Unraveling the Molecular Mechanisms Underlying Flat Stem Formation in Atractylodes lancea in Response to Phytoplasmas

Unraveling the Molecular Mechanisms Underlying Flat Stem Formation in Atractylodes lancea in Response to Phytoplasmas | 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 Unraveling the Molecular Mechanisms Underlying Flat Stem Formation in Atractylodes lancea in Response to Phytoplasmas Ling Gong, Lei Chen, Xiao Huang, Juan Deng, Xiaoyi Wu, Yating Hu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4281530/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 Atractylodes lancea (Thunb.) DC has been widely used as a medicinal herb for centuries. However, long-term artificial cultivation of A. lancea led to serious plant diseases, such as the flat branch disease caused by phytoplasmas. To explore the formation mechanism of flat stems, we measured the changes in physiological and biochemical indicators and related metabolic pathways in stems of A. lancea in response to phytoplasma. After infection by pathogen, significant changes were observed in the content of stress compounds H2O2 and MDA, as well as the activities of antioxidant enzymes APX, POD, PPO, and CAT. The contents of jasmonic acid and zeatin in the flat stem (FS) of A. lancea increased significantly, while auxin content decreased. High-throughput sequencing showed that differentially expressed genes (DEGs) are enriched in hormone biosynthesis, signal transduction, Ca2+ signaling, and other pathways. These results preliminary elucidate the molecular mechanism of flat stem development in A. lancea, providing a foundation for disease prevention in the future. Atractylodes lancea Flat stem Phytoplasma Morphology Physiology and Biochemistry Transcriptome Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key message Multiple regulatory pathways and differentially expressed genes in Atractylodes lancea to phytoplasmas infection were identified through RNA-seq analysis, providing valuable insights into the development of flat stems in A. lancea . Introduction Phytoplasmas are pleomorphic, wall-less intracellular bacteria that can cause devastating diseases in more than 1,000 plant species(Deng 1991). Phytoplasma-related diseases, such as witches'- broom, dwarfing disease, banding disease, and phyllody, are considered the main hindrance in many areas, resulting in enormous losses worldwide for agricultural, forestry, and pharmaceutical production. Phytoplasma causes damage to host plants by affecting physiological and biochemical processes (Catlin et al. 1975; Kartte and Seemüller 2008). Stem flattening is the typical symptom of phytoplasma-infected plants, which can enlarge the vegetative tissue and generate more vascular bundle networks. Flat stem often manifests as thickened and banded stems, disordered leaves, and abnormal inflorescence development, affecting plants' normal growth and development. At present, the occurrence of flat-stem disease has been reported in multiple countries and occurred on a variety of broad-leaved trees, including Sophora japonica (Liu et al. 2022), Amorpha fruticose (Huang et al. 2023), and Persimmon (Wang et al. 2017), etc. It also affects herbaceous plants such as Corn (Duduk and Bertaccini 2006), Cucumber (Wang et al. 2022), Arabidopsis thaliana (Bressan and Purcell 2005; Cettul and Firrao 2011), etc. Such physiological conditions were a result of the impact of phytoplasma on hormonal, developmental, and stress signaling pathways, as well as the intricate interplay between these factors. It was reported that phytoplasma infection affects hormone balance in plants, resulting in changes in levels of crucial hormones like auxin, cytokinin, jasmonic acid (JA) and ethylene (Dermastia et al. 2019). These alterations give rise to significant morphological modifications encompassin root architecture, stem growth, and floral development. Additionally, phytophthora infection triggers oxidative stress within plants, leading to the generation of reactive oxygen species (ROS). These ROS exert deleterious effects on cellular machinery and contribute to the manifestation of disease symptoms. A. lancea , also known as Cangzhu in Chinese, is a perennial herb that plays significant importance in traditional Chinese medicine. Its dried rhizomes have been widely used for dispelling wind and cold, drying dampness, strengthening the spleen, improving eyesight, and treating various gastrointestinal tract diseases and night blindness. With the increasing demand for A. lancea for medicinal purposes, there has been a shift towards artificially cultivated plants becoming the primary source of medicinal materials. Large-scale artificial planting of A. lancea in the field after long-term multi-generation asexual reproduction has brought about certain challenges such as the spread of diseases and pests. To gain insights into the development mechanism of stem flattening in A.lancea after infection by phytoplasma, the plant structure, stress compounds, antioxidant enzyme activity, endogenous hormone, and comparative transcriptome were analyzed. The results will provide new insight into the formation mechanism of flat stems in plants and help control the phytoplasma disease of A. lancea. Materials and methods Plant materials Both the normal stem (FNS) and flat stem (FS) exhibiting flattening symptoms on A. lancea , as well as the healthy stem (NS) from the same experimental field in the A. lancea cultivation base located in Yingshan County, Hubei Province (115°57′31″E, 30°58′16″N) were collected and stored at -80 ℃. The phytoplasma infection was further confirmed by nested PCR amplification of the 16S rRNA gene referring to Wan Qianyun's method (Wan et al. 2018). The gene-universal oligonucleotide primers P1/P7 and R16F2n/R16R2 were employed for Nested PCR analysis as detailed in Supplementary Table S1 (Hiruki 1991; Schneider et al. 1995). Phenotypic and microscope observation The phenotypic changes of A. lancea with flattening stem symptoms, including plant morphology, terminal bud (stem tip) shape, and phyllotaxy, were documented with a digital camera. The width (major axis) and thickness (minor axis) of the central stem were measured using vernier calipers in 12 biological replicates. Paraffin sections were prepared from the terminal bud and young stem for observation of their anatomical structure. Determination the content of H 2 O 2 , MDA, and the activities of antioxidant enzyme The extraction of hydrogen peroxide (H 2 O 2 ) and Malondialdehyde (MDA) was conducted based on the methods of Amin (Amin and Olson 1967; Satterfield and Bonnell 1955) and Spitz (Spitz and Oberley 1989). The extraction of ascorbate peroxidase (APX) followed the procedure described by Nakano (Nakano and Asada 1981). Peroxidase (POD) was extracted and detected according to Han's protocol (Zhao and Han 2011). Polyphenol oxidase (PPO) was extracted and detected using of Liu’ methods (Liu, et al. 2020). Catalase (CAT) extraction followed Johansson's procedure (Johansson and Borg 1988). The H 2 O 2 , MDA, CAT kits (Boxbio Biotechnology Co., Ltd, Beijing, China) and the APX kit (Elabscience Biotechnology Co., Ltd, Wuhan, China) were used respectively for the detection of the above metabolites/enzymes according to the manufacturer's instructions. The standard curve of H 2 O 2 was y=3.251x-0.01351 (R 2 =0.9997). Determination of endogenous hormone The NS, FS, FNS samples of A. lancea were ground into a powder with liquid nitrogen. Subsequently, 120 mg samples were dissolved in 80% methanol and subjected to vortexing for 0.5 h six times, followed by leaching at 4 ℃ for 12 h. The tube was vortexed and centrifuged at 12,000 r/min for 5 min. After centrifugation, the supernatant was filtered with a 0.22 µm polypropylene filter and directly injected into the LC-MS/MS system. Chromatographic separation was performed using Waters reversed-phase C18 chromatographic column (Waters, ACQUITY UPLC HSS T3 1.8 μm, 2.1×100 mm Column) with a mobile phase consisting of 0.04% formic acid (A) and acetonitrile (B) at a flow rate of 0.35 mL/min. The gradient was as follows: 0-1 min, 95%A, 1-8 min, 95%A-5%A, 8-9 min 5%A, 9-12 min 95%A. The column temperature was maintained at 40 ℃, and the injection volume was 2 µL. Detection wavelengths were set at different values depending on the analytes: ABA, IAA, JA were detected at λ=254 nm while zeatin was monitored at λ=270 nm using triple quadrupole mass spectrometry's multiple reaction monitoring modes. The reference standards of ABA, IAA, JA, and Zeatin were purchased from Shanghai Yuanye Bio-Technology Co., Ltd, Shanghai, China. RNA Sequencing, functional annotation and differentially expressed genes (DEGs) analysis The NS, FS, and FNS samples of A. lancea were ground with liquid nitrogen, followed by RNA extraction utilizing the TIANGEN polysaccharide and polyphenol plant total RNA extraction kit. After quantifying the total RNA content, a cDNA library was constructed and sequenced in Guangzhou Kedio Biotechnology Co., Ltd. The high-quality reads were assembled after following data filtration using the Trinity program. Functional annotation of these unigenes was conducted using protein databases including non-redundant protein (nr), SwissProt, Kyoto Encyclopedia of Genes and Genomes (KEGG), as well as Cluster of Orthologous Group/Eukaryotic Orthologous Group (COG/KOG), based on a blastx search with an E value threshold below 10 -5 . Unigene expression was determined using the fragments per kilobase of transcript per million mapped reads (FPKM) method. DEGs were identified with a |log2FC|>1 and a false discovery rate (FDR) ≤ 0.05. Subsequently, DEGs underwent GO and KEGG enrichment analysis, respectively. qRT‑PCR analysis 6 randomly selected DEGs related to hormone biosynthesis and signaling transduction were verified by qPCR using the SYBR method. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was employed as an internal reference gene. The primer sequences for the selected DEGs are listed in Supplementary Table S1. Purified RNA (1 μg) was reverse transcribed into cDNA using Evo M-MLV RT Mix Kit with gDNA Clean for qPCR Ver.2 (Accurate Biotechnology CO., LTD, Changsha, China) according to the manufacturer’s instruction. The qPCR reaction system consisted of a volume of 20 µL, which included 10 µL of 2 × SYBR Green Pro Taq HS Premix (ROX Plus), 2 µL of the cDNA template, 0.4 µL of each primer, and 7.2 µL ddH2O. The reaction conditions were pre-denaturation at 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s, and annealing at 60 °C for 30 s. The relative gene expression was calculated according to 2 -ΔΔCT (Liu et al. 2018; Stortenbeker and Bemer 2019). Statistical analysis The mean values and standard deviations (SD) were calculated using SPSS 17.0 software (SPSS Inc., Chicago, United States). Significant differences were determined using the independent-samples t-test. Each experiment was independently performed three times. Graphs were constructed using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, United States). Results Field observation showed that the NS exhibited a nearly cylindrical shape with alternate phyllotaxis, whereas the FS displayed a flatter and thicker structure characterized by disordered phyllotaxy, forming distinct bands (Fig. 1a, b, c). In contrast to the needle-like stem tip of NS (Fig. 1a), the FS possessed a broom-like morphology (Fig. 1b). Furthermore, in comparison to NS, the transverse section of FS exhibited more than twice the width (major axis) and thickness (minor axis), resulting in a significantly different aspect ratio (width/thickness) (Supplementary Fig. S1, Fig. 1d). To confirm phytoplasma infection, the amplification and sequencing of the 16S rRNA gene were performed. In comparison to NS and FNS, a 1245 bp fragment was amplified in FS, exhibiting 100% homology to the Aster yellows group (16SrI) phytoplasma (GenBank accession number: MG257945.3) after conducting a blast search on NCBI. The phylogenetic tree analysis revealed that the A. lancea phytoplasma clustered with subgroup 16SrI-B (AY101386.1) found in Epilobium hirsutum L., suggesting that phytoplasma infection may be responsible for inducing the flat stem variant in A. lancea . Microscope observation of shoot apical meristem and stems cross-section The microscope observation revealed that the stem tip of NS was composed of shoot apical meristem, leaf primordium, young leaves, pith, and axillary bud primordium (Fig. 2a). In contrast, the FS exhibited a significantly widened stem tip with young leaves arranged in a linear fashion on the shoot apical meristem and alterations in the distribution of leaf primordia. Although FS had the same anatomical structure as a normal stem, including the epidermis, cortex, phloem, cambium, xylem, and pith, its stem presented a flat shape (Fig. 2b). Furthermore, irregular protrusions were observed on the surface of FS resulting in an uneven epidermal texture compared to normal stems. Additionally, there was an evident increase in thickness for both cortex and vascular tissue to varying degrees (Fig. 2c). The determination of stress compound content, antioxidant enzyme activity and endogenous hormone content In addition to affecting stem structure, plants can also trigger the induction of stress biomarkers in response to phytoplasma infection. Our study revealed that both the FS and FNS exhibited significantly higher levels of H 2 O 2 accumulation compared to the NS group, while only the FS group showed a significant increase in MDA content. Meanwhile, there was an elevation in enzymatic activity of PPO and CAT in the FS. However, the APX and POD activities were markedly decreased when compared to those observed in the NS group (Fig. 3b, c). To investigate the potential association between hormone disorder and the development of a flat stem, the levels of four hormones in NS, FNS, and FS were quantified. Notably, FS exhibited significant differences compared to NS and FNS, characterized by elevated jasmonic acid and zeatin levels as well as reduced auxin (IAA) content (Fig. 3a). No significant difference was observed in abscisic acid concentration. Transcriptome sequencing and Unigene annotation RNA-seq sequencing analysis was performed to explore the mechanisms underlying the formation of flat stems. A total of 137,205 unigenes were annotated and blasted in four public databases of Nr, KOG/COG, Swiss Prot, and KEGG. The result showed that the Nr database, KEGG database, Swiss Prot, and KOG contained annotations for 50,592, 46,042, 27,999, and 225,234 unigenes respectively. The statistical results of all unigenes annotation and the Wayne diagram depicting transcriptome data for A. lancea were presented in Fig. 4a. By utilizing the Nr library for comparative analysis and annotation, we can ascertain the genetic similarity between A. lancea and its related species. The top four most closely related species were Cynara cardunculus (39.64%), Artemisia annua (14.05%), Lactuca sativa (9.84%), and Helianthus annuus (5.48%) (Supplemental Fig. S2). It is noteworthy that all these four species, along with A. lancea , belong to the Compositae family. Analysis of differentially expressed genes (DEGs ） in A. lancea The DEGs were screened with FDR1 as threshold value. Subsequently, volcano plot analysis was performed to visualize the significant differences in gene expression between each comparison group. Compared to NS, 114 genes were upregulated while 268 genes were downregulated in FS (Fig. 4b). Similarly, compared to FNS, FS exhibited upregulation of 77 genes and downregulation of 458 genes. Conversely, only a limited number of DEGs showed upregulation (8 genes) or downregulation (22 genes) between "NS vs. FNS" (Supplemental Fig. S3a, b). Given the small number of DEGs identified between "NS vs. FNS", our focus will be on analyzing the differences observed in "NS vs. FS" and "FNS vs. FS." Go enrichment analysis can be classified into three domains: biological processes, cellular components, and molecular functions. Regarding biological processes, both "NS vs. FS" and "FNS vs. FS" functional regions exhibited a significant enrichment of DEGs associated with metabolic, cellular, and single-organism processes. In terms of cellular components, the most prominent category of DEGs was involved in cell, cell part, organelle, and membrane localization. A substantial number of DEGs were annotated to molecular functions such as binding activity, catalytic activity, and transporter activity (Fig. 4c; Fig. S3c). The top 20 results obtained from KEGG pathway enrichment analysis are presented in Figure 4d and supplementary Figure S3d. Notably, the DEGs were significantly enriched in key pathways such as "metabolic pathways," "biosynthesis of secondary metabolites," and "zeatin biosynthesis," suggesting their potential involvement in phytoplasma response mechanisms. Differential expression of hormone metabolic pathway gene in A. lancea The DEGs associated with hormone biosynthesis and signal transduction between "NS vs. FS" and "FNS vs. FS" in A. lancea showed the same expression trend, including salicylic acid, jasmonate, brassinosteroid, ethylene, abscisic acid, gibberellins, cytokinin, and auxin. Notably, a majority of the DEGs enriched in the salicylic acid and jasmonate synthesis exhibited upregulation in the FS group compared to NS or FNS groups (Fig. 5a). This suggests that these two hormones may play crucial roles in response to stress conditions or pathogen attacks in A. lancea . On the other hand, DEGs enriched in brassinosteroid and cytokinin synthesis showed downregulation specifically in the FS group. This downregulation might indicate a suppression of growth-related processes regulated by these hormones under stressful conditions (Fig. 5a). The expression of 3 DEGs encoding 3-deoxy-7-phosphoheptulonate synthase (DAPH), the initial enzyme in a series of metabolic reactions, was downregulated in the FS. Auxin levels can influence plant growth and development, necessitating the presence of early response genes such as Auxin/Indole-3-Acetic Acid (Aux/IAA), auxin response factor (ARF) family, small auxin up RNA (SAUR), etc (Stortenbeker and Bemer 2019). In the study, 2 DEGs annotated to AUX/IAA and 2 DEGs annotated to SAUR were also found to be expressed at low levels in the FS, consistent with auxin content distribution. 1 DEG encoding adenylate isopentenyl-transferase (IPT), which acts as the first and rate-limiting synthase in cytokinin biosynthesis, exhibited upregulated in the FS. 2 allene oxide synthase (CYP74A) DEGs involved in jasmonic acid biosynthesis showed upregulation in the FS. 1 DEG encoding jasmonate ZIM-domain protein (JAZ), an essential repressor in the JA signaling pathway, displayed downregulation. These findings align with zeatin and jasmonic acid content distribution. Differential expression of plant-pathogen interaction pathway genes in A. lancea phytoplasma, a pathogenic bacteria, can invade the vascular tissue of plants and induce a cascade of physiological alterations. Elucidating the intricate interplay between pathogen and their host plants is pivotal for comprehending the flat stem developmental processes of A. lancea . In this study, we identified 10 DEGs that are involved in plant-pathogen interactions pathway (Fig. 6). Among these DEGs, 5 DEGs were implicated in Ca 2+ signaling, including calmodulin/calbindin (CaM/CML), respiratory burst oxidase homologs (CDPK) and respiratory burst oxidase homologs (Rboh). 1 DEGs encoding Rboh and 1 DEGs encoding CaM/CML were upregulated in the FS. In addition, 5 DEGs were annotated to β-Ketoacyl-CoA synthase (KCS), which played a vital role in determining the quantity and composition of very long chain fatty acids (VLCFAs) (Zhang et al. 2022). The transcription factor involved in the formation of flat stems Numerous transcription factors have been reported to play pivotal roles in plant growth and development, serving as crucial regulatory molecules involved in stress response and pathogen resistance. In comparison to NS and FNS, the DEGs were found to be associated with WRKY, ERF, and MYB transcription factors, which showed a decrease in expression levels in FS. This finding implies that plants might regulate the function of WRKY, ERF, and MYB transcription factors under phytoplasma infection conditions in order to cope with external stresses; however, this modulation could potentially lead to abnormal physiological processes like stem flattening. Validation of key gene expression To further validate the transcriptome results, a total of 6 genes were randomly selected for qPCR analysis (Supplementary Table S1). Among them, 3 genes were related to auxin metabolism, while the other 3 genes were associated with salicylic acid signal transduction (PR1), brassinosteroid signal transduction (TCH4), and jasmonic acid signal transduction regulator (JAZ) pathway, respectively. The expression patterns of these selected genes demonstrated a high degree of concordance with the transcriptome data (Fig. 7b). Discussion Phytoplasma infection induced stem flat in A. lancea The precise regulation of meristem activity governs the structural organization of plants. Imbalances in positive or negative maintenance signals within terminal buds can lead to phenotypic changes, such as thinning or enlargement of the meristem, resulting in enlarged vegetative bodies and inflorescence meristems. Our study revealed a broom-like appearance with widened meristems and systematically arranged leaves in rows at the terminal bud region of flatting symptomatic A.lancea , indicating that alterations in meristem activity have influenced stem morphology characteristics. Furthermore, molecular detection exclusively identified phytoplasma from FS samples belonging to 16SrI-B subgroup within Aster yellows group (Fig.1e), indicating that phytoplasma infection modifies meristem activity contributing to flat stem occurrence on A.lancea . Changes in stress compound content are involved in flat stem formation After infection by phytoplasma, plants have developed various basic defense mechanisms to protect themselves from pathogens. One such defense mechanism involves the rapid accumulation of ROS at the pathogen attack site (a phenomenon called oxidative burst) (Girodat et al. 2020), which can eliminate invading pathogens directly. In the study, the levels of H 2 O 2 in both FS and FNS were significantly higher compared to NS (Fig. 3). However, only FS exhibited a significant increase in malondialdehyde (MDA) content, indicating damage to the liposomes in FS but not in FNS. PPO catalyzes the formation of lignin and quinone compounds to form a protective shield, thus protecting cells from pathogens. Interestingly, PPO activity was significantly increased in the FNS compared with NS and FS (Fig. 3), suggesting FNS may have recovered from the phytoplasma infection to as a strategy to prevent stem flattening (Musetti et al. 2004). Various antioxidant enzymes, including POD, CAT, and APX, are involved in scavenging H 2 O 2 and reactive oxygen species metabolism during pathogen attacks with different mechanisms. However, contrary to expectations, the levels of APX and POD did not increase in the FS, indicating a potential disruption in the equilibrium between H 2 O 2 production and scavenging by antioxidant enzymes. Previous studies have reported that maintaining a dynamic balance of ROS played a role in keeping the balance of transition from cell proliferation to cell differentiation, which was a key factor for maintaining normal root morphology (Xu et al. 2020). Chen also found that appropriate levels of H 2 O 2 promoted axillary bud outgrowth in tomato plants (Chen et al. 2016). The results implied that the sharp increased H 2 O 2 induced by the phytoplasma infection led to the flat stem phenomenon in A. lancea . Changes in plant hormone content are involved in flat stem formation The establishment of stem cell niches and cell proliferation within the meristematic zone are governed by the plant hormone auxin (Beemster and Baskin 2001; Blilou et al. 2005). Altered auxin transport and distribution resulting from inhibited expression of an Aux/IAA gene (SHY2) effectively lead to changes in root meristem size (Mandal et al. 2023). In Solanaceae, inhibition of several Aux/IAA genes, such as IAA9 , reduces apical dominance and enhances hypocotyl/stem elongation(Wang et al. 2005). Our study reveals downregulation of DEGs encoding SAUR and Aux/IAA in the FS, accompanied by decreased auxin contents. Similar observations have been made in phytoplasma-related plants where an imbalance in auxin levels leads to morphological changes like witches' broom or dwarf symptoms(Christensen et al. 2005). In addition to auxin, cytokinin exerts an influence on root meristem activity(Ruzicka et al. 2009). Elevated levels of cytokinin achieved through exogenous application or over expression of the bacterial IPT gene(Medford et al. 1989; Kuderova et al. 2008) impede root growth and induce changes in meristem size. Increased endogenous cytokinin levels resulting from activation of plant IPT gene expression enhance or modify the morphogenic potential of transformed plant cells(Mens et al. 2018). In our study, one IPT was found to be upregulated with increased zeatin levels. These findings suggest that phytoplasma-induced imbalance between zeatin and auxin leads to expansion of stem apical meristem along with internal structural changes and stem flattening. Jasmonic acid (JA) and its methyl ester (MeJA) have the ability to stimulate cell expansion in plant medullary tissue. Exogenous application of jasmonic acid induces changes in apical meristem morphology of potato stolons(Cenzano et al. 2003). Following infection with phytoplasma, there was a significant increase in JA content along with upregulation observed for the JA synthesis-related gene CYP74A2 in the FS, which correlates well with citrus ulcer formation (Qin et al. 2019), suggesting that JA may also affect meristems leading to the development of flat stems. Recent studies have indicated a possible crosstalk among plant hormones in the regulation of vascular tissue development within the primary root meristem of Arabidopsis (Sun et al. 2023). Consequently, it is imperative to further investigate the intricate and precise hormonal regulatory network in A. lancea infected with phytoplasma and its consequential impact on flat stem formation. The different expressions of Ca 2+ signaling pathways associated genes are involved in flat stem formation Changes in Ca 2+ levels and signals significantly contribute to plant growth and development, including root morphogenesis. The functions of Ca 2+ generally rely on sensors, such as calmodulin-like proteins (CMLs) and calcium-dependent protein kinases (CDPKs), which decode and transduce Ca 2+ signals. In Arabidopsis thaliana , AtCML24 mutant caused the higher [Ca (2+)] cyt within germinated pollen and extended pollen tubes, thereby affecting pollen germination and pollen tube elongation (Yang et al. 2014). Similarly, OsCDPK5/13 negatively regulates aerenchyma formation of roots in rice (Yamauchi et al. 2017). It has been demonstrated that phytoplasma infection induces an elevation of intracellular Ca 2+ (Musetti et al. 2013). Subsequently, CDPK is upregulated along with the highly expressed key producers of reactive oxygen species (ROS) in plants known as RBOHs that can be activated by CDPKs. This suggests that phytoplasma infection induces an increase in calcium ion concentration to activate the expression of CDPK and RBOHs, resulting in elevated H 2 O 2 levels. The transcription factor involved in the formation of flat stems Transcription factors (TFs) directly regulate genes related to cell proliferation and cell elongation, thereby potentially influencing plant structural characteristics (Peng et al. 2023). The MYB family, one of the largest groups of transcription factors, is known to impact stem development as well as the biosynthesis of cell walls and cell cycle function. RNA interference BnMYB69 of Brassica napus presented substantial changes in morphology, anatomy, and metabolism (Lin et al. 2023). Overexpression of OsMPH1, a MYBLIKE GENE OF PLANT HEIGHT 1 in rice plants, led to increased plant height and seed yield by modulating the expression of genes related to cell wall formation and elongation (Zhang et al. 2017). Additionally, WRKY transcription factor also plays a role in regulating stem elongation and diameter. For instance, WRKY41a from herbaceous peonies promotes secondary cell wall thickening for enhanced stem strength (Tan, et al. 2023), while OsWRKY78 is crucial for controlling stem elongation in rice plants(Zhang et al. 2011). Furthermore, mutation of WRKY13 in A. thaliana can reduce stem diameter through repression of lignin synthesis(Li et al. 2015). Transcription factors also participate in the regulatory network of plant hormones. During plant growth, WRKY regulates auxin to influence the initiation of axillary meristem and the growth of new buds (Guo et al. 2015; Lan et al. 2020). Ethylene response factor (ERG) activates the expression of ASA1 (the rate-limiting enzyme of tryptophan synthesis), induces auxin biosynthesis and facilitates auxin accumulation in root tip, and consequently inhibits root elongation. The present study revealed significant differential expression of transcription factors WRKY, MYB, and ERG in the FS compared to NS and FNS, suggesting their potential regulatory role in the formation of flat stem in A. lancea . However, further confirmation is required to elucidate their precise functions. Conclusion Our studies revealed that the flat stem formation of A. lancea is a pathological phenomenon caused by phytoplasma infection, which alters the stem tip meristem. During phytoplasma infection, there are changes in stress compound content, antioxidant enzyme activity, and endogenous hormone levels. Notably, the H 2 O 2 content in the FS is significantly higher compared to NS, while the levels of antioxidant enzymes involved in scavenging H 2 O 2 are lower than those found in NS. Moreover, there is a substantial alteration in hormone content during phytoplasma infection, with differentially expressed genes (DEGs) associated with hormone biosynthesis and signal transduction identified through RNA-seq analysis. These DEGs include auxin, jasmonic acid, and zeatin-related genes. Additionally, our study characterizes several DEGs such as CML, CDPK, and RBOH that play crucial roles in Ca 2+ signaling pathways and ROS production along with transcription factors (TFs) related to stem flattening (Fig 8). This study provides new insights into the mechanisms underlying flat stem formation in A.lancea . Declarations Author contribution statement : Liu Changli and Yu Kun designed the project; Gong Ling and Chen Lei performed the research, analyzed the data, and wrote the paper. Huang Xiao, Deng Juan and Jiang Meiling participated in the research. Wu Xiaoyi and Hu Yating analyzed the RNA-seq data. All authors read and approved the final manuscript. Acknowledgments We would like to thank Prof. Zhang Xiuqiao for her help in experimental technique support. Funding This work was supported by the 71st General Project of China Postdoctoral Science Foundation (Grant number 2022M71223), the National Natural Science Foundation of China (Grant number 32000254, 31670341 and 81891014), and the Hubei Province Technology Innovation Special Major Project (Grant number 2018ACA124). Conflict of interest The authors declare no competing financial interest. References Amin VM, Olson NF (1967) Spectrophotometric Determination of Hydrogen Peroxide in Milk1. J DAIRY SCI 50:461-464 Beemster G, Baskin T (2001) STUNTED PLANT 1 mediates effects of cytokinin, but not of auxin, on cell division and expansion in the root of Arabidopsis. 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PLANT GROWTH REGUL Qin, Long, Xie, Yongrui, Qiang, Xiuping, Zou, Shanchun, Chen (2019) Abscisic Acid Promotes Jasmonic Acid Accumulation and Plays a Key Role in Citrus Canker Development. FRONT PLANT SCI 10:1634 Ruzicka K, Simaskova M, Duclercq J, Petrasek J, Zazimalova E, Simon S, Friml J, Van Montagu MC, Benkova E (2009) Cytokinin regulates root meristem activity via modulation of the polar auxin transport. P NATL ACAD SCI USA 106:4284-4289 Satterfield CN, Bonnell AH (1955) Interferences in Titanium Sulfate Method for Hydrogen Peroxide. ANAL CHEM 27:1174-1175 Schneider B, Seemueller E, Smart CD, Kirkpatrick BC (1995) PHYLOGENETIC CLASSIFICATION OF PLANT PATHOGENIC MYCOPLASMA-LIKE ORGANISMS OR PHYTOPLASMAS. Molecular and Diagnostic Procedures in Mycoplasmology I:369-380 Spitz DR, Oberley LW (1989) An assay for superoxide dismutase activity in mammalian tissue homogenates. ANAL BIOCHEM 179:8-18 Stortenbeker N, Bemer M (2019) The SAUR gene family: the plant's toolbox for adaptation of growth and development. J EXP BOT 70:17-27 Stortenbeker N, Bemer M (2019) The SAUR gene family: the plant's toolbox for adaptation of growth and development. J EXP BOT 70:17-27 Sun Y, Yang B, De Rybel B (2023) Hormonal control of the molecular networks guiding vascular tissue development in the primary root meristem of Arabidopsis. J EXP BOT 74:6964-6974 Tang Y, Lu L, Huang X, Zhao D, Tao J (2023) The herbaceous peony transcription factor WRKY41a promotes secondary cell wall thickening to enhance stem strength. PLANT PHYSIOL 191:428-445 Wan Q, Chen L, Ming S, Gong L, Huang X, Yu K, Che HY, Luo DQ (2018) First Report of 'Candidatus Phytoplasma asteris' Related Strain Associated with Atractylodes lancea Malformations in Hubei Province of China. PLANT DIS Wang H, Jones B, Li Z, Frasse P, Delalande C, Regad F, Chaabouni S, Latche A, Pech JC, Bouzayen M (2005) The tomato Aux/IAA transcription factor IAA9 is involved in fruit development and leaf morphogenesis. PLANT CELL 17:2676-2692 Wang J, Gao R, Yu XM, An M, Ai CX (2017) Morphological and molecular detection of phytoplasma associated with persimmon fasciation disease. Zhiwu Shengli Xuebao/Plant Physiology Journal 53:219-226 Wang X, Hu Q, Wang J, Lou L, Xu X, Chen X (2022) Comparative Biochemical and Transcriptomic Analyses Provide New Insights into Phytoplasma Infection Responses in Cucumber. GENES-BASEL 13: Xu P, Zhao PX, Cai XT, Mao JL, Miao ZQ, Xiang CB (2020) Integration of Jasmonic Acid and Ethylene Into Auxin Signaling in Root Development. FRONT PLANT SCI 11:271 Yamauchi T, Yoshioka M, Fukazawa A, Mori H, Nishizawa NK, Tsutsumi N, Yoshioka H, Nakazono M (2017) An NADPH Oxidase RBOH Functions in Rice Roots during Lysigenous Aerenchyma Formation under Oxygen-Deficient Conditions. PLANT CELL 29:775-790 Yang X, Wang SS, Wang M, Qiao Z, Bao CC, Zhang W (2014) Arabidopsis thaliana calmodulin-like protein CML24 regulates pollen tube growth by modulating the actin cytoskeleton and controlling the cytosolic Ca (2+) concentration. PLANT MOL BIOL 86:225-236 Zhang A, Xu J, Xu X, Wu J, Li P, Wang B, Fang H (2022) Genome-wide identification and characterization of the KCS gene family in sorghum (Sorghum bicolor (L.) Moench). PEERJ 10:e14156 Zhang CQ, Xu Y, Lu Y, Yu HX, Gu MH, Liu QQ (2011) The WRKY transcription factor OsWRKY78 regulates stem elongation and seed development in rice. PLANTA 234:541-554 Zhang Y, Yu C, Lin J, Liu J, Liu B, Wang J, Huang A, Li H, Zhao T (2017) OsMPH1 regulates plant height and improves grain yield in rice. PLOS ONE 12:e180825 Zhao, Y., Han S., 2011. Activity Determination and Property Analysis on Peroxydase in Onion. Journal of Anhui Agricultural Sciences, 39, 1275-1277. Supplementary Files SupplementaryMaterial.docx SupplementarySupplementaryTableS1.xls SupplementarySupplementaryFigure.pptx 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4281530","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":295601974,"identity":"bc550faf-83b2-4609-81c9-9c5a7e3df85f","order_by":0,"name":"Ling Gong","email":"","orcid":"","institution":"Capital Medical University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Gong","suffix":""},{"id":295601975,"identity":"995baba8-6858-47fd-adfe-881e2a79cd4e","order_by":1,"name":"Lei Chen","email":"","orcid":"","institution":"Hubei University of Chinese 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11:20:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4281530/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4281530/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":55714943,"identity":"36cc1722-dfde-42fb-9f83-f4cc4c9d1958","added_by":"auto","created_at":"2024-05-02 07:22:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":900185,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological observation of stem in \u003cem\u003eA. lancea\u003c/em\u003e. \u003cstrong\u003ea\u003c/strong\u003e Normal stem in healthy \u003cem\u003eA. lancea \u003c/em\u003e(NS). \u003cstrong\u003eb\u003c/strong\u003e Flat stemin\u003cem\u003e \u003c/em\u003esymptomatic\u003cem\u003e A. lancea \u003c/em\u003e(FS). \u003cstrong\u003ec\u003c/strong\u003e Flat stem\u003cem\u003e \u003c/em\u003e(FS) and normal stems (FNS) in the \u003cem\u003eA. lancea\u003c/em\u003e with flatting stem symptom. \u003cstrong\u003ed\u003c/strong\u003eThe aspect ratio of the cross-section of NS and FS. \u003cstrong\u003ee \u003c/strong\u003ePhylogenetic tree based on nucleotide sequence of phytoplasma 16S rDNA gene. \"-\" represents 1 cm; \"***\" indicates significant difference at P ≤ 0.001 (n = 12) according to the unpaired t-test.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-4281530/v1/9014d74fc0fba2650d2f2915.png"},{"id":55714945,"identity":"eae81ff2-d000-4cb3-8f2e-0b087ceacfc8","added_by":"auto","created_at":"2024-05-02 07:22:24","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":512431,"visible":true,"origin":"","legend":"\u003cp\u003eObservation of the shoot apical meristemand stem cross-section of \u003cem\u003eA. lancea \u003c/em\u003eby electron microscope. \u003cstrong\u003ea \u003c/strong\u003eterminal bud of NS. \u003cstrong\u003eb\u003c/strong\u003e terminal bud of FS\u003cem\u003e.\u003c/em\u003e \u003cstrong\u003ec\u003c/strong\u003e stems cross-section and enlarged view. \"-\" represents 0.2 mm.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-4281530/v1/d8e585064bffd7fda88a25f4.png"},{"id":55714950,"identity":"32a74f36-91b1-4073-91e0-0c96a3c1621e","added_by":"auto","created_at":"2024-05-02 07:22:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":111717,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea\u003c/strong\u003e Contents of stress compounds in \u003cem\u003eA. lancea\u003c/em\u003e. H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2 \u003c/sub\u003eHydrogen peroxide; MDA Malondialdehyde. \u003cstrong\u003eb\u003c/strong\u003e Activities of antioxidant enzymes in \u003cem\u003eA. lancea\u003c/em\u003e. APX Ascorbate peroxidase; POD Peroxidase; PPO Polyphenol oxidase; CAT Catalase. \u003cstrong\u003ec\u003c/strong\u003e Changes of endogenous hormone content in the NS, FS, and FNS in\u003cem\u003e A. lancea\u003c/em\u003e. The data in figure are \"mean ± standard deviation\"; different letters in the same group showed significant differences between groups, P\u0026lt;0.05 (n=3); the same below.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-4281530/v1/8cb73e93f0955180f7272e03.png"},{"id":55714941,"identity":"752c9a10-95ec-495d-85c3-e552e667e567","added_by":"auto","created_at":"2024-05-02 07:22:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":144914,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eThe\u003cstrong\u003e \u003c/strong\u003enumber of unigenes annotated in the four databases. \u003cstrong\u003eb\u003c/strong\u003e Volcano plot of DEGs between “NS vs. FS”. \u003cstrong\u003ec\u003c/strong\u003e GO enrichment classification of DEGs between “NS vs. FS”.\u003cstrong\u003e d\u003c/strong\u003e KO enrichment bubble diagram of DEGs between “NS vs. FS”.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-4281530/v1/c4d7ce96d998c5db9c62a98c.png"},{"id":55714946,"identity":"135cd4ce-f9fe-4777-92a2-196519b478f6","added_by":"auto","created_at":"2024-05-02 07:22:24","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":162364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eA\u003c/strong\u003e Distribution of DEGs related to hormone biosynthesis and signal transduction in \u003cem\u003eA. lancea\u003c/em\u003e. The x-axis represents the number of DEGs, and the y-axis represents the different hormone signals. \u003cstrong\u003eB\u003c/strong\u003eThe expression analysis of DEGs involved in plant hormone signaling (Ko04075) pathway.\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-4281530/v1/78213098b80a85af7642c034.png"},{"id":55715461,"identity":"0b4c9034-34d2-4587-8f4a-8d0c9082ee2a","added_by":"auto","created_at":"2024-05-02 07:30:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":133776,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression analysis of DEGs involved in plant-pathogen interaction (Ko04626) pathway.\u003c/p\u003e\n\u003cp\u003eDifferential expression analysis of transcription factors\u003c/p\u003e","description":"","filename":"Slide6.png","url":"https://assets-eu.researchsquare.com/files/rs-4281530/v1/19dc32fb408a860677d0e705.png"},{"id":55714949,"identity":"4ea5d52d-1036-45d9-bf49-d870608aa57b","added_by":"auto","created_at":"2024-05-02 07:22:24","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":90588,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea \u003c/strong\u003eThe expression analysis of transcription factors. \u003cstrong\u003eb \u003c/strong\u003eValidation of DEGs using qRT-PCR.\u003c/p\u003e","description":"","filename":"Slide7.png","url":"https://assets-eu.researchsquare.com/files/rs-4281530/v1/5724d123292d0197c5b1beed.png"},{"id":55714944,"identity":"b9b413bf-3f78-4b1e-84cb-1a1307cd9d17","added_by":"auto","created_at":"2024-05-02 07:22:24","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":87994,"visible":true,"origin":"","legend":"\u003cp\u003eThe presumptive mechanism of flat stem formation in \u003cem\u003eA. lancea\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"Slide8.png","url":"https://assets-eu.researchsquare.com/files/rs-4281530/v1/4a27bb35b60dc7dfc4e5076f.png"},{"id":57971464,"identity":"cbf73788-98bf-47d3-8ae0-1248251acd1c","added_by":"auto","created_at":"2024-06-08 09:41:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2820172,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4281530/v1/b3183d31-ffc9-4d61-aa88-87065db94d8a.pdf"},{"id":55714948,"identity":"af2a6698-ee2e-4170-ac9e-f29469cc85f0","added_by":"auto","created_at":"2024-05-02 07:22:24","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":12830,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-4281530/v1/76c95020cbd698a325342900.docx"},{"id":55714947,"identity":"27fd39e8-90b8-45db-affc-65b9af64289a","added_by":"auto","created_at":"2024-05-02 07:22:24","extension":"xls","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":42496,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarySupplementaryTableS1.xls","url":"https://assets-eu.researchsquare.com/files/rs-4281530/v1/5a7f0f9218bba81baa22dcc4.xls"},{"id":55714951,"identity":"23d98bd5-29e7-436a-b9ee-31846454d380","added_by":"auto","created_at":"2024-05-02 07:22:25","extension":"pptx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":11639139,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementarySupplementaryFigure.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4281530/v1/b6b1feec0b5cb4537dcc3c26.pptx"}],"financialInterests":"","formattedTitle":"Unraveling the Molecular Mechanisms Underlying Flat Stem Formation in Atractylodes lancea in Response to Phytoplasmas","fulltext":[{"header":"Key message ","content":"\u003cp\u003eMultiple regulatory pathways and differentially expressed genes in \u003cem\u003eAtractylodes lancea\u0026nbsp;\u003c/em\u003eto phytoplasmas\u003cem\u003e\u0026nbsp;\u003c/em\u003einfection were identified through RNA-seq analysis, providing valuable insights into the development of flat stems in \u003cem\u003eA. lancea\u003c/em\u003e.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003ePhytoplasmas are pleomorphic, wall-less intracellular bacteria that can cause devastating diseases in more than 1,000 plant species(Deng 1991). Phytoplasma-related diseases, such as witches\u0026apos;- broom, dwarfing disease, banding disease, and phyllody, are considered the main hindrance in many areas, resulting in enormous losses worldwide for agricultural, forestry, and pharmaceutical production. Phytoplasma causes damage to host plants by affecting physiological and biochemical processes\u0026nbsp;(Catlin et al. 1975; Kartte and Seem\u0026uuml;ller 2008).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eStem flattening is the typical symptom of phytoplasma-infected plants, which can enlarge the vegetative tissue and generate more vascular bundle networks. Flat stem often manifests as thickened and banded stems, disordered leaves, and abnormal inflorescence development, affecting plants\u0026apos; normal growth and development. At present, the occurrence of flat-stem disease has been reported in multiple countries and occurred on a variety of broad-leaved trees,\u0026nbsp;including \u003cem\u003eSophora japonica\u0026nbsp;\u003c/em\u003e(Liu et al. 2022), \u003cem\u003eAmorpha fruticose\u0026nbsp;\u003c/em\u003e(Huang et al. 2023), and \u003cem\u003ePersimmon\u0026nbsp;\u003c/em\u003e(Wang et al. 2017), etc. It also affects herbaceous plants such as \u003cem\u003eCorn\u0026nbsp;\u003c/em\u003e(Duduk and Bertaccini 2006), \u003cem\u003eCucumber\u0026nbsp;\u003c/em\u003e(Wang et al. 2022), \u003cem\u003eArabidopsis thaliana\u0026nbsp;\u003c/em\u003e(Bressan and Purcell 2005; Cettul and Firrao 2011), etc. Such physiological conditions were a result of the impact of phytoplasma on hormonal, developmental, and stress signaling pathways, as well as the intricate interplay between these factors.\u003c/p\u003e\n\u003cp\u003eIt was reported that phytoplasma infection affects hormone balance in plants, resulting in changes in levels of crucial hormones like auxin, cytokinin, jasmonic acid (JA) and ethylene (Dermastia\u0026nbsp;et al.\u0026nbsp;2019). These alterations give rise to significant morphological modifications encompassin root architecture, stem growth, and floral development. Additionally, phytophthora infection triggers oxidative stress within plants, leading to the generation of reactive oxygen species (ROS). These ROS exert deleterious effects on cellular machinery and contribute to the manifestation of disease symptoms.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eA. lancea\u003c/em\u003e, also known as Cangzhu in Chinese, is a perennial herb that plays significant importance in traditional Chinese medicine. Its dried rhizomes have been widely used for dispelling wind and cold, drying dampness, strengthening the spleen, improving eyesight, and treating various gastrointestinal tract diseases and night blindness.\u0026nbsp;With the increasing demand for \u003cem\u003eA. lancea\u003c/em\u003e for medicinal purposes,\u003cem\u003e\u0026nbsp;\u003c/em\u003ethere has been a shift towards artificially cultivated plants becoming the primary source of\u0026nbsp;medicinal materials.\u0026nbsp;Large-scale artificial planting of \u003cem\u003eA. lancea\u003c/em\u003e in the field after long-term multi-generation asexual reproduction has brought about certain challenges such as the spread of diseases and pests. To gain insights into the development mechanism of stem flattening in\u0026nbsp;\u003cem\u003eA.lancea\u003c/em\u003e after infection by phytoplasma,\u0026nbsp;the plant structure, stress compounds, antioxidant enzyme activity, endogenous hormone, and comparative transcriptome were analyzed. The results will provide new insight into the formation mechanism of flat stems in plants and help control the phytoplasma disease of \u003cem\u003eA. lancea.\u003c/em\u003e\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth the normal stem (FNS) and flat stem (FS) exhibiting flattening symptoms on \u003cem\u003eA. lancea\u003c/em\u003e, as well as the healthy stem (NS) from the same experimental field in the \u003cem\u003eA. lancea\u003c/em\u003e cultivation base located in Yingshan County, Hubei Province (115\u0026deg;57\u0026prime;31\u0026Prime;E, 30\u0026deg;58\u0026prime;16\u0026Prime;N) were collected and stored at -80 ℃. The phytoplasma infection was further confirmed by nested PCR amplification of the 16S rRNA gene referring to Wan Qianyun\u0026apos;s method\u0026nbsp;(Wan et al. 2018). The gene-universal oligonucleotide primers P1/P7 and R16F2n/R16R2 were employed for Nested PCR analysis as detailed in Supplementary Table S1\u0026nbsp;(Hiruki 1991; Schneider et al. 1995).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhenotypic and microscope observation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe phenotypic changes of \u003cem\u003eA. lancea\u0026nbsp;\u003c/em\u003ewith flattening stem symptoms, including plant morphology, terminal bud (stem tip) shape, and phyllotaxy, were documented with a digital camera. The width (major axis) and thickness (minor axis) of the central stem were measured using vernier calipers in 12 biological replicates. Paraffin sections were prepared from the terminal bud and young stem for observation of their anatomical structure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination the content of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, MDA, and the activities of antioxidant enzyme\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe extraction of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) and Malondialdehyde (MDA) was conducted based on the methods of Amin\u0026nbsp;(Amin and Olson 1967; Satterfield and Bonnell 1955)\u0026nbsp;and Spitz\u0026nbsp;(Spitz and Oberley 1989). The extraction of ascorbate peroxidase (APX) followed the procedure described by Nakano\u0026nbsp;(Nakano and Asada 1981). Peroxidase (POD) was extracted and detected according to Han\u0026apos;s protocol\u0026nbsp;(Zhao and Han 2011). Polyphenol oxidase (PPO) was extracted and detected using of Liu\u0026rsquo; methods\u0026nbsp;(Liu, et al. 2020). Catalase (CAT) extraction followed Johansson\u0026apos;s procedure\u0026nbsp;(Johansson and Borg 1988). The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, MDA, CAT kits (Boxbio Biotechnology Co., Ltd, Beijing, China) and the APX kit (Elabscience Biotechnology Co., Ltd, Wuhan, China) were used respectively for the detection of the above metabolites/enzymes according to the manufacturer\u0026apos;s instructions. The standard curve of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was y=3.251x-0.01351 (R\u003csup\u003e2\u003c/sup\u003e=0.9997).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDetermination of endogenous hormone\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe NS, FS, FNS samples\u0026nbsp;of \u003cem\u003eA. lancea\u003c/em\u003e were ground into a powder with liquid nitrogen. Subsequently, 120 mg samples were dissolved in 80% methanol and subjected to vortexing for 0.5 h six times, followed by leaching at 4 ℃ for 12 h. The tube was vortexed and centrifuged at 12,000 r/min for 5 min. After centrifugation, the supernatant was filtered with a 0.22 \u0026micro;m polypropylene filter and directly injected into the LC-MS/MS system. Chromatographic separation was performed using Waters reversed-phase C18 chromatographic column (Waters, ACQUITY UPLC HSS T3 1.8 \u0026mu;m, 2.1\u0026times;100 mm Column)\u0026nbsp;with a mobile phase consisting of 0.04% formic acid (A) and acetonitrile (B) at a flow rate of 0.35 mL/min. The gradient was as follows: 0-1 min, 95%A, 1-8 min, 95%A-5%A, 8-9 min 5%A, 9-12 min 95%A. The column temperature was maintained at 40 ℃, and the injection volume was 2 \u0026micro;L. Detection wavelengths were set at different values depending on the analytes: ABA, IAA, JA were detected at \u0026lambda;=254 nm while zeatin was monitored at \u0026lambda;=270 nm using triple quadrupole mass spectrometry\u0026apos;s multiple reaction monitoring modes. The reference standards of ABA, IAA, JA, and Zeatin were purchased from Shanghai Yuanye Bio-Technology Co., Ltd, Shanghai, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA Sequencing, functional annotation and differentially expressed genes (DEGs) analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe NS, FS, and FNS samples of \u003cem\u003eA. lancea\u003c/em\u003e were ground with liquid nitrogen, followed by RNA extraction utilizing the TIANGEN polysaccharide and polyphenol plant total RNA extraction kit. After quantifying the total RNA content, a cDNA library was constructed and sequenced in Guangzhou Kedio Biotechnology Co., Ltd.\u0026nbsp;The high-quality reads were assembled after following data filtration using the Trinity program. Functional annotation of these unigenes was conducted using protein databases including non-redundant protein (nr), SwissProt, Kyoto Encyclopedia of Genes and Genomes (KEGG), as well as Cluster of Orthologous Group/Eukaryotic Orthologous Group (COG/KOG), based on a blastx search with an E value threshold below 10\u003csup\u003e-5\u003c/sup\u003e. Unigene expression was determined using the fragments per kilobase of transcript per million mapped reads (FPKM) method. DEGs were identified with a |log2FC|\u0026gt;1 and a false discovery rate (FDR) \u0026le; 0.05. Subsequently, DEGs underwent GO and KEGG enrichment analysis, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eqRT‑PCR analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e6 randomly selected DEGs related to hormone biosynthesis and signaling transduction were verified by qPCR using the SYBR method. \u003cem\u003eGAPDH\u003c/em\u003e (glyceraldehyde-3-phosphate dehydrogenase) was employed as an internal reference gene. The primer sequences for the selected DEGs are listed in Supplementary Table S1. Purified RNA (1 \u0026mu;g) was reverse transcribed into cDNA using \u003cem\u003eEvo M-MLV\u003c/em\u003e RT Mix Kit with gDNA Clean for qPCR Ver.2 (Accurate Biotechnology CO., LTD, Changsha, China) according to the manufacturer\u0026rsquo;s instruction. The qPCR reaction system consisted of a volume of 20 \u0026micro;L, which included 10 \u0026micro;L of 2 \u0026times; SYBR Green \u003cem\u003ePro Taq\u003c/em\u003e HS Premix (ROX Plus), 2 \u0026micro;L of the cDNA template, 0.4 \u0026micro;L of each primer, and 7.2 \u0026micro;L ddH2O. The reaction conditions were pre-denaturation at 95 \u0026deg;C for 30 s, followed by 40 cycles of denaturation at 95 \u0026deg;C for 5 s, and annealing at 60 \u0026deg;C for 30 s. The relative gene expression was calculated according to 2\u003csup\u003e-\u0026Delta;\u0026Delta;CT\u003c/sup\u003e (Liu et al. 2018; Stortenbeker and Bemer 2019).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mean values and standard deviations (SD) were calculated using SPSS 17.0 software (SPSS Inc., Chicago, United States). Significant differences were determined using the independent-samples t-test. Each experiment was independently performed three times. Graphs were constructed using GraphPad Prism 8 software (GraphPad Software, San Diego, CA, United States).\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eField observation showed that the NS exhibited a nearly cylindrical shape with alternate phyllotaxis, whereas the FS displayed a flatter and thicker structure characterized by disordered phyllotaxy, forming distinct bands (Fig. 1a, b, c). In contrast to the needle-like stem tip of NS (Fig. 1a), the FS possessed a broom-like morphology (Fig. 1b). Furthermore, in comparison to NS, the transverse section of FS exhibited more than twice the width (major axis) and thickness (minor axis), resulting in a significantly different aspect ratio (width/thickness) (Supplementary Fig. S1, Fig. 1d). To confirm phytoplasma infection, the amplification and sequencing of the 16S rRNA gene were performed. In comparison to NS and FNS, a 1245 bp fragment was amplified in FS, exhibiting 100% homology to the Aster yellows group (16SrI) phytoplasma (GenBank accession number: MG257945.3) after conducting a blast search on NCBI. The phylogenetic tree analysis revealed that the \u003cem\u003eA. lancea\u003c/em\u003e phytoplasma clustered with subgroup 16SrI-B (AY101386.1) found in \u003cem\u003eEpilobium hirsutum\u003c/em\u003e L., suggesting that phytoplasma infection may be responsible for inducing the flat stem variant in \u003cem\u003eA. lancea\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicroscope observation of shoot apical meristem and stems cross-section\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe microscope observation revealed that the stem tip of NS\u003cem\u003e\u0026nbsp;\u003c/em\u003ewas composed of shoot apical meristem, leaf primordium, young leaves, pith, and axillary bud primordium (Fig. 2a). In contrast, the FS exhibited a significantly widened stem tip with young leaves arranged in a linear fashion on the shoot apical meristem and alterations in the distribution of leaf primordia. Although FS had the same anatomical structure as a normal stem, including the epidermis, cortex, phloem, cambium, xylem, and pith, its stem presented a flat shape (Fig. 2b).\u0026nbsp;Furthermore, irregular protrusions were observed on the surface of FS resulting in an uneven epidermal texture compared to normal stems. Additionally, there was an evident increase in thickness for both cortex and vascular tissue to varying degrees\u0026nbsp;(Fig. 2c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe determination of\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003estress compound content, antioxidant enzyme activity and endogenous hormone content\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn addition to affecting stem structure, plants can also trigger the induction of stress biomarkers in response to phytoplasma infection. Our study revealed that both the FS and FNS exhibited significantly higher levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e accumulation compared to the NS group, while only the FS group showed a significant increase in MDA content. Meanwhile, there was an elevation in enzymatic activity of PPO and CAT in the FS. However, the APX and POD activities were markedly decreased when compared to those observed in the NS group (Fig. 3b, c).\u003c/p\u003e\n\u003cp\u003eTo investigate the potential association between hormone disorder and the development of a flat stem, the levels of four hormones in NS,\u003cem\u003e\u0026nbsp;\u003c/em\u003eFNS, and FS were quantified. Notably, FS exhibited significant differences compared to NS and FNS, characterized by elevated jasmonic acid and zeatin levels as well as reduced auxin (IAA) content (Fig. 3a). No significant difference was observed in abscisic acid concentration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranscriptome sequencing and Unigene annotation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA-seq sequencing analysis was performed to explore the mechanisms underlying the formation of flat stems. A total of 137,205 unigenes were annotated and blasted in four public databases of Nr, KOG/COG, Swiss Prot, and KEGG. The result showed that the Nr database, KEGG database, Swiss Prot, and KOG contained annotations for 50,592, 46,042, 27,999, and 225,234 unigenes respectively. The statistical results of all unigenes annotation and the Wayne diagram depicting transcriptome data for \u003cem\u003eA. lancea\u0026nbsp;\u003c/em\u003ewere presented in Fig. 4a. By utilizing the Nr library for comparative analysis and annotation, we can ascertain the genetic similarity between \u003cem\u003eA. lancea\u003c/em\u003e and its related species. The top four most closely related species were \u003cem\u003eCynara cardunculus\u0026nbsp;\u003c/em\u003e(39.64%), \u003cem\u003eArtemisia annua\u003c/em\u003e (14.05%), \u003cem\u003eLactuca sativa\u0026nbsp;\u003c/em\u003e(9.84%), and \u003cem\u003eHelianthus annuus\u003c/em\u003e (5.48%) (Supplemental Fig. S2). It is noteworthy that all these four species, along with \u003cem\u003eA. lancea\u003c/em\u003e, belong to the Compositae family.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of differentially expressed genes (DEGs\u003c/strong\u003e\u003cstrong\u003e）\u003c/strong\u003e\u003cstrong\u003ein \u003cem\u003eA. lancea\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DEGs were screened with FDR\u0026lt;0.05 and |log2FC|\u0026gt;1 as threshold value. Subsequently, volcano plot analysis was performed to visualize the significant differences in gene expression between each comparison group. Compared to NS, 114 genes were upregulated while 268 genes were downregulated in FS (Fig. 4b). Similarly, compared to FNS, FS exhibited upregulation of 77 genes and downregulation of 458 genes. Conversely, only a limited number of DEGs showed upregulation (8 genes) or downregulation (22 genes) between \u0026quot;NS vs. FNS\u0026quot; (Supplemental Fig. S3a, b). Given the small number of DEGs identified between \u0026quot;NS vs. FNS\u0026quot;, our focus will be on analyzing the differences observed in \u0026quot;NS vs. FS\u0026quot; and \u0026quot;FNS vs. FS.\u0026quot;\u003c/p\u003e\n\u003cp\u003eGo enrichment analysis can be classified into three domains: biological processes, cellular components, and molecular functions. Regarding biological processes, both \u0026quot;NS vs. FS\u0026quot; and \u0026quot;FNS vs. FS\u0026quot; functional regions exhibited a significant enrichment of DEGs associated with metabolic, cellular, and single-organism processes. In terms of cellular components, the most prominent category of DEGs was involved in cell, cell part, organelle, and membrane localization. A substantial number of DEGs were annotated to molecular functions such as binding activity, catalytic activity, and transporter activity (Fig. 4c; Fig. S3c).\u003c/p\u003e\n\u003cp\u003eThe top 20 results obtained from KEGG pathway enrichment analysis are presented in Figure 4d and supplementary Figure S3d. Notably, the DEGs were significantly enriched in key pathways such as \u0026quot;metabolic pathways,\u0026quot; \u0026quot;biosynthesis of secondary metabolites,\u0026quot; and \u0026quot;zeatin biosynthesis,\u0026quot; suggesting their potential involvement in phytoplasma response mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential expression of hormone metabolic pathway gene in \u003cem\u003eA. lancea\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe DEGs associated with hormone biosynthesis and signal transduction between \u0026quot;NS vs. FS\u0026quot; and \u0026quot;FNS vs. FS\u0026quot; in \u003cem\u003eA. lancea\u003c/em\u003e showed the same expression trend, including salicylic acid, jasmonate, brassinosteroid, ethylene, abscisic acid, gibberellins, cytokinin, and auxin.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNotably, a majority of the DEGs enriched in the salicylic acid and jasmonate synthesis exhibited upregulation in the FS group compared to NS or FNS groups (Fig. 5a). This suggests that these two hormones may play crucial roles in response to stress conditions or pathogen attacks in \u003cem\u003eA. lancea\u003c/em\u003e. On the other hand, DEGs enriched in brassinosteroid and cytokinin synthesis showed downregulation specifically in the FS group. This downregulation might indicate a suppression of growth-related processes regulated by these hormones under stressful conditions (Fig. 5a).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe expression of 3 DEGs encoding 3-deoxy-7-phosphoheptulonate synthase (DAPH), the initial enzyme in a series of metabolic reactions, was downregulated in the FS. Auxin levels can influence plant growth and development, necessitating the presence of early response genes such as Auxin/Indole-3-Acetic Acid (Aux/IAA), auxin response factor (ARF) family, small auxin up RNA (SAUR), etc (Stortenbeker and Bemer 2019). In the study, 2 DEGs annotated to AUX/IAA and 2 DEGs annotated to SAUR were also found to be expressed at low levels in the FS, consistent with auxin content distribution. 1 DEG encoding adenylate isopentenyl-transferase (IPT), which acts as the first and rate-limiting synthase in cytokinin biosynthesis, exhibited upregulated in the FS. 2 allene oxide synthase (CYP74A) DEGs involved in jasmonic acid biosynthesis showed upregulation in the FS. 1 DEG encoding jasmonate ZIM-domain protein (JAZ), an essential repressor in the JA signaling pathway, displayed downregulation. These findings align with zeatin and jasmonic acid content distribution.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferential expression of plant-pathogen\u0026nbsp;interaction pathway genes in \u003cem\u003eA. lancea\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ephytoplasma, a pathogenic bacteria, can invade the vascular tissue of plants and induce a cascade of physiological alterations. Elucidating the intricate interplay between pathogen and their host plants is pivotal for comprehending the flat stem developmental processes of \u003cem\u003eA. lancea\u003c/em\u003e.\u0026nbsp;In this study, we identified 10 DEGs that are involved in plant-pathogen interactions pathway\u0026nbsp;(Fig. 6). Among these DEGs, 5 DEGs were implicated in Ca\u003csup\u003e2+\u003c/sup\u003e signaling, including calmodulin/calbindin (CaM/CML), respiratory burst oxidase homologs (CDPK) and respiratory burst oxidase homologs (Rboh). 1 DEGs encoding Rboh and 1 DEGs encoding CaM/CML were upregulated in the FS. In addition, 5 DEGs were annotated to \u0026beta;-Ketoacyl-CoA synthase (KCS), which played a vital role in determining the quantity and composition of very long chain fatty acids (VLCFAs) (Zhang et al. 2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe transcription factor\u0026nbsp;involved in the formation of flat stems\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNumerous transcription factors have been reported to play pivotal roles in plant growth and development, serving as crucial regulatory molecules involved in stress response and pathogen resistance. In comparison to NS and FNS, the DEGs were found to be associated with WRKY, ERF, and MYB transcription factors, which showed a decrease in expression levels in FS. This finding implies that plants might regulate the function of WRKY, ERF, and MYB transcription factors under phytoplasma infection conditions in order to cope with external stresses; however, this modulation could potentially lead to abnormal physiological processes like stem flattening.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eValidation of key gene expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further validate the transcriptome results, a total of 6 genes were randomly selected for qPCR analysis (Supplementary Table S1). Among them, 3 genes were related to auxin metabolism, while the other 3 genes were associated with salicylic acid signal transduction (PR1), brassinosteroid signal transduction (TCH4), and jasmonic acid signal transduction regulator (JAZ) pathway, respectively. The expression patterns of these selected genes demonstrated a high degree of concordance with the transcriptome data (Fig. 7b).\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003ePhytoplasma infection induced stem flat in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eA. lancea\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe precise regulation of meristem activity governs the structural organization of plants. Imbalances in positive or negative maintenance signals within terminal buds can lead to phenotypic changes, such as thinning or enlargement of the meristem, resulting in enlarged vegetative bodies and inflorescence meristems. Our study revealed a broom-like appearance with widened meristems and systematically arranged leaves in rows at the terminal bud region of flatting symptomatic \u003cem\u003eA.lancea\u003c/em\u003e, indicating that alterations in meristem activity have influenced\u0026nbsp;stem morphology characteristics. Furthermore, molecular detection exclusively identified phytoplasma from FS samples belonging to 16SrI-B subgroup within Aster yellows group (Fig.1e), indicating that phytoplasma infection modifies meristem activity contributing to flat stem occurrence on \u003cem\u003eA.lancea\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChanges in stress compound content are involved in flat stem formation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter infection by phytoplasma, plants have developed various basic defense mechanisms to protect themselves from pathogens. One such defense mechanism involves the rapid accumulation of ROS at the pathogen attack site (a phenomenon called oxidative burst)\u0026nbsp;(Girodat et al. 2020), which can eliminate invading pathogens directly. In the study, the levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in both FS and FNS were significantly higher compared to NS (Fig. 3). However, only FS exhibited a significant increase in malondialdehyde (MDA) content, indicating damage to the liposomes in FS but not in FNS. PPO catalyzes the formation of lignin and quinone compounds to form a protective shield, thus protecting cells from pathogens.\u0026nbsp;Interestingly, PPO activity was significantly increased in the FNS compared with NS and FS (Fig. 3), suggesting FNS may have recovered from the phytoplasma infection to as a strategy to prevent stem flattening\u0026nbsp;(Musetti et al. 2004).\u003c/p\u003e\n\u003cp\u003eVarious antioxidant enzymes, including POD, CAT, and APX, are involved in scavenging H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and reactive oxygen species metabolism during pathogen attacks with different mechanisms. However, contrary to expectations, the levels of APX and POD did not increase in the FS, indicating a potential disruption in the equilibrium between H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production and scavenging by antioxidant enzymes. Previous studies have reported that maintaining a dynamic balance of ROS played a role in keeping the balance of transition from cell proliferation to cell differentiation, which was a key factor for maintaining normal root morphology\u0026nbsp;(Xu et al. 2020). Chen also found that appropriate levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e promoted axillary bud outgrowth in tomato plants\u0026nbsp;(Chen et al. 2016). The results implied that the sharp increased H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e induced by the phytoplasma infection led to the flat stem phenomenon in\u0026nbsp;\u003cem\u003eA. lancea\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChanges in plant hormone content\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;are involved in flat stem formation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe establishment of stem cell niches and cell proliferation within the meristematic zone are governed by the plant hormone auxin\u0026nbsp;(Beemster and Baskin 2001; Blilou et al. 2005). Altered auxin transport and distribution resulting from inhibited expression of an \u003cem\u003eAux/IAA\u003c/em\u003e gene (SHY2) effectively lead to changes in root meristem size\u0026nbsp;(Mandal et al. 2023). In Solanaceae, inhibition of several \u003cem\u003eAux/IAA\u003c/em\u003e genes, such as \u003cem\u003eIAA9\u003c/em\u003e, reduces apical dominance and enhances hypocotyl/stem elongation(Wang et al. 2005). Our study reveals downregulation of DEGs encoding \u003cem\u003eSAUR\u003c/em\u003e and \u003cem\u003eAux/IAA\u003c/em\u003e in the FS, accompanied by decreased auxin contents. Similar observations have been made in phytoplasma-related plants where an imbalance in auxin levels leads to morphological changes like witches\u0026apos; broom or dwarf symptoms(Christensen et al. 2005).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition to auxin, cytokinin exerts an influence on root meristem activity(Ruzicka et al. 2009). Elevated levels of cytokinin achieved through exogenous application or over expression of the bacterial IPT gene(Medford et al. 1989; Kuderova et al. 2008)\u0026nbsp;impede root growth and induce changes in meristem size. Increased endogenous cytokinin levels resulting from activation of plant IPT gene expression enhance or modify the morphogenic potential of transformed plant cells(Mens et al. 2018). In our study, one IPT was found to be upregulated with increased zeatin levels. These findings suggest that phytoplasma-induced imbalance between zeatin and auxin leads to expansion of stem apical meristem along with internal structural changes and stem flattening.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Jasmonic acid (JA) and its methyl ester (MeJA) have the ability to stimulate cell expansion in plant medullary tissue. Exogenous application of jasmonic acid induces changes in apical meristem morphology of potato stolons(Cenzano et al. 2003). Following infection with phytoplasma, there was a significant increase in JA content along with upregulation observed for the JA synthesis-related gene CYP74A2 in the FS, which correlates well with citrus ulcer formation\u0026nbsp;(Qin et al. 2019), suggesting that JA may also affect meristems leading to the development of flat stems. Recent studies have indicated a possible crosstalk among plant hormones in the regulation of vascular tissue development within the primary root meristem of \u003cem\u003eArabidopsis\u003c/em\u003e(Sun et al. 2023). Consequently, it is imperative to further investigate the intricate and precise hormonal regulatory network in \u003cem\u003eA. lancea\u003c/em\u003e infected with phytoplasma and its consequential impact on flat stem formation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe different expressions of \u003cem\u003eCa\u003c/em\u003e\u003csup\u003e2+\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003esignaling pathways associated genes are involved in flat stem formation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChanges in Ca\u003csup\u003e2+\u003c/sup\u003e levels and signals significantly contribute to plant growth and development, including root morphogenesis. The functions of Ca\u003csup\u003e2+\u003c/sup\u003e generally rely on sensors, such as calmodulin-like proteins (CMLs) and calcium-dependent protein kinases (CDPKs), which decode and transduce Ca\u003csup\u003e2+\u003c/sup\u003e signals. In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, AtCML24 mutant caused the higher [Ca (2+)] cyt within germinated pollen and extended pollen tubes, thereby affecting pollen germination and pollen tube elongation\u0026nbsp;(Yang et al. 2014). Similarly, OsCDPK5/13 negatively regulates aerenchyma formation of roots in rice\u0026nbsp;(Yamauchi et al. 2017). It has been demonstrated that phytoplasma infection induces an elevation of intracellular Ca\u003csup\u003e2+\u0026nbsp;\u003c/sup\u003e(Musetti et al. 2013). Subsequently, CDPK is upregulated along with the highly expressed key producers of reactive oxygen species (ROS) in plants known as RBOHs that can be activated by CDPKs. This suggests that phytoplasma infection induces an increase in calcium ion concentration to activate the expression of CDPK and RBOHs, resulting in elevated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e levels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe transcription factor involved in the formation of flat stems\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTranscription factors (TFs) directly regulate genes related to cell proliferation and cell elongation, thereby potentially influencing plant structural characteristics\u0026nbsp;(Peng et al. 2023). The MYB family, one of the largest groups of transcription factors, is known to impact stem development as well as the biosynthesis of cell walls and cell cycle function. RNA interference\u0026nbsp;BnMYB69 of \u003cem\u003eBrassica napus\u003c/em\u003e presented substantial changes in morphology, anatomy, and metabolism\u0026nbsp;(Lin et al. 2023). Overexpression of OsMPH1, a MYBLIKE GENE OF PLANT HEIGHT 1 in rice plants, led to increased plant height and seed yield by modulating the expression of genes related to cell wall formation and elongation\u0026nbsp;(Zhang et al. 2017).\u003c/p\u003e\n\u003cp\u003eAdditionally, WRKY transcription factor also plays a role in regulating stem elongation and diameter. For instance, WRKY41a from herbaceous peonies promotes secondary cell wall thickening for enhanced stem strength\u0026nbsp;(Tan, et al. 2023), while OsWRKY78 is crucial for controlling stem elongation in rice plants(Zhang et al. 2011). Furthermore, mutation of WRKY13 in \u003cem\u003eA. thaliana\u003c/em\u003e can reduce stem diameter through repression of lignin synthesis(Li et al. 2015).\u003c/p\u003e\n\u003cp\u003eTranscription factors also participate in the regulatory network of plant hormones. During plant growth, WRKY regulates auxin to influence the initiation of axillary meristem and the growth of new buds\u0026nbsp;(Guo et al. 2015; Lan et al. 2020). Ethylene response factor (ERG) activates the expression of ASA1 (the rate-limiting enzyme of tryptophan synthesis), induces auxin biosynthesis and facilitates auxin accumulation in root tip, and consequently inhibits root elongation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe present study revealed significant differential expression of transcription factors WRKY, MYB, and ERG in the FS compared to NS and FNS, suggesting their potential regulatory role in the formation of flat stem in \u003cem\u003eA. lancea\u003c/em\u003e. However, further confirmation is required to elucidate their precise functions.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur studies revealed that the flat stem formation of\u003cem\u003e\u0026nbsp;A. lancea\u003c/em\u003e is a pathological phenomenon caused by phytoplasma infection, which alters the stem tip meristem. During phytoplasma infection, there are changes in stress compound content, antioxidant enzyme activity, and endogenous hormone levels. Notably, the H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content in the FS is significantly higher compared to NS, while the levels of antioxidant enzymes involved in scavenging H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e are lower than those found in NS. Moreover, there is a substantial alteration in hormone content during phytoplasma infection, with differentially expressed genes (DEGs) associated with hormone biosynthesis and signal transduction identified through RNA-seq analysis. These DEGs include auxin, jasmonic acid, and zeatin-related genes. Additionally, our study characterizes several DEGs such as CML, CDPK, and RBOH that play crucial roles in Ca\u003csup\u003e2+\u003c/sup\u003e signaling pathways and ROS production along with transcription factors (TFs) related to stem flattening (Fig 8). This study provides new insights into the mechanisms underlying flat stem formation in \u003cem\u003eA.lancea\u003c/em\u003e.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution statement\u003c/strong\u003e: Liu Changli and Yu Kun designed the project; Gong Ling and Chen Lei performed the research, analyzed the data, and wrote the paper. Huang Xiao, Deng Juan and Jiang Meiling participated in the research. Wu Xiaoyi and Hu Yating analyzed the RNA-seq data. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe would like to thank Prof. Zhang Xiuqiao for her help in experimental technique support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This work was supported by the 71st General Project of China Postdoctoral Science Foundation (Grant number 2022M71223), the National Natural Science Foundation of China (Grant number 32000254, 31670341 and 81891014), and the Hubei Province Technology Innovation Special Major Project (Grant number 2018ACA124).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors declare no competing financial interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAmin VM, Olson NF (1967) Spectrophotometric Determination of Hydrogen Peroxide in Milk1. 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Journal of Anhui Agricultural Sciences, 39, 1275-1277.\u003c/li\u003e\n\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":"Atractylodes lancea, Flat stem, Phytoplasma, Morphology, Physiology and Biochemistry, Transcriptome","lastPublishedDoi":"10.21203/rs.3.rs-4281530/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4281530/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Atractylodes lancea (Thunb.) DC has been widely used as a medicinal herb for centuries. However, long-term artificial cultivation of A. lancea led to serious plant diseases, such as the flat branch disease caused by phytoplasmas. To explore the formation mechanism of flat stems, we measured the changes in physiological and biochemical indicators and related metabolic pathways in stems of A. lancea in response to phytoplasma. After infection by pathogen, significant changes were observed in the content of stress compounds H2O2 and MDA, as well as the activities of antioxidant enzymes APX, POD, PPO, and CAT. The contents of jasmonic acid and zeatin in the flat stem (FS) of A. lancea increased significantly, while auxin content decreased. High-throughput sequencing showed that differentially expressed genes (DEGs) are enriched in hormone biosynthesis, signal transduction, Ca2+ signaling, and other pathways. These results preliminary elucidate the molecular mechanism of flat stem development in A. lancea, providing a foundation for disease prevention in the future.","manuscriptTitle":"Unraveling the Molecular Mechanisms Underlying Flat Stem Formation in Atractylodes lancea in Response to Phytoplasmas","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-02 07:22:19","doi":"10.21203/rs.3.rs-4281530/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":"0cb6bf82-1d71-45ac-bb64-daef6f33e210","owner":[],"postedDate":"May 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-08T09:33:39+00:00","versionOfRecord":[],"versionCreatedAt":"2024-05-02 07:22:19","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4281530","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4281530","identity":"rs-4281530","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}