the selected \u003cem\u003eMsNAC\u003c/em\u003e genes were consistent with the RNA-seq data (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These results strongly support the central role of these genes in mediating waterlogging stress responses in root tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study presents the first genome-wide analysis of the NAC transcription factor family in the waterlogging-tolerant woody species \u003cem\u003eM. sinostellata\u003c/em\u003e. While our findings provide strong correlative evidence linking specific \u003cem\u003eMsNAC\u003c/em\u003es to the stress response, we acknowledge that the functional characterization of these candidates remains to be experimentally validated.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEvolutionary Expansion and Diversification of the\u003c/b\u003e \u003cb\u003eMsNAC\u003c/b\u003e \u003cb\u003eFamily\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe identified 96 \u003cem\u003eMsNAC\u003c/em\u003e genes in \u003cem\u003eM. sinostellata\u003c/em\u003e, a number comparable to tomato (93) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and cassava (96) [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], but smaller than in species like poplar (270) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e] and oat (333) [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. This variation is often attributed to differential whole-genome duplication (WGD) and segmental duplication events during plant evolution [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The presence of 27 duplicated gene pairs within the \u003cem\u003eMsNAC\u003c/em\u003e family (Supplementary Table S6), coupled with Ka/Ks ratios all significantly less than 1 (purifying selection), indicates that small-scale duplication events, particularly tandem duplications, have been a significant force in its expansion, with strong selective pressure to maintain essential functions. The extreme conservation observed in the \u003cem\u003eMsNAC65\u003c/em\u003e-\u003cem\u003eMsNAC14\u003c/em\u003e pair (Ka/Ks\u0026thinsp;\u0026asymp;\u0026thinsp;0) suggests these genes perform critical, non-redundant functions.\u003c/p\u003e \u003cp\u003ePhylogenetic analysis classified most \u003cem\u003eMsNAC\u003c/em\u003es into 17 subgroups conserved with A. thaliana [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], indicating the early establishment of major NAC functional clades. However, the 14 unclassified \u003cem\u003eMsNAC\u003c/em\u003e genes are of particular interest. Similar lineage-specific NAC members have been reported in other species [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e], and we hypothesize that these unclassified \u003cem\u003eMsNAC\u003c/em\u003es may represent evolutionary innovations contributing to the unique waterlogging tolerance of \u003cem\u003eM. sinostellata\u003c/em\u003e.\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eStructural and Promoter Analysis Suggests Complex Regulatory Capacity\u003c/h2\u003e \u003cp\u003eThe diversity in gene structure (intron number 3\u0026ndash;11) and the identification of additional functional domains in proteins like \u003cem\u003eMsNAC\u003c/em\u003e11, \u003cem\u003eMsNAC\u003c/em\u003e36, and \u003cem\u003eMsNAC\u003c/em\u003e96 point to functional diversification. Promoter analysis revealed a complex regulatory landscape, dominated by light-, ABA-, and MeJA-responsive elements. The co-occurrence of these elements suggests the existence of a sophisticated light-hormone-MYB regulatory network governing \u003cem\u003eMsNAC\u003c/em\u003e expression. This integration may be crucial for \u003cem\u003eM. sinostellata\u003c/em\u003e to coordinate energy metabolism (via light signaling) with stress defense programs (via ABA/JA) in its natural shady, humid, and potentially waterlogged understory habitat.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTissue-Specific and Dynamic Expression Under Waterlogging Stress\u003c/h2\u003e \u003cp\u003eExpression profiling revealed a spatiotemporally complex response to waterlogging, with the most pronounced changes in roots, the primary organ sensing hypoxia.\u003c/p\u003e \u003cp\u003eIn roots, the sustained upregulation of MsNAC66 strongly suggests its potential as a key regulator of long-term tolerance, analogous to ANAC013 in Arabidopsis [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. In contrast, genes like \u003cem\u003eMsNAC\u003c/em\u003e14, \u003cem\u003eMsNAC\u003c/em\u003e16, \u003cem\u003eMsNAC\u003c/em\u003e63, \u003cem\u003eMsNAC\u003c/em\u003e65, and \u003cem\u003eMsNAC\u003c/em\u003e70 showed transient induction, characteristic of early-response regulators that initiate defense programs. Notably, a set of root-preferential genes (\u003cem\u003eMsNAC\u003c/em\u003e3, \u003cem\u003eMsNAC\u003c/em\u003e4, \u003cem\u003eMsNAC\u003c/em\u003e29, \u003cem\u003eMsNAC\u003c/em\u003e67, \u003cem\u003eMsNAC\u003c/em\u003e85, \u003cem\u003eMsNAC\u003c/em\u003e91, \u003cem\u003eMsNAC\u003c/em\u003e92) was persistently downregulated, likely representing an energy-saving strategy by repressing non-essential growth processes during stress, a strategy also observed in other species [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. In leaves, sustained upregulation of \u003cem\u003eMsNAC\u003c/em\u003e41, \u003cem\u003eMsNAC\u003c/em\u003e60, and \u003cem\u003eMsNAC96\u003c/em\u003e may mediate systemic responses, while transient patterns in others (e.g., \u003cem\u003eMsNAC\u003c/em\u003e38, \u003cem\u003eMsNAC\u003c/em\u003e79) might relate to modulating senescence. In stems, the delayed strong activation of \u003cem\u003eMsNAC\u003c/em\u003e88 points to a potential role in longer-term acclimation, such as aerenchyma or adventitious root formation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eA Proposed Model for\u003c/b\u003e \u003cb\u003eMsNAC\u003c/b\u003e \u003cb\u003eFunction in Waterlogging Tolerance\u003c/b\u003e\u003c/p\u003e \u003cp\u003eSynthesizing our findings, we propose a root-centric model for the role of \u003cem\u003eMsNAC\u003c/em\u003es in \u003cem\u003eM. sinostellata\u003c/em\u003e's waterlogging tolerance: (1) Immediate Alert \u0026amp; Defense: Early-responsive \u003cem\u003eMsNAC\u003c/em\u003es (e.g., \u003cem\u003eMsNAC\u003c/em\u003e14/16/63/65/70) are transiently induced to activate initial hypoxic responses (e.g., fermentation, ROS scavenging); (2) Sustained Core Defense: A subset (e.g., \u003cem\u003eMsNAC\u003c/em\u003e66) is stably upregulated to maintain core tolerance mechanisms for prolonged survival. (3) Energy Economy Management: Root-preferential developmental \u003cem\u003eMsNAC\u003c/em\u003es (e.g., \u003cem\u003eMsNAC\u003c/em\u003e3/4) are repressed to conserve energy. (4) Systemic Coordination \u0026amp; Acclimation: Specific \u003cem\u003eMsNAC\u003c/em\u003es in aerial parts adjust photosynthesis, and potentially promote aerenchyma formation (stems) and adventitious root development, improving overall aeration. This model highlights the functional diversification and specialization within the \u003cem\u003eMsNAC\u003c/em\u003e family, facilitating a coordinated response from initial signaling to long-term anatomical adaptation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLimitations and Future Perspectives\u003c/h2\u003e \u003cp\u003eIt is important to note that the roles proposed for the \u003cem\u003eMsNAC\u003c/em\u003e candidates in our working model are primarily inferred from their expression patterns and promoter analyzes. While these correlational data are highly suggestive, direct functional evidence is required to confirm their biological roles. Future work should focus on functional validation using approaches such as heterologous expression in model plants (e.g., \u003cem\u003eA. thaliana\u003c/em\u003e) to assess their impact on waterlogging tolerance phenotypes, and/or the use of techniques like EMSA and dual-luciferase assays to identify their direct downstream targets. The candidate genes prioritized in this study, particularly the root-specific MsNAC66 and the transiently induced cluster, provide a high-value starting point for such in-depth mechanistic investigations.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, this study provides the first genome-wide identification and characterization of the NAC transcription factor family in \u003cem\u003eM. sinostellata\u003c/em\u003e. We identified 96 \u003cem\u003eMsNAC\u003c/em\u003e genes that have evolved under strong purifying selection, with lineage-specific expansions potentially contributing to waterlogging adaptation. The complex cis-regulatory landscape of these genes suggests an advanced capacity to integrate light, hormone, and stress signals. Most importantly, the spatiotemporally dynamic expression profiles under waterlogging stress prioritize key candidate genes for future functional validation, such as the persistently upregulated \u003cem\u003eMsNAC66\u003c/em\u003e and the transiently induced \u003cem\u003eMsNAC14\u003c/em\u003e/\u003cem\u003e16/63/65\u003c/em\u003e/\u003cem\u003e70\u003c/em\u003e. The resources and insights generated here lay a solid foundation for unraveling the molecular mechanisms of waterlogging tolerance in woody perennials and for the molecular breeding of resilient Magnolia cultivars.This study fills a critical knowledge gap regarding stress-responsive transcription factors in the Magnoliaceae family and provides valuable genetic resources for the molecular breeding of resilient ornamental trees, which is increasingly important in an era of climate change marked by frequent flooding events.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eABA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Abscisic acid\u003c/p\u003e\n\u003cp\u003eFPKM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Fragments Per Kilobase of transcript per Million mapped reads\u003c/p\u003e\n\u003cp\u003eGO\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Gene Ontology\u003c/p\u003e\n\u003cp\u003eHMM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Hidden Markov Model\u003c/p\u003e\n\u003cp\u003eJA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Jasmonic acid\u003c/p\u003e\n\u003cp\u003eMeJA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Methyl jasmonate\u003c/p\u003e\n\u003cp\u003eNAC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;NAM, ATAF1/2, CUC2\u003c/p\u003e\n\u003cp\u003eROS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Reactive oxygen species\u003c/p\u003e\n\u003cp\u003eRT-qPCR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Reverse transcription quantitative polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eSA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Salicylic acid\u003c/p\u003e\n\u003cp\u003eTF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Transcription factor\u003c/p\u003e\n\u003cp\u003eWGD \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Whole-genome duplication\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all our colleagues for providing useful discussions and technical assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eF.X. conceived and designed the study, conducted the bioinformatics analysis, prepared the experimental materials, performed the experiments and data analysis, wrote the manuscript, and prepared the figures and tables. F.L., Z.H., Y.H., and D.F. were involved in sample collection and data analysis. L.R., L.H., and X.B. participated in the study design and revised the manuscript. W.Y. conceived and designed the study, contributed to data analysis, wrote and revised the manuscript, and provided funding support. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by the Youth Project of the Basic Research Program of Shaanxi Academy of Sciences (grant no. 2024k-20); Xi\u0026rsquo;an Agricultural Technology Research Project (grant no. 24NYGG0071); Applied Technology R\u0026amp;D Project of Shaanxi Academy of Sciences (grant no. 2025k-09); Shaanxi Provincial Innovation Capability Support Plan-Regional Science and Technology Innovation Demonstration Station (grant no. 2023XYSF-08); Platform Project of Shaanxi Academy of Sciences (grant no. 2024j-03); General Project of Shaanxi Key Research and Development Program, 2025SF-YBXM-502 (grant no. 2025SF-YBXM-502); Innovation Talent Promotion Program of Shaanxi Provincial Department of Science and Technology-Science and Technology Innovation Team Project (grant no. 2021TD-33); The Industrialization project of \u0026ldquo;One Institute, One Product\u0026rdquo; under the Science and Technology Plan of Shaanxi Academy of Sciences (grant no. 2019K-02). The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw RNA-seq data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number PRJNA1274043. All genome assembly data and raw transcriptome sequencing reads of\u003cem\u003e\u0026nbsp;M. sinostellata\u003c/em\u003e in this study have been deposited in NCBI under accession number PRJNA1169943. The genome annotation information of \u003cem\u003eM. sinostellata\u003c/em\u003e has been uploaded to the figshare database (https://figshare.com/account/items/27184485/edit). The genome assembly data of \u003cem\u003eM. sinostellata\u003c/em\u003e from this study have also been stored in Science DB under accession number CSTR 31253.11.sciencedb.31088, with a DOI of 10.57760/sciencedb.31088. All other data generated or analyzed during this study are included in this published article and its supplementary information files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe confirm that all experimental research on \u003cem\u003eM. sinostellata\u003c/em\u003e, including the collection of plant material, complied with relevant institutional, national, and international guidelines and legislation. The \u003cem\u003eM. sinostellata\u003c/em\u003e plants used in this experiment were authorized by Xi\u0026apos;an Botanical Garden of Shaanxi Province, Institute of Botany of Shaanxi Province.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eXi\u0026rsquo;an Botanical Garden of Shaanxi Province, Institute of Botany of Shaanxi Province, Xi\u0026rsquo;an, 710061, China\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e2\u003c/sup\u003eCollege of Tropical Agriculture and Forestry, Hainan University, Haikou, 570100, China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eVoesenek LAC, Bailey-Serres J. Flood adaptive traits and processes: an overview. New Phytol. 2015;206(1):57\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFukao T, Barrera-Figueroa BE, Juntawong P, Pe\u0026ntilde;a-Castro JM. Submergence and waterlogging stress in plants: a review highlighting research opportunities and understudied aspects. Front Plant Sci. 2019;10:340.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGibbs DJ, Lee SC, Md Isa N, Gramuglia S, Fukao T, Bassel GW, et al. Homeostatic response to hypoxia is regulated by the N-end rule pathway in plants. Nature. 2011;479(7373):415\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNuruzzaman M, Sharoni AM, Kikuchi S. Roles of NAC transcription factors in the regulation of biotic and abiotic stress responses in plants. Front Microbiol. 2013;4:248.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePuranik S, Sahu PP, Srivastava PS, Prasad M. NAC proteins: regulation and role in stress tolerance. Trends Plant Sci. 2012;17(6):369\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan K, Zhao Y, Sun Y, Li Y. NACs, generalists in plant life. Plant Biotechnol J. 2023;21(12):2433\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXiong H, He H, Chang Y, Miao B, Liu Z, Wang Q, et al. Multiple roles of NAC transcription factors in plant development and stress responses. J Integr Plant Biol. 2025;67(1):1\u0026ndash;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan K, Zhao Y, Liu J, Tian Y, El-Kassaby YA, Qi Y, et al. Genome-wide investigation and analysis of NAC transcription factor family in \u003cem\u003ePopulus tomentosa\u003c/em\u003e and expression analysis under salt stress. Plant Biol. 2024;26(5):764\u0026ndash;76.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOoka H, Satoh K, Doi K, Nagata T, Otomo Y, Murakami K, et al. Comprehensive analysis of NAC family genes in \u003cem\u003eOryza sativa\u003c/em\u003e and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. DNA Res. 2003;10(6):239\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNuruzzaman M, Manimekalai R, Sharoni AM, Satoh K, Kondoh H, Ooka H, et al. Genome-wide analysis of NAC transcription factor family in rice. Gene. 2010;465(1\u0026ndash;2):30\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuerin C, Roche J, Allard V, Ravel C, Mouzeyar S, Bouzidi MF. Genome-wide analysis, expansion and expression of the NAC family under drought and heat stresses in bread wheat (\u003cem\u003eT. aestivum\u003c/em\u003e L). PLoS ONE. 2019;14(3):e0213390.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu M, Sun QP, Zhang DF, Wang TY, Pan JB. Identification of stress-related NAC transcription factor members in maize (\u003cem\u003eZea mays\u003c/em\u003e L.) and characterization of the expression pattern of these genes. Biochem Biophys Res Commun. 2015;462(2):144\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu M, Ma Z, Sun W, Huang L, Wu Q, Tang Z, et al. Genome-wide analysis of the NAC transcription factor family in Tartary buckwheat (\u003cem\u003eFagopyrum tataricum\u003c/em\u003e). BMC Genomics. 2019;20(1):113.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJin JF, Wang ZQ, He QY, Wang JY, Li PF, Xu JM, et al. Genome-wide identification and expression analysis of the NAC transcription factor family in tomato (Solanum lycopersicum) during aluminum stress. BMC Genomics. 2020;21(1):288.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGong X, Zhao L, Song X, Lin Z, Gu B, Yan J, et al. Genome-wide analyses and expression patterns under abiotic stress of NAC transcription factors in white pear (\u003cem\u003ePyrus bretschneideri\u003c/em\u003e). BMC Plant Biol. 2019;19(1):251.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNing Y, Liu J, Song B, Xu H, Liu Z, Chen L. Genome-wide analyses of the NAC transcription factor family to reveal the potential candidate genes responding to powdery mildew in balsam pear. Plant Biotechnol Rep. 2023;17(6):917\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArroyo-\u0026Aacute;lvarez E, Chan-Le\u0026oacute;n A, Gir\u0026oacute;n-Ram\u0026iacute;rez A, Fuentes G, Estrella-Maldonado H, Santamar\u0026iacute;a JM. Genome-Wide analysis of WRKY and NAC transcription factors in \u003cem\u003eCarica papaya\u003c/em\u003e L. and their possible role in the loss of drought tolerance by recent cultivars through the domestication of their wild ancestors. Plants. 2023;12(15):2775.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBennett T, van den Toorn A, Sanchez-Perez GF, Campilho A, Willemsen V, Snel B, et al. SOMBRERO, BEARSKIN1, and BEARSKIN2 regulate root cap maturation in Arabidopsis. Plant Cell. 2010;22(3):640\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie Q, Frugis G, Colgan D, Chua NH. Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development. Genes Dev. 2000;14(23):3024\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang C, Wang Y, Zhu Y, Tang J, Hu B, Liu L, et al. OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice. Proc Natl Acad Sci U S A. 2014;111(28):10013\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAida M, Ishida T, Fukaki H, Fujisawa H, Tasaka M. Genes involved in organ separation in Arabidopsis: An analysis of the cup-shaped cotyledon mutant. Plant Cell. 1997;9(6):841\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakeda S, Hanano K, Kariya A, Shimizu S, Zhao L, Matsui M, et al. CUP-SHAPED COTYLEDON1 transcription factor activates the expression of LSH4 and LSH3, two members of the ALOG gene family, in shoot organ boundary cells. Plant J. 2011;66(6):1066\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu H, Dai M, Yao J, Xiao B, Li X, Zhang Q, et al. Overexpressing a NAC transcription factor enhances drought resistance and salt tolerance in rice. Plant Physiol. 2006;141(3):1159\u0026ndash;75.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu ZY, Kim SY, Hyeon DY, Kim DH, Dong T, Park Y, et al. The Arabidopsis NAC transcription factor \u003cem\u003eANAC096\u003c/em\u003e cooperates with bZIP-type transcription factors in dehydration and osmotic stress responses. Plant Cell. 2013;25(11):4708\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng L, Hu Y, Yang T, Wang Z, Wang D, Jia L, et al. A root cap-localized NAC transcription factor controls root halotropic response to salt stress in Arabidopsis. Nat Commun. 2024;15(1):2061.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYarra R, Wei W. The NAC-type transcription factor \u003cem\u003eGmNAC20\u003c/em\u003e improves cold, salinity tolerance, and lateral root formation in transgenic rice plants. Funct Integr Genomics. 2021;21(3\u0026ndash;4):473\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMei C, Yang J, Mei Q, Jia D, Yan P, Feng B, et al. MtNAC104 positively regulates apple cold tolerance via CBF-dependent and CBF-independent pathways. Plant Biotechnol J. 2023;21(10):2057\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShan W, Kuang JF, Lu WJ, Chen JY. Banana fruit NAC transcription factor \u003cem\u003eMaNAC1\u003c/em\u003e is a direct target of \u003cem\u003eMaICE1\u003c/em\u003e and involved in cold stress through interacting with \u003cem\u003eMaCBF1\u003c/em\u003e. Plant Cell Environ. 2014;37(9):2116\u0026ndash;27.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRauf M, Arif M, Fisahn J, Xue GP, Balazadeh S, Mueller-Roeber B. NAC transcription factor speedy hyponastic growth regulates flooding-induced leaf movement in Arabidopsis. Plant Cell. 2013;25(12):4941\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie Z, Jin L, Sun Y, Zhan C, Tang S, Qin T, et al. OsNAC120 balances plant growth and drought tolerance by integrating GA and ABA signaling in rice. Plant Commun. 2024;5(1):100782.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu A, Allu AD, Garapati P, Siddiqui H, Dortay H, Zanor MI, et al. JUNGBRUNNEN1, a reactive oxygen species-responsive NAC transcription factor, regulates longevity in Arabidopsis. Plant Cell. 2012;24(2):482\u0026ndash;506.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeng LF, Su L, Fu LF, Lin S, Zhang JM, Liu QH, et al. Genome-wide analysis of the rose (\u003cem\u003eRosa chinensis\u003c/em\u003e) NAC family and characterization of \u003cem\u003eRcNAC091\u003c/em\u003e. Plant Mol Biol. 2022;108(4\u0026ndash;5):605\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhuo X, Zheng T, Zhang Z, Zhang Y, Jiang L, Ahmad S, et al. Genome-wide analysis of the NAC transcription factor gene family reveals differential expression patterns and cold-stress responses in the woody plant \u003cem\u003ePrunus mume\u003c/em\u003e. Genes. 2018;9(10):494.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang ZQ, Ni LJ, Liu DN, Fu ZK, Hua JF, Lu ZG, et al. Genome-wide identification and characterization of NAC family in \u003cem\u003eHibiscus hamabo\u003c/em\u003e Sieb. et Zucc. under various abiotic stresses. Int J Mol Sci. 2022;23(6):3055.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Q, Zhou L, Yuan M, Peng F, Zhu X, Wang Y. Genome-wide identification of NAC gene family members of tree peony (\u003cem\u003ePaeonia suffruticosa\u003c/em\u003e Andrews) and their expression under heat and waterlogging stress. Int J Mol Sci. 2024;25(17):9312.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang CT, Ye CL. The reasons for the endangerment of Endemic wild Precious plants in Zhejiang Province and the protection countermeasures. Fujian Forestry Sci Technol. 2007;34(2):202\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu YF, Xu JN, Shen YM, Shi CG, Ren MJ, Qiu JX, et al. Analysis of the distribution characteristics of the endangered plant \u003cem\u003eMagnolia sinostellata\u003c/em\u003e and its association with the properties of Habitat Soil. For Resour Manage. 2023;1:62\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang J, Wang YL, Wu YF. Establishment and application of evaluation system of morphological waterlogging tolerance of magnoliaceae. Landsc Archit Plants. 2022;5(44):210.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEl-Gebali S, Mistry J, Bateman A, Eddy SR, Luciani A, Potter SC, et al. The Pfam protein families database in 2019. Nucleic Acids Res. 2019;47(D1):D427\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFinn RD, Clements J, Eddy SR. HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 2011;39(suppl2):W29\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLetunic I, Khedkar S, Bork P. SMART: recent updates, new developments and status in 2020. Nucleic Acids Res. 2021;49(D1):D458\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu S, Wang J, Chitsaz F, Derbyshire MK, Geer RC, Gonzales NR, et al. CDD/SPARCLE: the conserved domain database in 2020. Nucleic Acids Res. 2020;48(D1):D265\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMarchler-Bauer A, Bo Y, Han L, He J, Lanczycki CJ, Lu S, et al. CDD/SPARCLE: functional classification of proteins via subfamily domain architectures. Nucleic Acids Res. 2017;45(D1):D200\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, et al. Protein Identification and Analysis Tools on the ExPASy Server. In: Walker JM, editor. The Proteomics Protocols Handbook. Totowa, NJ: Humana; 2005. pp. 571\u0026ndash;607.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChou KC, Shen HB. Cell-PLoc 2.0: an improved package of web-servers for predicting subcellular localization of proteins in various organisms. Nat Sci. 2010;2(10):1090\u0026ndash;103.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu B, Jin J, Guo AY, Zhang H, Luo J, Gao G. GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics. 2015;31(8):1296\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBailey TL, Johnson J, Grant CE, Noble WS. The MEME Suite. Nucleic Acids Res. 2015;43(W1):W39\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33(7):1870\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSubramanian B, Gao S, Lercher MJ, Hu S, Chen WH. Evolview v3: a webserver for visualization, annotation, and management of phylogenetic trees. Nucleic Acids Res. 2019;47(W1):W270\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLescot M, D\u0026eacute;hais P, Thijs G, Marchal K, Moreau Y, Van de Peer Y, et al. 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(1):325\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen C, Wu Y, Li J, Wang X, Zeng Z, Xu J, et al. TBtools-II: A one for all, all for one bioinformatics platform for biological big-data mining. Mol Plant. 2023;16(11):1733\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang Z, Xiao J, Wu J, Zhang H, Liu G, Wang X, et al. ParaAT: A parallel tool for constructing multiple protein-coding DNA alignments. Biochem Biophys Res Commun. 2012;419(4):779\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang D, Zhang Y, Zhang Z, Zhu J, Yu J. KaKs_Calculator 2.0: a toolkit incorporating gamma-series methods and sliding window strategies. Genomics Proteom Bioinf. 2010;8(1):77\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019;37(8):907\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30(7):923\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLove MI, Huber W, Anders S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014;15(12):550.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThe Gene Ontology Consortium. The Gene Ontology resource: enriching a GOld mine. Nucleic Acids Res. 2021;49(D1):D325\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu T, Hu E, Xu S, Chen M, Guo P, Dai Z, et al. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. Innov (Camb). 2021;2(3):100141.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLivak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2\u0026thinsp;\u0026ndash; ∆∆CT method. Methods. 2001;25(4):402\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu W, Wei Y, Xia Z, Yan Y, Hou X, Zou M, et al. Genome-Wide Identification and Expression Analysis of the NAC Transcription Factor Family in Cassava. PLoS ONE. 2015;10(8):e0136993.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu Y, Cheng J, Hu H, Yan L, Jia J, Wu B. Genome-Wide Identification of NAC Family Genes in Oat and Functional Characterization of \u003cem\u003eAsNAC109\u003c/em\u003e in Abiotic Stress Tolerance. Plants. 2024;13(7):1017.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh AK, Sharma V, Pal AK, Acharya V, Ahuja PS. Genome-wide organization and expression profiling of the NAC transcription factor family in potato (\u003cem\u003eSolanum tuberosum\u003c/em\u003e L). DNA Res. 2013;20(4):403\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie WL, Wang YQ. Genome-wide identification of the NAC gene family in Apocynum pictum and its expression analysis under drought stress. Agric Res Arid Areas. 2025;43(2):54\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEysholdt-Derzso E, Renziehausen T, Frings S, Frohn S, von Bongartz K, Igisch CP, et al. Endoplasmic reticulum-bound ANAC013 factor is cleaved by RHOMBOID-LIKE 2 during the initial response to hypoxia in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Proc Natl Acad Sci U S A. 2023;120(19):e2221308120.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing Q, Ran J, Chen X, Gao Z, Qian X, Zhang C, et al. Identification of the NAC Family and a Functional Analysis of \u003cem\u003eNoNAC36a\u003c/em\u003e Under Flooding Stress in Watercress (\u003cem\u003eNasturtium officinale\u003c/em\u003e R. Br). Horticulturae. 2024;10(11):1219.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Magnolia sinostellata, NAC transcription factor, Genome-wide analysis, Waterlogging stress","lastPublishedDoi":"10.21203/rs.3.rs-8390673/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8390673/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe NAC transcription factors are master regulators of plant responses to abiotic stresses; however, their functions in woody ornamentals, particularly under waterlogging, remain poorly explored. \u003cem\u003eMagnolia sinostellata\u003c/em\u003e, a rare and waterlogging-tolerant species, provides an ideal system to decipher these mechanisms.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, a genome-wide analysis identified 96 \u003cem\u003eMsNAC\u003c/em\u003e genes in \u003cem\u003eM. sinostellata\u003c/em\u003e, which were phylogenetically classified into 17 distinct subgroups. Gene structure and motif analyzes revealed considerable diversity, with several members harboring auxiliary functional domains beyond the conserved NAC domain, suggesting potential functional diversification. Promoter cis-acting element analysis uncovered an abundance of stress-responsive elements, among which abscisic acid (ABA, 19.3%) and methyl jasmonate (MeJA, 13.7%) responses were the most prominent, highlighting a complex regulatory landscape intertwined with hormonal and light signaling pathways. Collinearity and evolutionary pressure analyzes demonstrated that the expansion of the \u003cem\u003eMsNAC\u003c/em\u003e family was primarily driven by gene duplication events under strong purifying selection (Ka/Ks\u0026thinsp;\u0026lt;\u0026thinsp;1). Transcriptomic and RT-qPCR analyzes further revealed that \u003cem\u003eMsNAC\u003c/em\u003e genes exhibit tissue-specific and temporally dynamic expression patterns under waterlogging stress. Notably, roots, as the primary sensing organ, mounted the most pronounced response. The key candidate \u003cem\u003eMsNAC\u003c/em\u003e66 was significantly and persistently upregulated, while others, including \u003cem\u003eMsNAC\u003c/em\u003e14, \u003cem\u003eMsNAC\u003c/em\u003e16, \u003cem\u003eMsNAC\u003c/em\u003e63, \u003cem\u003eMsNAC\u003c/em\u003e65, and \u003cem\u003eMsNAC\u003c/em\u003e70, displayed transient induction patterns.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur study provides the first comprehensive genomic characterization of the NAC family in \u003cem\u003eM. sinostellata\u003c/em\u003e and identifies several core candidate genes with putative roles in the root-centric adaptive response to waterlogging, as determined by their expression dynamics. These findings not only advance our understanding of NAC-mediated stress tolerance in woody plants but also offer valuable genetic resources for the molecular breeding of waterlogging-resistant magnolia cultivars.\u003c/p\u003e","manuscriptTitle":"Genome-Wide Identification and Expression Profiling of the NAC Transcription Factor Family in the Waterlogging-Tolerant Tree Magnolia sinostellata","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-25 09:57:56","doi":"10.21203/rs.3.rs-8390673/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"68a35c00-6638-4dbc-b512-33f19f64322a","owner":[],"postedDate":"December 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T21:11:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-25 09:57:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8390673","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8390673","identity":"rs-8390673","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}