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. 2025 Sep 25;25:1221. doi: 10.1186/s12870-025-07196-0

Transcriptional landscape of Atractylodes chinensis contracting root rot

Yingzhe Li 1,#, Yang Liu 2,3,#, Tian Zhang 4,#, Shuai Ma 1, Yueyue Wang 1, Chunhan Qiao 1, Yu Cao 1,✉, Chunying Zhao 1,5,✉
PMCID: PMC12465743  PMID: 40999336

Abstract

Atractylodes chinensis is among the most economically valuable medicinal plants, serving as the primary source of Atractylodis rhizoma. Its pharmacological activities in treating digestive and urinary disorders have been officially recognized in both the Chinese and Japanese Pharmacopoeias. Whereas, the natural scarcity of Atractylodes chinensis necessitates enhanced cultivation strategies, particularly targeting root rot resistance to ameliorate crop yield and sustainable production. This study implemented controlled cultivation of Atractylodes chinensis and collected rhizome samples exhibiting early-stage root rot symptoms, advanced necrosis, and healthy controls. Ultra-high throughput sequencing was employed to detect differentially expressed genes (DEGs). Key dysregulated genes under root rot infection were systematically screened through Reactome pathway enrichment analysis, followed by clarification of cascade signaling. Further findings were validated through chromosomal mapping, correlation analysis, co-expression profiling, and dynamic protein interaction studies. Four critical genes annotated as probable sucrose-phosphate synthase 3, UDP-glucose 6-dehydrogenase 1-like, UDP-D-apiose/UDP-D-xylose synthase 2, and granule-bound starch synthase 1 demonstrated incipient infection expression patterns. Their dysregulation correlated with reduced nodule formation, diminished nitrogenase activity, disrupted cell wall integrity, and elevated reactive oxygen species levels. Twenty-nine progressive DEGs revealed functional connections to critical physiological processes including one-carbon metabolism, transmethylation-feedback regulation, polarity along the adaxial-abaxial axis, and synthesis of amino acid and phytoalexin. Notably, ten DEGs exhibited continuous dysregulation throughout infection progression, coupled with impaired response to metal stresses, potassium loss, and pathogens infection. This investigation systematically delineates the stage-specific pathological gene network underlying Atractylodes chinensis root rot pathogenesis, providing molecular targets for future disease-resistant cultivar development.

Supplementary information

The online version contains supplementary material available at 10.1186/s12870-025-07196-0.

Keywords: Atractylodes chinensis, Root rot, Dysregulated genes, Cascade signaling

Introduction

Atractylodes chinensis (A. chinensis), a dicotyledonous perennial herb characterized by hypogeal germination, constitutes a cornerstone medicinal species in East Asian traditional medicine [1]. Its dried rhizome, designated as Atractylodis rhizoma in the Chinese Pharmacopoeia and Sojutsu in the Japanese Pharmacopoeia, contains bioactive compounds with demonstrated efficacy against gastrointestinal and urinary disorders. Recognized for its dual pharmacological and nutritional value, this plant has emerged as a high-demand commercial resource in nutraceutical markets [2, 3]. Contemporary research reveals its multifaceted therapeutic potential: polysaccharide fractions exhibit hypoglycemic properties and expedite apatinib-induced apoptosis in gastric cancer cells [4, 5], while volatile oils ameliorate diabetic gastroparesis via IGF-1/IGF-1R signaling activation [6]. Notably, atractylon demonstrates marked anti-inflammatory effects, significantly decreasing acetic acid-induced writhing, carrageenan-triggered paw edema, and hot-plate latent pain in murine models [7]. Furthermore, A. chinensis-based formulations, including modified sanmiao pills for gouty arthritis management [8] and anti-aging creams for So-eum Sasang phenotypes [9], underscore its clinical versatility.

The escalating depletion of wild A. chinensis populations has compelled the need for intensified cultivation efforts to meet pharmaceutical demands [10]. However, rhizome utilization faces enormous challenges from root rot pathogens, particularly Fusarium, which severely compromise plant viability and yield [11]. Pathogen invasion typically manifests through progressive symptomatology including root blackening, microsclerotia formation, stunted growth, foliar chlorosis, and eventual plant mortality [12]. Current mitigation strategies primarily target microbial actions. For instance, endophytic Rhodococcus strains enhance Atractylodes macrocephala’s root rot resistance via reactive oxygen species (ROS) scavenging and cell wall reinforcement [13]. Similarly, Trichoderma harzianum biocontrol improves Atractylodes lancea’s disease resilience while elevating concentration of medicinal atractylon, atractylodin, hinesol and beta-eudesmol in rhizome [14]. Rhizospheric Paenibacillus polymyxa presents antifungal activity through cell wall-degrading enzymes [15], and Bacillus amyloliquefaciens and Bacillus subtilis effectively suppress proliferation of Fusarium and Volutella [11].

Root rot progression is divided into distinct pathomorphological stages. In Eucalyptus grandis, early Armillaria infection correlates with threitol accumulation during root colonization [16], while Fusarium pathogenesis in Cucumis sativus progresses from basal stem discoloration to advanced mycelial proliferation and systemic root necrosis [17]. Root rot disease in A. chinensis is frequently caused by mixed pathogenic strains, including Fusarium and Volutella species [18, 19]. Despite these insights, the molecular mechanisms underlying stage-specific pathological changes in cultivated A. chinensis remain poorly characterized. This study therefore aims to delineate the transcriptional dynamics of root rot development through phase-resolved analysis of dysregulated genes, ultimately informing targeted strategies for disease management and sustainable cultivation.

Materials and methods

A. chinensis cultivation and rhizome sampling

The cultivation of A. chinensis was conducted in Longhua County, Hebei Province, a state-level regional fine variety breeding base certified by the Ministry of Agriculture and Rural Affairs of the People’s Republic of China (http://www.zys.moa.gov.cn/gzdt/202204/t20220407_6395684.htm). Based on the phasic characteristics of root rot [20], A. chinensis individuals appearing brown and wilted leaves were classified into the primary root rot group (PRR), while those showing leaf abscission and stem decay were categorized into the advanced root rot group (ARR). The control group (CT) consisted of individuals without visible root rot symptoms. Rhizomes were collected using the five-spot sampling method (Fig. 1A, B). Each experimental group consisted of three biological replicates, with no technical replicates employed. Samples were stored in 10 mL frozen tubes, immediately frozen in liquid nitrogen (−196 °C) and preserved in −80 °C refrigerator.

Fig. 1.

Fig. 1

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Root rot-sparked genetic abnormalities. A Root states of Atractylodes chinensis. B Root sample collection. C Number of differentially expressed genes in PRR comparing with CT. D Locus of differential genes. E Reactome pathway mapped by up-regulated genes (left) and down-regulated genes (right) between PRR and CT. F Thermal expression pattern, G correlation, and H ontology term of genes including L6452_29839 (probable sucrose-phosphate synthase 3), L6452_06931 (UDP-glucose 6-dehydrogenase 1-like), L6452_11144 (UDP-D-apiose/UDP-D-xylose synthase 2), and L6452_36719 (granule-bound starch synthase 1, chloroplastic/amyloplastic) primarily affected by incipient root rot. I Average content of amylose and amylopectin in ARR, PRR, CT samples. ARR, PRR and CT indicate advanced root rot group, primary root rot group and control group, respectively

RNA extraction and quality control

RNA was extracted from A. chinensis rhizomes using TRIzol reagent (Invitrogen, USA) and treated with RNase-free DNase I (Takara, Japan). RNA quantification, quality, and integrity were assessed using the Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and NanoDrop spectrophotometer (Thermo Scientific, USA).

RNA library construction and transcriptome sequencing

A total of 1.5 µg RNA per sample was used for mRNA library construction. Libraries were prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s instructions. Library quality was evaluated on the Agilent 2100 Bioanalyzer system, and sequencing was performed on the Illumina NovaSeq 6000 platform (paired-end, 150 bp).

Quality control of RNA sequencing data

Raw base sizes were 6.37 G, 7.26 G, 6.6 G, 5.92 G, 6.26 G, 7.25 G, 6.12 G, 6.2 G, and 6.28 G for PRR1, PRR2, PRR3, ARR1, ARR2, ARR3, CT1, CT2, and CT3, respectively. Clean data were obtained by removing adapter-containing reads, poly-N sequences, and low-quality reads. Quality metrics, including Q20, Q30, GC content, and sequence duplication levels, were calculated. High-quality clean data were used for downstream analyses.

Differential expression of protein-coding genes

De novo transcriptome assembly was exerted using Trinity and CD-HIT [21, 22]. Due to the lack of reference genome for A. chinensis, data were mapped to the closely related genome of Arctium lappa (ASM2352574v1) using STAR (2.5.2b) [23]. Pathogen-related gene discrimination was accomplished by NCBI NR database. Read counts for each gene were obtained using HTSeq (0.5.4p3) [24]. Gene expression levels were quantified as fragments per kilobase of transcript per million fragments mapped (FPKM). The statistical power of this experimental design, calculated in DESeq R package is 1.10.1 [25], with p value less than 0.05 considered significant. Adjusted p value (padj) less than 0.05, calculated using the Benjamini and Hochberg method, was used to identify differentially expressed genes (DEGs).

Functional classification and pathway analysis

DEGs were analyzed for pathway enrichment, ontology term, protein interaction, and weighted gene co-expression network (WGCNA) using OMICSEE, Plant Reactome, Metascape (3.5.20240901), STRING (12.0), and ImageGP, with annotations based on Oryza sativa [26–29].

Determination of amylose and amylopectin content

Amylose and amylopectin contents in ARR, PRR, and CT samples were quantified using Amylose Content and Amylopectin Content Assay Kits (Solarbio, China) according to manufacturer protocols. Absorbance measurements were performed with a Multiskan GO 1510 spectrophotometer (Thermo Fisher Scientific, Finland). Between-group comparisons of mean amylose/amylopectin content were analyzed using Student’s t-test, with statistical significance defined as p value less than 0.05.

Results

Dysregulation of transcriptional dynamics in the initial phase of root rot pathogenesis

Comparative transcriptomics showed 366 significantly up-regulated and 225 down-regulated genes (padj < 0.05; fold change > 4) in PRR versus CT (Fig. 1C and Table S1). Chromosomal mapping demonstrated consistent enrichment of DEGs among ARR, PRR, and CT groups on chromosome CM042048.1 (Fig. 1D). Plant Reactome analysis identified 65 pathways significantly enriched by up-regulated genes, including phenylpropanoid biosynthesis (p = 0.042), and 38 pathways enriched by down-regulated genes such as UDP-D-apiose biosynthesis (p = 0.036) (Fig. 1E). Key hub genes responsive to early root rot infection were functionally characterized to down-regulated L6452_29839 (probable sucrose-phosphate synthase 3), L6452_06931 (UDP-glucose 6-dehydrogenase 1-like), L6452_11144 (UDP-D-apiose/UDP-D-xylose synthase 2), and up-regulated L6452_36719 (granule-bound starch synthase 1) (Figs. 1F and 2). Strong co-expression patterns emerged among these regulators. The down-regulated enzyme genes exhibited high positive correlations (r ≥ 0.7), while L6452_36719 showed inverse correlation (Fig. 1G). L6452_06931 was significantly enriched (p < 0.05) in carboxylic acid and carbohydrate derivative metabolic processes (Fig. 1H). Analysis revealed significantly higher amylose content in CT compared to PRR (p < 0.05) and ARR (p < 0.001), while amylopectin content was significantly lower in CT than in PRR (p < 0.05) (Fig. 1I).

Fig. 2.

Fig. 2

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Cascade signal maladjustment during root rot period

Aberrant gene expression profiles in the advanced stage of root rot disease

Transcriptional dysregulation intensified with root rot progression. Comparative analysis of ARR and PRR stages identified 551 up-regulated and 759 down-regulated genes (padj < 0.05; fold change > 4) (Fig. 3A and Table S1). Pathway-centric screening revealed 29 hub genes functionally linked to disease advancement, such as up-regulated ethylene biosynthesis enzyme (L6452_04007, 1-aminocyclopropane-1-carboxylate synthase 7-like) and down-regulated methionine metabolism regulator (L6452_24444, 5-methyltetrahydropteroyltriglutamate–homocysteine methyltransferase) (Figs. 2 and 3B and C). WGCNA co-expression network analysis clustered 27 down-regulated metabolic genes into a conserved module, while excluding up-regulated L6452_04007 and L6452_20382 (Fig. 3D). Protein interaction prediction indicated strong interplay among hub genes encoding: (i) central carbon metabolism enzymes including cytosolic glyceraldehyde-3-phosphate dehydrogenase-like (L6452_17015) and mitochondrial succinate-semialdehyde dehydrogenase (L6452_00095); (ii) nucleotide-sugar converter with UDP-glucose 6-dehydrogenase 3-like (L6452_10785); (iii) sucrose metabolism regulators containing probable sucrose-phosphate synthase 1 (L6452_26647), sucrose-phosphatase 2 (L6452_11321), and sucrose-phosphatase 2-like (L6452_29411); (iv) specialized metabolism components referring to chloroplastic anthranilate synthase beta subunit 2 (L6452_13603), cytosolic fructose-bisphosphate aldolase 6 (L6452_29185), and S-adenosylmethionine synthase 2 (L6452_24645) (Fig. 3E). Dysregulation of five core metabolic enzymes (L6452_17015, L6452_10785, L6452_00095, L6452_29185, and L6452_26647) significantly perturbed 9 ontology terms (p < 0.001) like embryo development ending in seed dormancy and glutamate metabolic process (Fig. 3F).

Fig. 3.

Fig. 3

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Analysis of differential genes between ARR and PRR. A Number and B assembled pathway of genes according to up-regulation (left) and down-regulation (right). C Expression difference of candidate genes including L6452_28255 (bifunctional dihydrofolate reductase-thymidylate synthase 1), L6452_44605 (serine hydroxymethyltransferase 3, chloroplastic), L6452_09860 (glutathione synthase), L6452_11294 (formate–tetrahydrofolate ligase), L6452_13603 (anthranilate synthase beta subunit 2, chloroplastic), L6452_21574 (aspartate aminotransferase, cytoplasmic), L6452_24444 (5-methyltetrahydropteroyltriglutamate–homocysteine methyltransferase), L6452_24445 (5-methyltetrahydropteroyltriglutamate–homocysteine methyltransferase), L6452_11345 (5-methyltetrahydropteroyltriglutamate–homocysteine methyltransferase-like), L6452_24645 (S-adenosylmethionine synthase 2), L6452_27433 (methionine S-methyltransferase), L6452_20511 (cycloartenol-C-24-methyltransferase), L6452_04007 (1-aminocyclopropane-1-carboxylate synthase 7-like), L6452_20382 (1-aminocyclopropane-1-carboxylate synthase 2-like), L6452_26647 (probable sucrose-phosphate synthase 1), L6452_29411 (sucrose-phosphatase 2-like), L6452_11321 (sucrose-phosphatase 2), L6452_29185 (fructose-bisphosphate aldolase 6, cytosolic), L6452_17015 (glyceraldehyde-3-phosphate dehydrogenase, cytosolic-like), L6452_38554 (phospho-2-dehydro-3-deoxyheptonate aldolase 1, chloroplastic-like), L6452_43701 (phospho-2-dehydro-3-deoxyheptonate aldolase 2, chloroplastic), L6452_00095 (succinate-semialdehyde dehydrogenase, mitochondrial), L6452_40878 (succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial), L6452_24453 (cytochrome P450 98A2-like), L6452_03960 (protein STRICTOSIDINE SYNTHASE-LIKE 4-like), L6452_09449 (biotin synthase), L6452_21682 (3-hydroxyisobutyryl-CoA hydrolase-like protein 3, mitochondrial), L6452_10785 (UDP-glucose 6-dehydrogenase 3-like), L6452_24203 (UDP-D-apiose/UDP-D-xylose synthase 2) dislocated by malignant root rot. D Gene co-expression module, E protein synergism, and F function cluster on candidates. ARR and PRR severally refer to advanced root rot group and primary root rot group

Conserved transcriptomic reprogramming across pathogenic progression of root rot

Cross-stage comparative analysis identified 196 consistently up-regulated and 188 down-regulated genes (padj < 0.05; fold change > 1) across all root rot conditions (ARR/PRR/CT) (Fig. 4A, B and Table S1). Pathway enrichment revealed two dominant mechanisms, pyridoxamine anabolism as the top pathway for up-regulated genes and removal of superoxide radicals most significantly enriched in down-regulated genes (Fig. 4C). Integrative screening pinpointed hub genes encoding pyridoxal metabolism regulator (L6452_41016, pyridoxal kinase), nucleotide-sugar interconversion enzyme (L6452_31567, bifunctional UDP-glucose 4-epimerase and UDP-xylose 4-epimerase 1-like), cell wall biosynthesis machinery (L6452_05034/L6452_18564/L6452_25201, cellulose synthase A catalytic subunit), ROS scavenging system-related superoxide dismutase [Cu-Zn] (L6452_25713) and L-ascorbate peroxidase (L6452_38883/L6452_03621), as well as specialized metabolism mediators including homogentisate 1,2-dioxygenase (L6452_28564) and probable sucrose-phosphate synthase 1 (L6452_26647) (Fig. 4D, E). Chromosomal mapping showed spatial clustering of common core regulators, with up-regulated gene density peaks on CM042047.1 and down-regulated peaks on CM042051.1 (Fig. 4F). Pathogen-derived L6452_14497 (padj < 0.05; fold change > 4) labelled as ATP synthase subunit beta was consistently detected in the entire cohort, and its progressive up-regulation correlated with disease advancement (Fig. 4G).

Fig. 4.

Fig. 4

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Ongoing gene expression disturbance with root rot development. A Number of differential genes between ARR, PRR and CT following to reformulated threshold fold change. B Common gene and C corresponding reactome pathway congregated by up-regulated genes (left) and down-regulated genes (right). D Read count and E FPKM of candidate constantly shaped by root rot progresses in common genes including L6452_41016 (pyridoxal kinase), L6452_31567 (bifunctional UDP-glucose 4-epimerase and UDP-xylose 4-epimerase 1-like), L6452_28564 (homogentisate 1,2-dioxygenase), L6452_05034 (cellulose synthase A catalytic subunit 2 [UDP-forming]-like), L6452_18564 (cellulose synthase A catalytic subunit 1 [UDP-forming]), L6452_25201 (cellulose synthase A catalytic subunit 1 [UDP-forming]), L6452_26647 (probable sucrose-phosphate synthase 1), L6452_25713 (superoxide dismutase [Cu-Zn]), L6452_38883 (probable L-ascorbate peroxidase 6, chloroplastic/mitochondrial), L6452_03621 (L-ascorbate peroxidase, cytosolic). F Density of common up-regulated genes (left) and down-regulated genes (right) in genomic positions. G The difference in pathogen-derived L6452_14497 expression between three groups. ARR, PRR and CT separately mean advanced root rot group, primary root rot group, and control group

Discussion

Pathogenic infections frequently elicit symptomatic disease phenotypes in host plants, primarily through resource competition and interference, which disrupt a wide array of genes as symptoms progress [30]. The genomic locus analysis unveiled a coincidental enrichment of DEGs on chromosome CM042048.1 comparing PRR, ARR, and CT.

Four pivotal genes experiencing transcriptional dysfunction in early root rot

The initial stages of root rot infection in A. chinensis elicited prominent transcriptional changes, with 366 up-regulated genes and 225 down-regulated genes relative to the control. Through comprehensive gene annotation and pathway enrichment analysis, 4 hub genes were identified as central to the pathogenesis of root rot: the down-regulated L6452_29839, L6452_06931, and L6452_11144, along with the up-regulated L6452_36719. The gene L6452_29839, annotated as probable sucrose-phosphate synthase 3, is implicated in sucrose-phosphate synthase activity, which is positively linked to sink production and strength as well as osmotic stress responses of roots [31]. Previous investigation has confirmed that overexpression of sucrose-phosphate synthase in alfalfa enhances nodule formation, nitrogenase activity, and overall plant growth [32]. L6452_06931 (UDP-glucose 6-dehydrogenase 1-like) and L6452_11144 (UDP-D-apiose/UDP-D-xylose synthase 2) are involved in conversion of UDP-D-glucose to UDP-alpha-D-xylose and UDP-alpha-D-apiose. Notably, transcriptional down-regulation of UDP-glucose 6-dehydrogenase has been observed in soybean under flooding stress, contrasting with its up-regulation during normal growth conditions [33]. D-Apiose, a vital component for plant cell wall development, is synthesized via UDP-D-apiose/UDP-D-xylose synthase. Depletion of these synthases results in pronounced growth defects, abnormal cell wall architecture characterized by excessive thickening and wall gaps, alongside increased ROS production, culminating in cell death [34]. The up-regulated gene L6452_36719, annotated as granule-bound starch synthase 1, is instrumental in amylopectin synthesis. The Chinese Pharmacopoeia (2000) specifies a maximum daily medicinal dose of 9 g for A. chinensis rhizome. Compared to CT, amylose content was significantly reduced in PRR and ARR, while amylopectin content was significantly increased in PRR. Glycemic index, a key dietary factor in managing diabetes and mitigating chronic disease risk, was negatively correlated with amylose content [35, 36]. Higher amylopectin content was associated with faster glycemic responses and a higher glycemic index [36]. Notably, high amylopectin levels in plants have been associated with rapid postprandial glucose spikes, posing health risks for individuals with obesity or diabetes [37]. Collectively, these findings indicated that early root rot infection in A. chinensis disrupted the expression of 4 hub genes, potentially compromising nodule formation, nitrogenase activity, cell wall integrity, ROS homeostasis, and amylopectin metabolism.

Gene dysregulation and cascading metabolic impacts in advanced root rot

Gene dysregulation becomes more evident with the progression of root rot. Six genes including L6452_28255 (bifunctional dihydrofolate reductase-thymidylate synthase 1), L6452_44605 (serine hydroxymethyltransferase 3, chloroplastic), L6452_09860 (glutathione synthase), L6452_11294 (formate-tetrahydrofolate ligase), L6452_13603 (anthranilate synthase beta subunit 2, chloroplastic), and L6452_21574 (aspartate aminotransferase, cytoplasmic) were tightly related to cascading catalytic reactions. Serine biosynthesis, which relies on tetrahydrofolate-dependent activities and serine hydroxymethyltransferase, is crucial for primary root growth [38, 39]. Glutamate and glycine act synergistically to modulate calcium signaling and hypocotyl elongation [40]. Glutathione synthase bolsters plant tolerance to biotic and abiotic stresses by detoxifying ROS [41, 42]. Formate-tetrahydrofolate ligase facilitates the reversible synthesis of 10-formyltetrahydrofolate, a key step in one-carbon metabolism that underpins nucleotide synthesis and energy metabolism for plant growth [43, 44]. Anthranilate synthase beta subunit initiating tryptophan biosynthesis serves to the production of indole-3-acetic acid, thereby influencing physiological processes in plants [45]. Moreover, anthranilic acid derivatives exhibit promising therapeutic potential, containing neuroprotective effects and regulation of alpha-glucosidase activity in the context of obesity and diabetes [46]. Besides L-glutamate, pyruvate and oxaloacetate are intricately linked to the tricarboxylic acid cycle through the actions of anthranilate synthase beta subunit and aspartate aminotransferase, respectively [47, 48].

Several genes associated with methylation and ethylene biosynthesis pathways were significantly impacted, containing L6452_24444/L6452_24445/L6452_11345 (5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase), L6452_27433 (methionine S-methyltransferase), L6452_20511 (cycloartenol-C-24-methyltransferase), and L6452_24645 (S-adenosylmethionine synthase 2), L6452_04007 (1-aminocyclopropane-1-carboxylate synthase 7-like), L6452_20382 (1-aminocyclopropane-1-carboxylate synthase 2-like). Drought stress represents a critical environmental constraint on the growth and productivity of agricultural crops such as A. chinensis, particularly during the seedling stage [1]. The increase of 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase has been observed in drought-tolerant cotton varieties, highlighting its role in stress adaptation [49]. Methionine S-methyltransferase contributes to barley germination and retains high activity in root tissues [50]. Mutations in S-adenosylmethionine synthase 2 cause methionine accumulation and impaired root elongation [51]. Cycloartenol-C-24-methyltransferase exhibits increased expression under nitrogen-deficient conditions, influencing the feedback regulation of transmethylation reactions [52, 53]. Its catalytic product, 24-methylenecycloartenol, has been evidenced to improve glycemic control and serum biochemical markers in type II diabetic rats [54]. Ethylene biosynthesis, mediated by 1-aminocyclopropane-1-carboxylate synthase, accelerates shoot growth inhibition and abscisic acid accumulation [55].

Genes involved in glycolysis and sucrose metabolism, embracing L6452_26647 (probable sucrose-phosphate synthase 1), L6452_29411 (sucrose-phosphatase 2-like), L6452_11321 (sucrose-phosphatase 2), L6452_29185 (fructose-bisphosphate aldolase 6, cytosolic), L6452_17015 (glyceraldehyde-3-phosphate dehydrogenase, cytosolic-like), L6452_38554 (phospho-2-dehydro-3-deoxyheptonate aldolase 1, chloroplastic-like), and L6452_43701 (phospho-2-dehydro-3-deoxyheptonate aldolase 2, chloroplastic), emerged significant dysregulation. Elevated expression of sucrose phosphatase genes has been shown to confer drought tolerance in maize by modulating sucrose metabolism [56]. Conversely, the down-regulation of sucrose-phosphatase 2-like or sucrose-phosphatase 2 genes probably impair sucrose production, aligning with previous findings that root rot reduces sugar yield in field-grown sugar beet [57]. Fructose-bisphosphate aldolase, a glycolytic enzyme, promotes root growth through the activation of glycolytic pathways [58]. Glycerone phosphate, which reversibly converts to glyceraldehyde-3-phosphate, acts a fundamental role in chloroplast development and seedling establishment [59]. In Arabidopsis, glyceraldehyde-3-phosphate dehydrogenase is indispensable in roots and heterotrophic cells of the aerial part, particularly in the root meristem and cap during normal primary root growth, and serves as a metabolic bridge linking glycolysis to developmental pathways like the phosphorylated serine biosynthesis pathway [60]. Phospho-2-dehydro-3-deoxyheptonate aldolase has been implicated in raising the production of medicinal compounds in plants. For instance, its expression is observably higher in wild plant tissues, stimulating salidroside synthesis compared to tissue-cultured plants [61]. In Lotus japonicus, the synthesis of 7-phospho-2-dehydro-3-deoxy-D-arabino-heptonic acid supports root hair development by stabilizing cell wall dynamics and the actin cytoskeleton, while also facilitating arbuscular mycorrhizal and rhizobial symbioses [62].

Six genes encompassing L6452_00095 (succinate-semialdehyde dehydrogenase, mitochondrial), L6452_40878 (succinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial), L6452_24453 (cytochrome P450 98A2-like), L6452_03960 (protein STRICTOSIDINE SYNTHASE-LIKE 4-like), L6452_09449 (biotin synthase), and L6452_21682 (3-hydroxyisobutyryl-CoA hydrolase-like protein 3, mitochondrial) also were determined as significantly dysregulated in worsening root conditions. Succinate-semialdehyde dehydrogenase ensures plant morphogenesis through maintaining polarity along the adaxial-abaxial axis [63], while succinate dehydrogenase [ubiquinone] flavoprotein subunit regulates stomatal closure, minimizing water loss and preventing tissue desiccation during early dehydration in Ilex paraguariensis [64]. Fumaric acid, a product of enzymatic activity, is integral to plant energy metabolism and carbon skeleton formation [65]. Beyond its metabolic roles, fumaric acid has anti-inflammatory, antioxidant, analgesic, immunomodulatory, neuroprotective, and chemopreventive properties [66]. Ubiquinol, another enzymatic product, not only inhibits low-density lipoprotein peroxidation but also mitigates lesion size in aortic tissues [67]. Biotin synthase is used to generate biotin, a vitamin that strengthens plant resilience to carbonate stress [68]. Strictosidine synthase and cytochrome P450 participate in the biosynthesis of specialized metabolites: 3alpha(S)-strictosidine (a intermediate in camptothecin biosynthesis) and 3beta-Hydroxy-9beta-pimara-7,15-diene-19,6beta-olide (a precursor for phytoalexin momilactone A), respectively [69, 70]. Additionally, UDP-glucose 6-dehydrogenase and UDP-D-apiose/UDP-D-xylose synthase had been mentioned on above L6452_10785 (UDP-glucose 6-dehydrogenase 3-like) and L6452_11144 (UDP-D-apiose/UDP-D-xylose synthase 2).

These findings revealed that, distinct from the early phase, acute root rot triggered the dysregulation of 29 hub genes, whose misalignment severely compromised the physiological rhythm of A. chinensis. This disruption spaned multiple pathways, including one-carbon metabolism, transmethylation feedback regulation, adaxial-abaxial axis polarity, amino acid and phytoalexin biosynthesis, as well as glycolytic and ethylene biosynthetic pathways.

Insights into gene expression disorder persisting throughout root rot

Given the limited overlap of DEGs, the fold change threshold was adjusted downward to identify 10 common genes consistently dysregulated throughout the progression of root rot. These included 7 down-regulated genes (L6452_25713, L6452_18564, L6452_25201, L6452_05034, L6452_03621, L6452_38883, L6452_41016) and 3 up-regulated genes (L6452_41016, L6452_31567, L6452_28564). L6452_25713 encodes superoxide dismutase [Cu-Zn], a gene whose expression and enzymatic activity are significantly augmented in root rot-resistant varieties of Capsicum annuum and Glycine max compared to susceptible lines [71, 72]. Cellulose synthase A catalytic subunits, encoded by L6452_18564, L6452_25201, and L6452_05034, are critical for cellulose biosynthesis. Mutations in these genes lead to root cell wall defects, impaired cell elongation, and gapped walls due to cellulose deficiency [73]. Notably, high beta-D-glucan content, mediated by cellulose synthase A, is concerned with salt tolerance in Triticum aestivum [74]. L6452_03621 and L6452_38883 encode L-ascorbate peroxidase and probable L-ascorbate peroxidase 6, respectively. These enzymes are vital for scavenging hydrogen peroxide and enhancing plant resistance to environmental stressors such as copper and cadmium through bolstering the antioxidant defense system [75, 76]. L6452_41016 encodes pyridoxal kinase, a family member of which, salt overly sensitive 4, is implicated in nitric oxide-induced potassium loss in roots [77]. As a product of pyridoxal kinase activity, pyridoxal 5’-phosphate is recruited by tryptophan synthase being desired for growth and replication of pathogenic microorganisms such as Salmonella bacteria, Chlamydiae bacteria, Mycobacterium tuberculosis, Francisella tularensis, and Streptococcus pneumoniae [78]. Reduced levels of pyridoxamine 5’-phosphate, another catalytic product, correlate with increased osmotic resistance to sodium stress in Arabidopsis [79]. L6452_31567 encodes bifunctional UDP-glucose 4-epimerase and UDP-xylose 4-epimerase 1-like which catalyzes the conversion of UDP-galactose to UDP-glucose [80], however its downstream pathway appeared disrupted in the present work. L6452_28564 encodes homogentisate 1,2-dioxygenase, a key enzyme in homogentisate catabolism. Impairing this pathway increases the production of lipid-soluble antioxidants like vitamin E, and herbicide tolerance in soybean [81]. Together with L6452_26647, previously discussed, the dysregulation of these 8 common genes permeated the entire developmental course of A. chinensis root rot, inducing widespread dysfunction in cell wall integrity, nodule formation and mass, nitrogenase activity, hydrogen peroxide scavenging, resistance to environmental stress, prevention of potassium loss, pathogen defense, and production of lipid-soluble antioxidants.

Eukaryotes secrete extracellular vesicles into their surrounding milieu to preserve and transport high concentrations of biomolecules over long distances [82]. These vesicles encapsulate DNA, RNA, proteins, and metabolites, with their abundance and composition varying in response to environmental conditions [82]. For instance, Hyaloperonospora arabidopsidis, the oomycete pathogen causing downy mildew disease in Arabidopsis, manipulates host plant homeostasis through the secretion of effector proteins [83]. This includes RxLR effectors such as HaRxL106 which recruits the Arabidopsis transcription factor BIM1 to induce transcriptional activation of brassinosteroid-responsive genes, leading to alterations in plant growth patterns that phenocopy shade avoidance syndrome [83]. In our study, comparative analysis of pathogen genes using the NR database revealed significant up-regulation (padj < 0.05; fold change > 4) of the gene encoding ATP synthase subunit beta in both PRR and ARR. Previous research evidences that the LRR domain of a blast resistance gene interacts with ATP synthase subunit beta, promoting its degradation through the 26 S proteasome pathway [84]. Introgression of this blast resistance gene into two japonica rice varieties conferred broad-spectrum resistance and substantially increased yields [84]. Under natural blast disease nursery conditions, these introgression lines exhibited yield increases of approximately 51.59% and 79.31% compared to their parental lines [84]. Furthermore, citral demonstrates potent bacteriostatic activity against rice blast and suppresses ATP synthase subunit beta at the protein level in Magnaporthe oryzae spores [85]. Notably, genes annotated as ATP synthase subunit beta were also significantly up-regulated in the causative agents of PRR and ARR, implicating this subunit in root rot pathogenesis. Additionally, cross-kingdom RNA interference represents an interspecies communication mechanism wherein small RNAs are transported between organisms; these RNAs silence target genes in trans by loading into host Argonaute proteins [86]. In Medicago truncatula, fungal small RNA (e.g. Rhizophagus irregularis Rir2216)-mediated silencing of MtWRKY69 is coupled with arbuscular mycorrhizal symbiosis [86]. However, our RNA extraction protocol for plant mRNA, lacking lytic enzymes (e.g. glucanases) or detergents (e.g. Triton) [87, 88], limited our ability to investigate pathogen-derived RNA (including non-coding species) in plant-pathogen crosstalk mechanisms.

The present study clarified unique and overlapped genes dysregulating during the onset and progression of A. chinensis root rot. These genes orchestrate cascading signal transductions affecting cell architecture, biochemical processes, and stress responses.

Supplementary information

12870_2025_7196_MOESM1_ESM.xlsx (1.7MB, xlsx)

Supplementary Material 1. Table S1. List of differentially expressed genes

Authors’ contributions

Y.L. conceived and designed the study. Y.L. and S.M. carried out the experiments. Y.L., T.Z., and Y.C. analyzed the data. C.Q. and Y.C. wrote the manuscript. Y.W. and C.Z. provided valuable insights in the discussion and revision of the manuscript. All authors read and approved the final manuscript.

Funding

This work was supported by Hebei Natural Science Foundation (H2021406030 and H2023406026), and also funded by Science Research Project of Hebei Education Department (QN2022082), and Chengde Medical University Scientific Research Fund of Senior Talent (202104 and 202403). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data availability

The raw data of this study have been deposited into China National GeneBank Sequence Archive (CNSA) with accession number CNP0007787 (https://db.cngb.org/data_resources/project/CNP0007787/).

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Yingzhe Li, Yang Liu and Tian Zhang contributed equally to this work.

Contributor Information

Yu Cao, Email: yucao@cdmc.edu.cn.

Chunying Zhao, Email: zhaochunying1908@163.com.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

12870_2025_7196_MOESM1_ESM.xlsx (1.7MB, xlsx)

Supplementary Material 1. Table S1. List of differentially expressed genes

Data Availability Statement

The raw data of this study have been deposited into China National GeneBank Sequence Archive (CNSA) with accession number CNP0007787 (https://db.cngb.org/data_resources/project/CNP0007787/).

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