Transcriptome profiling of Rehmannia glutinosa uncovers ABA/GA antagonism mechanisms in tuberous root initiation

Transcriptome profiling of Rehmannia glutinosa uncovers ABA/GA antagonism mechanisms in tuberous root initiation | 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 Transcriptome profiling of Rehmannia glutinosa uncovers ABA/GA antagonism mechanisms in tuberous root initiation Ruixue Yang, Yiying Du, Zhenyang Fang, Heyang Wang, Qingxiang Yang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7635435/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 Rehmannia glutinosa tuberous roots are the critical determinant of its medicinal and economic values. The swelling of its adventitious roots is a prerequisite for tuberous roots initiation, which is antagonistically regulated by abscisic acid (ABA) and gibberellins (GA). However, the antagonistic mechanisms between ABA and GA remain poorly understood. In this study, exogenous ABA significantly promoted adventitious roots swelling, whereas GA exerted an inhibitory effect. To investigate the potential mechanisms, transcriptome analysis was carried out on R. glutinosa roots treated with ABA or GA. Compared to control samples, 7,416 differentially expressed genes (DEGs) (ABA) and 9,034 DEGs (GA) were identified in adventitious roots. The expression patterns of DEGs involved in ABA or GA metabolism and signal transduction revealed the dynamic regulation of ABA/GA equilibrium. Kyoto Encyclopedia of Genes and Genomes and Gene set enrichment analysis showed enhanced starch accumulation and cell wall biosynthesis and modification in ABA-treated adventitious roots. The lignification induced by GA and key regulators in the antagonistic action of ABA and GA, including DREBs, ERFs and PERs, were identified using weighted gene co-expression network analysis and protein-protein interaction network analysis. And the transient expression analysis of RgERF017 and RgPER10 in tobacco further demonstrated that these two genes are involved in ABA/GA antagonism through the control of ABA and GA metabolism. This study provides a comprehensive analysis of the antagonistic regulatory network between ABA and GA during R. glutinosa tuberous roots initiation, deepening our understanding of the molecular mechanisms underlying tuberous root formation. Transcriptome analysis Rehmannia glutinosa ABA/GA antagonism tuberous root initiation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Key Message Downstream responses to ABA/GA antagonism and novel key regulators in its network were identified, among which RgERF017 and RgPER10 induce ABA/GA balance shift through regulating ABA/GA metabolism. Introduction Rehmannia glutinosa is a perennial plant with significant medicinal and dietary properties. Its tuberous roots are widely used in traditional Chinese medicine due to richness of bioactive compounds. These bioactive compounds accumulate gradually along with the development of R. glutinosa roots (Zhang et al., 2008 ; Huang et al., 2010 ; Chen et al., 2021b ). Therefore, R. glutinosa tuberous root development decides the values of R. glutinosa . R. glutinosa tuberous roots originate from adventitious roots, undergoing five developmental stages: adventitious roots, fibrous roots, initiated tuberous roots, medium tuberous roots, and late tuberous roots (Li et al., 2015 ). R. glutinosa is usually propagated vegetatively using the enlarged roots in the later developmental stage, which we call mother roots. Adventitious roots stem from mother roots. And the transition from adventitious roots to fibrous roots is prerequisite for tuberous root formation and is intricately regulated by plant hormones (Sun et al., 2015 ; Chen et al., 2022a ). Abscisic acid (ABA) and gibberellins (GA) are two essential plant hormones that play pivotal roles in plant growth and development (Liu & Hou 2018 ; Shu et al., 2018 ; Zhang et al., 2024 ). Numerous studies have demonstrated their antagonistic interactions in regulating tuberous root development (Liu et al., 2010 ; Lin et al., 2020 ). Generally, GA inhibits the initiation of tuberous root by promoting cell elongation and lignification (Chen et al., 2022a ), while ABA stimulates tuberous root formation through enhancing starch biosynthesis, cell division, and expansion (Li et al., 2020 ; Wang et al., 2024a ) and thus antagonizes the effect of GA (Chen et al., 2022a ). The antagonistic regulation of ABA and GA in tuberous root development depends on the dynamic change of the content ratio of ABA/GA. When the ratio of ABA/GA increases, it was beneficial to the initiation of tuberous roots (Chen et al., 2022a ). The determination of endogenous hormones level in R. glutinosa showed the increased ratio of ABA/GA in the early developmental stage, during which adventitious roots had initiated expansion and been transformed into fibrous roots (Sun et al., 2015 ). This demonstrated the antagonism of ABA/GA might be involved in the early stage of R. glutinosa tuberous roots development. The antagonistic crosstalk between ABA and GA mainly involves two layers (Liu & Hou 2018 ). Firstly, their antagonistic effects are evident in the reciprocal regulation of the metabolism pathways (Liu & Hou 2018 ). Key enzymes involved in ABA metabolism include 9- cis -epoxycarotenoid dioxygenase (NCED) and the abscisic acid 8'-hydroxylase (CYP707A). Gibberellin 20-oxidase (GA20ox), gibberellin 3-oxidase (GA3ox), and gibberellin 2-oxidase (GA2ox) are the classical enzymes of GA metabolism. The trade-off of the expression of these key enzymes was observed in diverse plant biological processes antagonistically mediated by ABA and GA (Oh et al., 2004 ; Oh et al., 2006 ; Seo et al., 2006 ; Oh et al., 2007 ; Gubler et al., 2008 ; Kashiwakura et al., 2016 ; Sun et al., 2025 ). The modulation triggers the shift of ratio of endogenous ABA/GA. The higher level of GA detected in ABA-deficient mutants (Seo et al., 2006 ) and the enhancement of NCEDs expression and repression of CYP707A s observed in GA-deficient mutants (Oh et al., 2007 ) are two typical examples. Secondly, the crosstalk between ABA and GA signal transduction pathways further underpins the antagonistic effects of the two hormones. ABA and GA signaling intersect at different transcription factors (TFs), which are involved in both ABA and GA signaling and metabolism. Abscisic acid-insensitive 5 (ABI5) is a basic leucine zipper TF, which is viewed as the central factor in ABA/GA antagonism during seed germination (Li et al., 2022a ). It can control the expression of many ABA and GA signaling and metabolism genes (Li et al., 2022a ). The expression of ABI5 can be regulated by an AP2-domain-containing TF, abscisic acid-insensitive 4 (ABI4) (Finkelstein et al., 1998 ; Muñiz García et al., 2013 ), which plays a critical role in ABA/GA antagonism as well (Li et al., 2022a ; Xian et al., 2024 ). It not only interacts with and stabilizes DELLA, the master negative regulator in GA signaling pathway, but also controls the expression of DELLA , GA2ox7 , and NCED6 (Xian et al., 2024 ). Overexpression of ABI4 will result in a higher ratio of ABA/GA (Shu et al., 2016 ). In R. glutinosa , the growing ratio of ABA/GA during the transition from adventitious roots to fibrous and initiated tuberous roots is also mainly attributed to the improved expression of ABA biosynthetic and signaling genes and the reduced expression of CYP707A s (Li et al., 2015 ; Sun et al., 2015 ). However, the specific mechanisms underlying the dynamic balance of ABA/GA and ultimately the initiation of tuberous roots are still totally obscure so far in R. glutinosa . To explore the mechanisms, we performed transcriptomic analysis of R. glutinosa roots exposed to ABA and GA to elucidate the antagonistic regulatory network of ABA and GA in tuber development. Exogenous ABA or GA disrupted the original ABA/GA homeostasis. The one with a higher concentration activated its corresponding signaling pathway, while the other signaling was triggered soon as well due to the crosstalk of ABA/GA signaling. Some DREBs (dehydration-responsive element binding proteins), ERFs (ethylene-responsive transcription factors) and PERs (peroxidases) were identified as the key hub elements in the ABA/GA signaling crosstalk, which orchestrated ABA/GA antagonism by integrating ABA/GA metabolism. And according to the analysis, the expansion of adventitious roots arose from the promotion of starch and sucrose metabolism, cell wall biogenesis and modification, cell division and expansion. Inversely, the suppression of these biological processes plus the induction of lignification led to the inhibition of root enlargement. Taken together, these findings not only offer novel entry points and candidate molecules for in-depth investigations of the antagonism of ABA/GA, but aid in uncovering the fundamental principles of tuber development in R. glutinosa . Materials and methods Plant materials R. glutinosa (cultivar Jinjiu) was grown in 11cm×11cm pots filled with a mixture of vermiculite and humus soil (1:1.5, V: V) in climate chambers at 28°C, 70% relative humidity, with a photoperiod of 16 hours of light and 8 hours of darkness. R. glutinosa 20-day-old seedlings were divided into three groups: control group (CK), ABA-treated group (ABA), and GA-treated group (GA). The seedlings in the three groups were irrigated continuously with distilled water, 40 µM ABA, and 150 µM GA for two weeks respectively. Then, both newly grown and mother roots were collected for transcriptome sequencing. All samples were frozen in liquid nitrogen immediately after collection and stored at − 80°C for subsequent experiments. Transcriptome sequencing and data analysis Total RNA was extracted from samples using an RNA extraction kit. One µg of RNA per sample was utilized for sequencing library construction. The sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Preparation Kit (NEB, USA). Paired-end sequencing was then performed on the Illumina Hiseq 2000 platform. Q20, Q30, GC content, and sequence repetition levels were calculated and the clean reads with high quality were screened from raw reads through in-house perl scripts. Transcriptome assembly was accomplished using Trinity (Grabherr et al., 2011 ). The data obtained were further processed with the BMKCloud online platform ( www.biocloud.net ). Based on all expressed genes, principal component analysis (PCA) was performed to elucidate the correlation between the samples. Pearson's Correlation Coefficient was employed to evaluate the correlation between the samples. NR, Pfam, KOG/COG/eggNOG, Swiss-Prot, and KEGG were used to annotate the unigenes. Differential expression analysis was performed using the DESeq R package. A fold change (FC) ≥ 2 and a false discovery rate (FDR) < 0.05 were used to identify differentially expressed genes (DEGs). Gene Ontology (GO) enrichment analysis of the DEGs was implemented by the topGO R packages based Kolmogorov–Smirnov test. KOBAS software was used to detect the statistical enrichment of DEGs in the KEGG pathway (Kanehisa et al., 2004 ; Xie et al., 2011 ). Gene Set Enrichment Analysis (GSEA) analysis of all genes for their role in GO and KEGG-related biological processes are carried out using the BMKCloud web platform ( www.biocloud.net ), with a p -value less than 0.01. Weighted gene co-expression network analysis (WGCNA) WGCNA was conducted using the DEGs contained in ARM or GRM (Figure S2 , S3). The threshold criteria included an average FPKM value of 1 for gene expression, an inter-module similarity threshold of 0.5, and a minimum of 30 genes per module. The protein-protein interaction (PPI) network of genes in key modules, derived from the WGCNA analysis, were analyzed using the STRING website ( https://cn.string-db.org/ ), and visualized using Cytoscape (version 3.8.2) software for visualization. RT-qPCR analysis Total RNA was extracted from R. glutinosa roots or tobacco leaves using RNAprep Pure Plant Kit (Vazyme Biotech, Nanjing, China). The first strand cDNA was synthesized with 1 µg total RNA using the first strand cDNA synthesis kit (Vazyme Biotech, Nanjing, China). And qRT-PCR was performed on the LightCycler®96 instrument system (Roche, Switzerland) using SYBR qPCR Mix (Vazyme Biotech, Nanjing, China). Specific primers were designed using Primer Premier 5.0, with RgGAPDH or NtActin as the internal reference primer. The expression level was analyzed by 2 − ΔΔCt. And the specific primers were shown in supplementary file 14. Transient transformation of RgERF017 and RgPER10 in tobacco The sequences of RgERF017 ( Unigene_033855 ) and RgPER10 ( Unigene_155852 ) were cloned into the pMD19-T vector (TaKaRa) and verified by colony PCR using M13-F/R primers followed by sequencing. Correctly sequenced CDS fragments were cloned into the Super1300-GFP expression vector using the ClonExpress II One Step Cloning Kit (Vazyme). The recombinant constructs and the empty Super1300-GFP vector were subsequently transformed into Agrobacterium tumefaciens strain GV3101 competent cells. One-month-old tobacco was used for agroinfiltration, with the left side of each leaf infiltrated with Agrobacterium harboring the empty Super1300-GFP vector and the right side infiltrated with Agrobacterium containing the recombinant construct. After 48 h of dark incubation, infiltrated leaf tissues were collected for other analyses. Results Morphological responses of R. glutinosa adventitious roots to exogenous ABA and GA The development of R. glutinosa tuberous root begins with the swelling of adventitious roots. During this process, the antagonistic regulation of ABA and GA plays a critical role. To assess the effects of ABA and GA on the morphological changes of R. glutinosa adventitious roots, 20-day-old seedlings were continuously irrigated with distilled water, 40 µM ABA, and 150 µM GA respectively for two weeks. In the control group, adventitious roots showed no swelling. However, ABA treatment led to a significant enlargement of these roots. In contrast, GA treatment did not induce swelling but resulted in a reduction in root length and a significant decrease in the number of adventitious roots (Fig. 1 ). No significant differences were observed in the mother roots across the three treatments, nor were any noted before or after treatment (Fig. 1 ). These results suggest that ABA promotes the development of R. glutinosa adventitious roots, whereas GA exerts an inhibitory effect. The two hormones exhibit antagonism in regulating the early transition of adventitious roots, but no effect on the morphology of mother roots. Quality control of RNA-Seq data and function annotations To investigate the mechanisms underlying the antagonistic effects of ABA and GA on R. glutinosa tuberous root initiation, RNA-seq technology was employed to analyze both adventitious roots (CR: control roots, AR: ABA-treated roots and GR: GA-treated roots) and mother roots (CM: control mother roots, AM: ABA-treated mother roots and GM: GA-treated mother roots). Twelve cDNA libraries were constructed for Illumina sequencing, and the effects of exogenous ABA and GA on the development of R. glutinosa adventitious root were comprehensively analyzed at the transcriptional level. As a result, a total of 258,732,013 clean reads were obtained. The average content of GC was 44.74%. The average values of Q20 and Q30 were 98.14 and 94.60, respectively (Table S1 ). The PCA results reveal distinct differences among the groups (Fig. 2 A). And the roots treated with GA were notably farther from the CK groups than those treated with ABA, especially for adventitious roots. This suggests that the GA treatment groups exhibit greater variability, and the endogenous ABA level should increase in the early development stage under normal conditions. The inference agrees with the result of Sun et al. ( 2015 ), who determined the level of endogenous hormones during R. glutinosa tuberous root development. The Pearson correlation coefficient among adventitious root replicates ranged from 0.914 to 0.991, while those across various treatments varied from 0.332 to 0.620. The correlations among mother root replicates ranged from 0.844 to 0.955, whereas those among different treatments ranged from 0.592 to 0.870. The higher values of different treatments in mother root groups indicated that mother root was insensitive to plant hormones (Fig. 2 B). Based on the NR, Swiss-Prot, COG, KOG, eggNOG, GO, KEGG, and Pfam databases, 30,432 unigenes were annotated totally. And 28,332 (93.10%), 21,203 (69.67%), 9,451 (31.06%), 17,034 (56.00%), 24,881 (81.76%), 24,432 (80.29%), 20,435 (67.15%), and 23,137 (76.03%) unigenes were annotated across these eight databases, respectively. Furthermore, 6,033 (19.82%) unigenes were found to be homologous across all eight databases (Table S2 ). DEGs involved in ABA or GA metabolism and signal transduction FC ≥ 2 and FDR < 0.05 were used to identify DEGs. A total of 12608 DEGs were identified from adventitious roots in response to exogenous ABA and GA. The number of DEGs both in AR vs. CR and GR vs. CR was 7,416 (3,293 up-regulated and 4,123 down-regulated) (Table S3; Figure S1 ), and 9,034 (3,492 up-regulated and 5,542 down-regulated) (Table S4; Figure S1 ), respectively (Fig. 3 A). The results indicated a higher number of DEGs in response to GA treatment, with a greater proportion of genes being down-regulated under ABA or GA treatments. Among these genes, 55 DEGs related to ABA metabolism and signal transduction pathways and 24 DEGs involved in GA metabolism and signal transduction pathways were chosen for further analysis (Figure. 3B C). The expression levels of ABA biosynthesis genes zeaxanthin epoxidase s ( ZEPs ) and NCEDs were increased following ABA treatment. However, the expression of another ABA biosynthesis gene abscisic acid deficient 2 ( ABA2 ) was reduced after ABA treatment, but up-regulated by exogenous GA. Moreover, an overwhelmingly majority of ABA catabolism-associated genes CYP707A s were up-regulated post-ABA treatment, while down-regulated under GA treatment. XERICOs (XER s ) act as inducers of ABA biosynthesis (Ko et al., 2006 ). By and large, both ABA and GA stimulated the expression of XERs , especially the latter. The expression of ABA signaling negative regulators PP2Cs (clade A protein phosphatases of type 2Cs) were elevated remarkably in AR group. Under GA treatment, only a small subset of PP2C s were up-regulated. In contrast, most pyrabactin resistance-like protein s ( PYL s), which encode ABA receptors and inhibit the effect of PP2Cs (Zhao et al., 2020 ), were down-regulated under ABA treatment. And a subset of PYL s was significantly up-regulated under GA treatment. Both ABFs and SnRK2 play positive roles in ABA signaling process (Zhao et al., 2020 ; Collin et al., 2021 ; Ali et al., 2022 ; Liu et al., 2024 ). They also exhibited significantly up-regulated expressions under both ABA and GA treatment. Copalyl diphosphate synthase (CPS) is involved in GA synthesis (Bouré & Arnaud 2023 ). It was down-regulated following GA treatment, but up-regulated under ABA treatment. Ent-Kaurenes (KS), ent-Kaurene oxidase (KO) and ent-Kaurenoic acid oxidase (KAO) are entailed in the production of the common GA precursor GA 12 (Bouré & Arnaud 2023 ). However, they showed different expression patterns. KSs increased after GA treatment but decreased under ABA treatment. KO showed minimal changes following GA treatment but was up-regulated under ABA treatment. KAO s expression was up-regulated under both GA and ABA treatments, with a more pronounced increase observed under ABA treatment. The biosynthesis of bioactive GAs requires GA20ox s and GA3ox s (Shani et al., 2024 ), which were down-regulated after GA treatment (Figure. 3C). GA2oxs , as GA-deactivating enzymes, exhibited a significant down-regulation following GA treatment as well (Figure. 3C). DELLA / GAI , the key negative regulator in the GA signaling pathway, was predominantly up-regulated following GA treatment, while it was down-regulated under ABA treatment. And the expression of GA receptor Gibberellin-Insensitive Dwarf 1 ( GID1 ) was suppressed by both ABA and GA. Although these genes displayed differential expression patterns in the presence of exogenous ABA and GA, it is still noticeable that the exogenous ABA or GA prevented the biosynthesis and signaling of itself and facilitated that of antagonist. Consequently, the antagonistic regulation of ABA/GA balance was actuated by excess ABA or GA. And this antagonism may depend more on ABA metabolism and signal transduction pathways according to Fig. 3 B and C. KEGG pathway enrichment analysis The KEGG enrichment analysis of DEGs revealed the top 10 pathways based on their q values (Table S5). The metabolic pathways enriched in both AR and GR included the "Plant MAPK signaling pathway" (Ko04016), "Plant hormone signal transduction" (Ko04075), "Phenylpropanoid biosynthesis" (Ko00940), "Plant-pathogen interaction" (Ko04626), and "Isoquinoline alkaloid biosynthesis" (Ko00950) (Fig. 4 A, B; Table S5). Notably, among the top five pathways, "Starch and sucrose metabolism" (Ko00500) was significantly enriched in AR but not in GR, with a total of 140 DEGs involved in this pathway. To further examine the modulation of the starch and sucrose metabolism pathway under ABA or GA treatment, a GSEA was conducted. The GSEA result for AR indicated a normalized enrichment score (NES) of 1.413, with 122 core genes (Fig. 4 C; Table S6). In contrast, GSEA for GR revealed an NES of -1.291, with 82 core genes and a relatively higher p -value (Fig. 4 D; Table S6). The result suggested that the starch and sucrose metabolism pathway was regulated antagonistically by ABA and GA. It was up-regulated significantly in AR and down-regulated slightly in GR. Starch and sucrose metabolism response to ABA and GA To further elucidate the regulatory roles of ABA and GA in starch and sucrose metabolism, we examined the expression profiles of relevant DEGs within this pathway. It was evident that the metabolism pathway from sucrose to starch was up-regulated by ABA, as all enzymes involved were virtually accumulated transcriptionally under ABA treatment (Fig. 5 ; Table S7). These enzymes included sucrose synthase (SUS), ectonucleotide pyrophosphatase/phosphodiesterase family member 1/3 (ENPP1_3), glucose-1-phosphate adenylyltransferase (glgC), granule-bound starch synthase (WAXY), starch synthase (glgA), 1,4-alpha-glucan branching enzyme (GBE1), and isoamylase (ISA). However, GA did not negatively regulate these enzymes. GBE1 , WAXY , and glgC showed minimal changes in GR. And others were up-regulated by GR. This demonstrated that the antagonism of ABA/GA was not reflected in starch biosynthesis. Cell wall biogenesis and organization GO analysis was performed using GSEA with a p -value threshold of 0.01. The result showed that the top 5 biological processes ranked by p -value in AR were activated (Table S8). However, the top 5 biological processes in GR were repressed, including carbohydrate metabolic process, defense response, response to biotic stimulus, lipid metabolic process, and cell wall organization (Table S8). And given the aforesaid analysis on cellulose metabolism, we paid more attention to the biological processes associated with cell wall. It was found that the GO terms associated with the cell wall exhibited contrasting patterns under GA and ABA treatments. In AR, significant enrichment was observed for the gene sets of "cell wall biogenesis" (GO:0042546) and "cell wall organization" (GO:0071555). The NES for "cell wall biogenesis" and "cell wall organization" in AR were 1.562 and 1.678, respectively (Fig. 6 A; Table S8). The two biological processes were primarily up-regulated in AR. More biological processes related to cell wall were enriched in GR (Fig. 6 B; Table S8), and they were all suppressed. In sum, ABA and GA showed antagonism in the biological processes of "cell wall biogenesis" and "cell wall organization". To further explore the effects of ABA and GA on cell wall-related genes, a heatmap of the DEGs was plotted (Fig. 6 C). Among the DEGs, cellulose synthase s ( CESA s) involved in cellulose synthesis were significantly down-regulated by GA, while their expression in AR showed minimal change. Another cellulose-related gene cellulose synthase-like protein s ( CSLs ) showed the different expression profile in the presence of ABA and GA, most elevated in AR but reduced in GR. Xyloglucan endotransglucosylase/hydrolase proteins (XTHs) and robable beta-1,4-xylosyltransferase (IRX) are associated with hemicellulose synthesis. XTHs were antagonistically regulated by ABA and GA, whereas probable beta-1,4-xylosyltransferase ( IRX ) genes were up-regulated in response to both ABA and GA. Genes related to pectin synthesis and modification, including polygalacturonase s ( PG s), pectin methylesterases ( PME s), pectin acetylesterase s ( PAE s), and galacturonosyltransferase s ( GAUT s), were predominantly up-regulated in AR and down-regulated in GR. Additionally, the expression patterns of another two important genes affecting the cell wall structure were also depicted in Fig. 6 . Expansins ( EXPs ) were up-regulated in AR but showed little change in GR. Wall-associated receptor kinases (WAKs) belong to receptor-like protein family. They are cross-linked pectins and involved in cell wall integrity signaling pathway. The heatmap showed that ABA predominantly down-regulated WAKs and most WAK s in GR showed minimal change with only a few being down-regulated. Overall, the DEGs directly implicated in cell wall biosynthesis, cell wall remodeling and modification were up-regulated in AR and down-regulated in GR (Fig. 6 C). Key genes for tuberous roots initiation antagonistically regulated by ABA/GA identified using WGCNA and PPI network Despite the antagonistic regulation of ABA and GA on the initiation of R. glutinosa tuberous root, neither ABA nor GA induced any morphological alterations in mother roots (Fig. 1 ). The insensitivity of R. glutinosa mother roots to ABA and GA were also manifested in Pearson correlation coefficient as well (Fig. 2 B). This implied that the variation at transcriptomics level in mother roots was inadequate for the root enlargement initiated by ABA/GA, and the DEGs in AM vs. CM or GM vs. CM did not contain the key genes for the induction of root swelling regulated by ABA/GA. Therefore, the DEGs in the overlapping regions of AM vs. CM and AR vs. CR were excluded and the rest of DEGs in AR vs. CR were referred to as ARM (Figure S2 ). The DEGs in GR vs. CR were processed similarly and designated as GRM (Figure S3). To investigate the regulatory effects of ABA and GA on tuberous root initiation and identify the related key genes, we audaciously performed WGCNA on the 9,897 DEGs in ARM or GRM (Table S9; Figure S4). This analysis resulted in the identification of 11 distinct modules (Fig. 7 A; Table S10). The module-sample correlation analysis was then conducted to identify specific modules that strongly correlate with ABA and GA treatment. The darkred module containing 348 genes and the turquoise module containing 1055 genes were finally highlighted in bright red (Fig. 7 B; Table S10). In the darkred module, NCED3 was identified as a hub gene in the PPI network, along with other highly connected genes such as DREB1B , PP2CA , DREB1C , RD22 , protein phosphatase 2C 7 ( HAB2 ), and XTH22 (Fig. 7 C). Similarly, the turquoise module PPI network highlighted flavone 3'-O-methyltransferase 1 ( OMT1 ) as a hub gene, with other highly connected genes, including cinnamyl alcohol dehydrogenase 5 ( CAD5 ), aldehyde dehydrogenase family 2 member C4 ( ALDH2C4 ), probable nicotianamine synthase 4 ( NAS4 ), glycerol kinase ( GLPK ), protein PHR1-LIKE 1 ( PHL1 ), and serine-glyoxylate aminotransferase ( AGT1 ) (Fig. 7 D; Table S11). Based on FPKM values, 160 DEGs with opposite expression patterns in ABA and GA treatments were selected from GRM and ARM (Table S12; Figure S5). Among these, 95 DEGs showed up-regulated expression in AR but down-regulated expression in GR, while 65 DEGs exhibited the opposite pattern. DEGs with contrasting expression patterns are denoted in yellow in the PPI interaction map. In the PPI network analysis, seven genes including DREB1B , DREB1C , DREB1D , ERF025 , ERF017 , ROH1 ( F6A14.15 ), and protein phosphatase 2C 63 ( F17I5.110 ) displayed reduced expression patterns in GR (Fig. 7 C). Conversely, in the PPI network of GR, 11 genes, including probable ubiquitin-conjugating enzyme E2 24 ( UBC24 ), PER47 , PER10 , PER59 , NAS4 , probable serine/threonine-protein kinase PBL15 ( PBL15 ), type IV inositol polyphosphate 5-phosphatase 9 ( IP5P9 ), phosphatidylinositol/phosphatidylcholine transfer protein SFH2 ( SFH2 ), WAT1-related protein At1g43650 ( UMAMIT22 ), and WAT1-related protein At5g07050 ( UMAMIT9 ), exhibited decreased expression patterns in AR (Fig. 7 D). These genes labeled yellow may play crucial roles in the antagonism of ABA/GA and affect the expression of the forementioned hub genes potentially associated with tuberous root initiation. The gene expression pattern was verified by qRT-PCR To confirm the RNA-Seq precision, 12 genes were selected for qRT-PCR assessment. The results revealed a resemblance between the expression patterns of these 12 genes tested by qRT-PCR and those detected by RNA-Seq, thus substantiating the reliability of RNA-Seq (Fig. 8 ; Table S13). Transient expression of RgERF017 or RgPER10 in tobacco affected the expression of ABA and GA metabolism genes To initially explore their roles in ABA/GA antagonism, the identified hub genes, DREB s, ERF s and PER s, were selected. We examined the expression of these genes during R. glutinosa early development stage. The elevated expression was detected in fibrous roots, with ERFs and PERs showing particularly notable increase (Fig. 9 A). Two representative candidates with significant changes, RgERF017 and RgPER10 , were then cloned and used for the transient transformation analysis (Figure S6). The significant transcript accumulation of key ABA biosynthetic genes NCEDs and ABA-responsive genes RD22s and the downregulation of ABA catabolic genes CYP707As were detected in the tobacco leaves transiently expressing RgERF017 or RgPER10 (Fig. 9 B). This revealed an elevation in endogenous ABA levels in tobacco leaves. GA biosynthetic genes GA20oxs and GA3ox were downregulated (Fig. 9 B), but GA catabolic genes GA2oxs showed different responses. The transient expression of RgPER10 caused the upregulation of GA2ox s, suggesting a reduction in GA levels. However, RgERF017 led to the reduced accumulation of GA2ox s transcripts. These results suggested that RgERF017 and RgPER10 are involved in ABA/GA antagonism through the regulation of ABA and GA metabolism genes. Discussion ABA and GA antagonistically regulate the initiation of R. glutinosa root expansion The antagonistic actions of ABA and GA in tuberization were first found in potato (Vreugdenhil et al., 1994 ; Xu et al., 1998 ). The ABA-deficient potato lines can recover tuberization with the inhibition of GA biosynthesis (Vreugdenhil et al., 1994 ). Endogenous GA level was negatively correlated with tuber formation and exogenous ABA stimulated tuberization in potato (Xu et al., 1998 ). It was thus deemed that the positive regulatory effect of ABA on potato tuberization was achieved by counteracting GA (Xu et al., 1998 ). Afterwards, the roles of ABA or GA were identified in various stem/root tubers, including yam stem tuber and bulbil (Kim et al., 2003 ; Zhou et al., 2021 ), carrot, turnip and radish taproot (Wang et al., 2015 ; Wang et al., 2017 ; Liu et al., 2021 ; Meng et al., 2024 ), Gladiolus hybridus corm (Li et al., 2021b ) and Pseudostellaria heterophylla tuberous root (Wang et al., 2024a ). Except for yam tuber and bulbil, ABA stimulates, and GA represses the initiation of tuberization in general. To ascertain the roles of ABA and GA in R. glutinosa tuberization, we treated R. glutinosa seedlings with ABA and GA respectively. Phenotypically, exogenous ABA significantly promoted root expansion, whereas GA treatment inhibited tuberization and root growth, resulting in reduced root length and quantity (Fig. 1 ). Therefore, it is evident that ABA and GA antagonistically regulate the initiation of R. glutinosa root expansion, with ABA acting as a positive regulator. The feedback regulation on metabolism involves the antagonistic action of ABA and GA Excess phytohormones tips the balance between ABA and GA. It actuates the mediation of ABA/GA homeostasis, which involves ABA and GA metabolism and signaling pathways. The endogenous ABA levels are directly associated with both ABA synthesis and catabolism. The ABA biosynthetic genes, ZEP s and NCED s, were up-regulated following ABA treatment, while ABA2 was repressed (Fig. 3 B). ABA2 functions in plastids after ZEP and NCED (Finkelstein, 2013 ). The suppression of ABA2 prevented ABA biosynthesis. However, ABA catabolism was promoted, because most catabolic genes CYP707A s were upregulated by exogenous ABA (Fig. 3 B). And in terms of ABA accumulation, catabolism outweighs biosynthesis (Finkelstein, 2013 ), so the expression pattens of ABA metabolic genes demonstrated that the content of ABA may decrease after the sharp increase caused by ABA treatment. ABA metabolism is tightly regulated through the feedback signaling mechanisms (Finkelstein, 2013 ), in which ABFs play a vital role (Wang et al., 2018 ; Collin et al., 2021 ). ABFs are activated by the canonical ABA signaling pathway at both transcriptional and protein level in the presence of ABA (Lopez-Molina et al., 2001 ; Wang et al., 2018 ). The active ABFs then induce the expression of ABA-responsive genes, including NCED s, and ABA signaling negative regulator PP2C s (Zong et al., 2016 ; Wang et al., 2018 ; Collin et al., 2020 ). The elevated expression of ABF s, NCED s and PP2C s was detected in our study and shown in Fig. 3 B. PYLs usually count as ABA receptors. They are also the targets of ABFs (Zhao et al., 2020 ), but ABFs may mainly contribute to the maintenance of PYL s expression (Zhao et al., 2020 ). PYLs possess unique functions in various biological processes, which implies their diverse expression patterns (Zhao et al., 2020 ). Santiago et al. ( 2009 ) and Zhao et al. ( 2020 ) observed that some PYL s were up-regulated by exogenous ABA but some were depressed or exhibited no obvious response. In R. glutinosa , most PYL s showed decreased expression but some kept no change in post-ABA situation (Fig. 3 B). The expression of ABF s and the other two core ABA signaling components, PP2C s and PYL s, gave a hint that the feedback regulation of ABA pathway was already activated and the core ABA signaling was suppressed. Like ABA, GA synthesis and catabolism directly dictate endogenous GA levels, which are controlled by feedback loop (Bouré & Arnaud 2023 ). CPS, KS KO, and KAO are the essential enzymes for the biosynthesis of bioactive GA precursor GA 12 (Shani et al., 2024 ). After GA treatment, these enzymes other than KS were all repressed at transcriptional level in R. glutinosa roots (Fig. 3 C). Although some of the genes responded similarly to GA application in other plants and were thought to be involved in the feedback regulation of GA (Zhang et al., 2016 ; Guan et al., 2019 ), the unchanged bioactive GA in Arabidopsis overexpressing AtCPS and AtKS (Fleet et al., 2003 ) indicated that the enzymes may be incapable of influencing bioactive GA content straightly. The key enzymes remarkably affecting the biologically active GAs are 2-oxoglutarate-dependent dioxygenases (2-ODDs), namely the biosynthetic GA20ox, GA3ox, and catabolic GA2ox (Bouré & Arnaud 2023 ; Shani et al., 2024 ). There are numerous reports of repressed GA20ox and GA3ox and enhanced GA2ox by exogenous GA (Hernández-García et al., 2021 ; Bouré & Arnaud 2023 ; Shani et al., 2024 ). However, not only the biosynthetic GA20ox s and GA3ox but the majority of GA2ox s as well were downregulated by GA in R. glutinosa (Fig. 3 C). The inhibited expression of GA2ox s was also reported in GA-treated yam, which actually took place 30 days after GA treatment (Zhou et al., 2021 ). So, it is speculated that the feedback regulation started by GA treatment may move into the later stage. The feedback responses on GA metabolism modulated by GA signaling, in which DELLA proteins function as critical repressors (Shani et al., 2024 ). DELLA proteins are degraded after GA binding to its receptor GID1(Shani et al., 2024 ). The GA biosynthetic genes GA20ox2 , GA3ox1 and GA receptor GID1 are early DELLA-responsive genes (Zentella et al., 2007 ). Their transcript levels are reduced after GA treatment, which resulted from the reduced accumulation of DELLA proteins (Silverstone et al., 2001 ; Griffiths et al., 2006 ; Zentella et al., 2007 ; Weston et al., 2008 ). The expression patterns of these genes in R. glutinosa (Fig. 3 C) were consistent with the finding and implied the decreased DELLA at protein level. Nevertheless, long-term treatment of GA led to the increased expression of DELLA at transcriptional level in tubers (Zhou et al., 2021 ; Meng et al., 2024 ). This result was also detected in R. glutinosa (Fig. 3 C). The up-regulated DELLA is not indicative of the accumulation of DELLA proteins. The accumulated transcript of DELLA may serve as a reservoir for the timely up-regulation of DELLA proteins, which indicated that GA signaling would be repressed. ABA/GA homeostasis is not merely regulated by individual ABA or GA signaling pathways, but by the interplay between the two signaling pathways (Bouré & Arnaud 2023 ). DELLAs and ABFs are their respective key factors and serve as the crosstalk interface as well. The ABA/GA antagonism in seed germination and potato tuberization correlates with DELLAs and ABFs (Muñiz García et al., 2013 ; Li et al., 2022a ). However, DELLA s were antagonistically regulated by exogenous ABA and GA, while both ABA and GA promoted the expression of ABF s in R. glutinosa (Fig. 3 B, C). It is notable that the degree of up-regulated expression of ABF s varies among individuals following ABA or GA treatment. This may lead to an antagonistic effect on the expression of ABF targets, NCED s and PYL s (Fig. 3 B). ABI5, a member of ABFs, is recruited and activated by DELLA (Lim et al., 2013 ). It also negatively controls the expression of GA3ox (Lim et al., 2013 ), to which the observation in Fig. 3 C conformed. Furthermore, XER is another crosstalk point. It is conducive to the accumulation of ABA through transcriptional upregulation of NCED (Ko et al., 2006 ). And it was also identified as one DELLA-responsive gene (Zentella et al., 2007 ). The expression of XER kept in line with DELLA s, but NCED s displayed the opposite expression patterns (Fig. 3 B). It is because NCEDs are modulated by multiple factors, such as ABI5 (Lim et al., 2013 ) and ABI4 (Shu et al., 2016 ), besides XER. In Arabidopsis, RGL2 (a DELLA protein) stimulates ABI5 activity and ABA formation through the XER protein (Piskurewicz et al., 2008 ). Therefore, the expression of DELLA s, ABF s and XER s after GA treatment suggested that ABA levels would be elevated. This hypothesis about ABA levels can be reinforced by the suppressed expression of CYP707A s under GA treatment (Fig. 3 B). Overall, most genes presented in Fig. 3 B and C produced antagonistic responses to ABA and GA treatment. And the expression of these genes denotes the declined ABA levels post-ABA treatment and elevated ABA levels after GA application according to the discussion above. Compared to ABA, the trend of GA metabolism is not evident following exposure to the two hormones. But as mentioned above, GA signaling would be suppressed following GA treatment. Shu et al. ( 2017 ) proposed a model of the antagonism of ABA and GA, which suggests that ABA and GA tend to exhibit a negative correlation (Shu et al., 2017 ). This view is drawn from the endogenous concentrations of ABA and GA, while the exogenous ABA and GA appear to elicit different responses in our study. The persistent excessive exogenous ABA or GA suppressed respective biosynthesis and signaling and stimulated reciprocally through feedback regulation and crosstalk. These responses reset ABA/GA homeostasis, which remains true to the core tenets of the model. Moreover, given more antagonistic expression patterns present in key genes related to ABA metabolism and signaling, it is potential that the ABA/GA balance mainly depends on ABA metabolism. Alternatively, it is also plausible that feedback responses implicating ABA levels persists for a longer duration, or that the feedback associated with GA metabolism is more sensitive to exogenous phytohormones. In sum, ABA/GA homeostasis depends on the antagonistic action of ABA and GA on their metabolism and signaling crosstalk. The potential key regulators in the antagonistic action of ABA and GA during R. glutinosa tuberous root initiation Exposure to ABA or GA leads to thousands of gene expression changes (Table S9), among which there are key regulators involved in the regulatory antagonism of ABA and GA. After removing mother root DEGs not relevant to this biological process, those from ARM or GRM were subjected to WGCNA for key regulator identification (Fig. 7 ). To date, as per the existing studies the regulators involving ABA/GA balance primarily encompass TFs (Yaish et al., 2010 ; Lee et al., 2015 ; Lee et al., 2016 ; Tang et al., 2017 ; Li et al., 2022b ; Jin et al., 2023 ; Xian et al., 2024 ; Xie et al., 2024 ), reactive oxygen species (ROS) (Chen et al., 2019 ; Liu et al., 2010 ; Li et al., 2021a ), and proteins related to ubiquitin and ubiquitin-like modification (Miura et al., 2009 ; Lin et al., 2020 ). According to the WGCNA and PPI visualized in Fig. 7 C and D, the hub TFs antagonistically regulated by ABA and GA were only shown in the distinctive module with a robust correlation to ABA. These hub TFs are DREB1C, DREB1B, DREB1D, ERF017 and ERF025. They are all induced by ABA but suppressed by GA (Fig. 7 ). And under normal growing conditions the significant accumulation of DREB1C, ERF017 and ERF025 transcripts occurred during the tuberous root initiation (Fig. 9 A). Both DREBs and ERFs belong to AP2/ERF super-family. AP2/ERF super-family is well-recognized for their regulatory functions in hormonal signaling pathways and various aspects of plant development (Feng et al., 2020 ). Shu et al. ( 2017 ) proposed that the research on AP2-domain-containing TFs may be helpful to disclose the interplay of ABA and GA and understand the mechanisms underlying ABA/GA antagonism. And a series of investigations have confirmed this opinion, among which ABI4 has been the most thoroughly studied (Yaish et al., 2010 ; Shu et al., 2013 ; Shu et al., 2016 ; Li et al., 2022b ; Xian et al., 2024 ). Although the hub TFs identified in Fig. 7 are from different subfamilies, DREB1C, DREB1B, and DREB1D falling into DREB subfamily, ERF017 and ERF025 belonging to ERF subfamily, some DREBs and ERFs still exhibit an association with ABA or GA metabolism or signaling (Tang et al., 2017 ; Zhou et al., 2016 ; Sun et al., 2021 ; Chen et al., 2022b ; Li et al., 2022b ; Ma et al., 2022 ; Vonapartis et al., 2022 ). We investigated the expression of ABA and GA metabolism genes in the tobacco levels transiently expressing RgERF017. The expression levels of both ABA and GA metabolism genes were significantly affected, which also resulted in the accumulated endogenous ABA. Therefore, these TFs are the candidates participating in the modulation of ABA/GA antagonism despite their obscure role in ABA/GA antagonism crosstalk (Shu et al., 2017 ). In addition to AP2/ERF TFs, some ROS-related PERs were also identified as hub genes in the turquoise module (Fig. 7 D). Thereinto, PER10, PER47 , and PER59 inversely responded to exogenous ABA and GA (Fig. 7 ). And their transcripts were also significantly accumulated in R. glutinosa early development stage under normal conditions (Fig. 9 A). ROS are the inevitable by-products of metabolism (Wang et al., 2024b ). The accumulation of ROS is toxic to organisms, but low-level ROS still serve as key signaling molecules, implicating in plant development (Wang et al., 2024b ). In Arabidopsis, ROS mediate seed dormancy and germination through regulating the balance of ABA/GA (Liu et al., 2010 ; Li et al., 2021a ). ABA catabolic gene CYP707A s and GA biosynthesis are stimulated by ROS (Liu et al., 2010 ). Hence the genes for peroxidases capable of eliminating ROS appear to affect ABA/GA homeostasis. AtPER1 is such a gene that has been experimentally demonstrated to be involved in the trade-off between ABA and GA (Chen et al., 2019 ). In our study, the transient expression of RgPER10 in tobacco also induced the change of ratio of ABA/GA through regulating ABA and GA metabolism (Fig. 9 B). But interestingly its ROS-scavenging capacity appears to be less potent than that of RgEFR017 (Figure S7). The results demonstrated that the involvement of RgERF017 in ABA/GA antagonism may also be associated with but not limited to the scavenging of ROS. Furthermore, the PERs were predicted to interact with CADs, MEE, OMT etc. in PPI analysis (Fig. 7 D). These proteins, including PERs, are closely associated with lignin biosynthesis (Liu et al., 2022 ; Zhao et al., 2022 ; Martin et al., 2023 ). And their expression was all elevated by GA (Fig. 7 D). It is reported that exogenous GA promotes lignin biosynthesis through positively regulating these lignin biosynthetic genes (Wang et al., 2017 ; Liu et al., 2022 ). And the increased lignin content in roots can arrest root growth (Wang et al., 2017 ). Taken together, our results on PER s suggest that on the one hand PER s in R. glutinosa may be involved in ROS-mediated feedback loop of ABA/GA homeostasis, and on the other hand PER s also exert direct influence on root development through mediating lignin biosynthesis. Apart from TFs and ROS, other proteins, including UBC24 and NAS4, were also identified as hub genes antagonistically regulated by ABA and GA in our WGCNA (Fig. 7 ). UBC24, a ubiquitin-conjugating E2 enzyme, and its interacting proteins are more closely related to phosphate homeostasis (Liu et al., 2012 ). NAS4 and its predicted interacting proteins predominantly participate in the transport of metal ions (Wang et al., 2020 ; Yang et al., 2024 ). So far, no studies have shown that they have a crucial function in the biological processes regulated by ABA/GA antagonism, and the relationship between them and ABA/GA antagonism as well as R. glutinosa root development calls for further investigation. The downstream responses to ABA/GA antagonism Downstream responses and plant development are controlled by the ratio of ABA/GA. The prevailing one with higher content dominates the expression of downstream key genes and thus determines the direction of development. Starch and sucrose metabolism are closely linked to tuber development, as these carbohydrates serve as vital sources of energy and structural materials for tuber growth and expansion (Sojikul et al., 2015 ; Pan et al., 2021 ; Cai et al., 2022 ). Dynamic transcriptional profiling of R. glutinosa tuberous root showed that starch and sucrose metabolism is the most overrepresented group in carbohydrate metabolism during root development (Sun et al., 2015 ). In analogy with R. glutinosa roots under normal developmental conditions, starch and sucrose metabolism was also enriched in the swelling fibrous roots induced by the addition of ABA, and this pathway was upregulated in its entirety, especially the biosynthetic process from sucrose to starch (Fig. 4 , 5 ). The upregulation of genes encoding starch synthases, including GBE1 s, glgA s and WAXY s (Fig. 5 ), demonstrated that ABA actuated the accumulation of starch. Starch biosynthesis and metabolism are regulated by sucrose (Yoon et al., 2021 ). It can provide energy, processed materials and even hexose-based sugar signals for sinks development after its cleavage (Yoon et al., 2021 ; Wang et al., 2023 ). Sucrose cleavage can be catalyzed by INVs or SUSs. These two enzymes channel sucrose into two distinct metabolic pathways, with SUSs starting the pathways for starch synthesis (Fig. 5 ). The silencing of SUS s in tuber plants, potato (Herbers & Sonnewald 1998 ) and Gladiolus hybridus (Li et al., 2021b ), gave rise to the decreased starch content and tuber yield. In R. glutinosa , the remarkably elevated expression of SUS s was observed in the swelling roots treated by ABA, and meanwhile all the INV s showed significantly lower expression (Fig. 5 ). This may be one reason for the accumulation of starch in R. glutinosa roots. Thus, it is reasonable to conjecture that SUS and INV may be key genes for the downstream responses. However, SUSs and INVs were not obviously regulated antagonistically by ABA and GA. GA also induced the expression of most SUS s, but to a lower degree than ABA (Fig. 5 ). Only unigene_0941071 was repressed by exogenous GA (Fig. 5 ). As for INV s, the declined expression was also detected in most homologous unigenes post-GA treatment, except for unigene_154406 and unigene_131992 (Fig. 5 ). Probably, only certain genes, those subject to antagonistic regulation, are the pivotal nodes in the ABA/GA antagonistic network, determining the direction of sucrose cleavage and eventually even the root development. Alternatively, SUS s and even starch biosynthesis are more sensitive to ABA than to GA, as the regulatory effect of GA on starch biosynthesis was weaker overall. Cell wall biosynthesis and modification underpin the tuber expansion and shaping (Jarvis, 1992 ; Liu et al., 2007 ). During tuberous root development, the metabolic process related to cell wall metabolism was enriched (Li et al., 2015 ; Cai et al., 2022 ). And in R. glutinosa , those DEGs associated with cell wall metabolism were mainly identified in the initiation stage of fibrous roots expansion (Li et al., 2015 ). Similarly, Our GSEA analysis also detected up-regulated cell wall-related biological processes during the tuberous root initiation induced by ABA (Fig. 6 ; Table S8). By contrast, all biological processes related to cell wall were significantly downregulated under GA treatment. Cell walls are tied to cell division and expansion (Delmer et al., 2024 ). Plus, the GESA analysis also revealed that the biological processes related to cell cycle were only significantly enriched in AR group, rather than in GR group (Table S8), so cell proliferation and expansion may occur extensively during the initiation of tuberous root in AR group. Cell walls are mainly composed of cellulose, hemicellulose, pectin, wall-associated proteins, etc. (Chen et al., 2020 ; Delmer et al., 2024 ). Cell wall biogenesis and expansion growth cannot proceed without the involvement of cell wall polymers and their synthesis enzymes, wall-modifying enzymes and other important wall-associated proteins (Chen et al., 2020 ; Delmer et al., 2024 ). It is obvious that the enzymes responsible for the synthesis of cellulose and pectin, CESAs and GAUTs (Anderson & Kieber 2020 ; Delmer et al., 2024 ), maintained high expression levels in both CR and AR groups (Fig. 6 ). In contrast, they are significantly suppressed by GA (Fig. 6 ). This indicated that the synthesis of cellulose and pectin remains highly active during the normal developmental stage, likely priming cells for division and expansion. And ABA sustains this synthetic activity, while GA suppresses it. Moreover, important wall-modifying enzymes, XTHs and PMEs, hemicellulose synthase CSLs and cell wall signal transducers (WAKs) (Chen et al., 2021a ) were antagonistically regulated by ABA and GA (Fig. 6 ). XTHs cleave or reorganize the backbone of xyloglucan, the most abundant hemicellulose in the primary walls (Delmer et al., 2024 ). PMEs demethylesterify pectic backbones of homogalacturonan, which is initially generated in a highly methyl-esterified form by GAUTs (Delmer et al., 2024 ). The higher expression of these enzymes suggests enhanced wall remodeling for cell expansion takes place in the presence of ABA. And this conclusion can be supported by the expression pattern of EXPs as well. The overwhelming majority of EXPs exhibited significantly upregulated expressions exclusively in the AR group (Fig. 6 ). EXPs facilitate cell wall-loosening through disrupting the noncovalent interactions between laterally aligned polysaccharides and thus help to cell enlargement (Cosgrove, 2024 ). Consequently, the specific expression of wall-associated enzymes and proteins further disclosed that exogenous ABA promotes cell expansion to a greater extent compared with normal conditions, and GA arrests tuberous root initiation by retarding cell division and expansion. Previous studies (Xu et al., 1998 ; Carrera et al., 2001 ; Bou-Torrent et al., 2011 ; Cheng et al., 2013 ; Roumeliotis et al., 2013 ; Davière & Achard 2016 ; Chen et al., 2022a ) have shown that the inhibitory effect of GA on tuber formation is associated with enhanced cell elongation, manifested as stimulation of stolon elongation or promotion of aerial growth. The elongated overground stem observed after GA treatment (not shown) supports the latter part of the conclusion, but as we argued above, GA directly suppresses cell division and expansion in R. glutinosa roots. Furthermore, the stimulation of lignification in roots by GA also contributes to the inhibition of tuber expansion (Chen et al., 2022a ). The increased lignification induced by exogenous GA, as identified through WGCNA, supports this opinion. Conclusion In this study, we elucidated the molecular mechanisms underlying the antagonistic regulation of adventitious root swelling in R. glutinosa by ABA and GA through metabolic, signaling pathway, and transcription factor network analyses, complemented by phenotypic observations and gene expression profiling. Exogenous ABA or GA disrupts ABA/GA homeostasis and the one with a higher concentration will then activate its corresponding signaling pathway. The other signaling will be triggered soon due to the crosstalk of ABA/GA signaling. The key hub elements in ABA/GA signaling crosstalk, such as ABFs, DELLAs, DERBs and ERFs, orchestrate ABA/GA antagonism by integrating ABA/GA metabolism and downstream responses. In general, higher ratio of ABA/GA results in the promotion of starch and sucrose metabolism, cell wall biogenesis and modification, cell division and expansion, while lower ratio of ABA/GA suppresses these biological processes and induces lignification. The outputs of these downstream responses, like ROS, also function as feedback regulators of ABA/GA metabolism. Both ABA/GA signaling and the regulators from downstream responses modulate ABA/GA metabolism mainly through regulating the expression of key metabolic genes. The modulation of ABA/GA metabolism finally contributes to the trade-off between ABA and GA. Meanwhile, the downstream responses triggers or inhibits the initiation of R. glutinosa tuberous roots. This proposed model was depicted in Fig. 10 . Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Fundings: This work was supported by Natural Science Foundation of Henan Province (No. 242300421579 and No. 252300421280), Key Research Development and Promotion Project (in Science and Technology) of Henan Province (No. 232102110028), and Program for Innovative Research Team (in Science and Technology) in University of Henan Province (No. 23IRTSTHN022). Author contributions Ruixue Yang and Peilei Chen conceived and designed the experiments. Ruixue Yang, Yiying Du, Zhenyang Fang and Heyang Wang performed the experiments and analyzed the data. Ruixue Yang and Peilei Chen wrote the manuscript. Qingxiang Yang complemented the manuscript. Peilei Chen and Hongying Duan supervised the work and corrected the manuscript. Acknowledgements The authors acknowledge financial support from the Natural Science Foundation of China [grant numbers 32372746], and the support of Henan Province University’s Engineering and Technology Center of Conservation and Utilization for Genuine Chineses Medicinal Herbs, and Xinxiang Engineering and Technology Center of Conservation and Utilization for Chinese Medicinal Herbs. Data availability statement All data supporting the findings of this study are available within the manuscript and within its supplementary materials published online. References Ali, F., Qanmber, G., Li, F. & Wang, Z. 2022. "Updated role of ABA in seed maturation, dormancy, and germination." Journal of Advanced Research, 35 : 199-214. https://doi.org/10.1016/j.jare.2021.03.011. Anderson, C. T. & Kieber, J. J. 2020. "Dynamic Construction, Perception, and Remodeling of Plant Cell Walls." Annual Review of Plant Biology, 71 : 39-69. https://doi.org/10.1146/annurev-arplant-081519-035846. 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Zong, W., Tang, N., Yang, J., Peng, L., Ma, S., Xu, Y., Li, G. & Xiong, L. 2016. "Feedback Regulation of ABA Signaling and Biosynthesis by a bZIP Transcription Factor Targets Drought Resistance Related Genes." Plant Physiology, 171 : 2810-2825. https://doi.org/10.1104/pp.16.00469. Supplementary Files SupplementaryTables.xlsx Supplemental Figure 1. DEGs in the adventitious roots and mother roots of R. glutinosa treated with ABA or GA. Supplemental Figure 2. Venn diagram of DEGs in AM vs. CM and AR vs. CR. Supplemental Figure 3. Venn diagram of DEGs in GM vs. CM and GR vs. CR. Supplemental Figure 4. Venn diagram of DEGs in ARM and GRM. Supplemental Figure 5. 160 DEGs with opposite expression patterns between ARM and GRM. Supplemental Figure 6. PCR verification of RgERF017 and RgPER10 expression in tobacco leaves infiltrated with recombinant plasmids. Supplemental Figure 7. NBT staining was performed to detect reactive oxygen species (ROS) accumulation in tobacco leaves. SupplementalFigures.docx Supplementary Information Supplemental Table 1. Quality control of RNA-Seq data. Supplemental Table 2. Unigenes annotated statistical table. Supplemental Table 3. 7,416 DEGs in AR vs. CR. Supplemental Table 4. 9,034 DEGs in GR vs. CR. Supplemental Table 5. KEGG pathway analysis of DEGs from AR vs. CR and GR vs. CR. Supplemental Table 6. GSEA analysis of KEGG pathways from AR vs. CR and GR vs. CR. Supplemental Table 7. Unigenes involved in the starch and sucrose metabolism pathway. Supplemental Table 8. GSEA analysis of Biological Processes from AR vs. CR and GR vs. CR. Supplemental Table 9. 9897 DEGs in ARM and GRM. Supplemental Table 10. Unigenes from different modules in WGCNA. Supplemental Table 11. Unigenes information of the darkred and turquoise modules in the STRING database. Supplemental Table 12. Expression and annotation of 160 genes in transcriptome analysis. Supplemental Table 13. The primers used in this study. 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11:49:47","extension":"png","order_by":36,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1787565,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFigure.9.png","url":"https://assets-eu.researchsquare.com/files/rs-7635435/v1/b99022222ef2d3bdeb7f59cc.png"},{"id":92858017,"identity":"4409cce8-54d2-4ff7-b58d-4de6728e370e","added_by":"auto","created_at":"2025-10-06 11:49:47","extension":"xml","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":283536,"visible":true,"origin":"","legend":"","description":"","filename":"PCRED25010790structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7635435/v1/8aecef12d4ffc11e205b2f1a.xml"},{"id":92858015,"identity":"50e9e7ae-60b1-4cc3-8d6e-ae7fec596d7e","added_by":"auto","created_at":"2025-10-06 11:49:47","extension":"html","order_by":38,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":302359,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7635435/v1/19690acaad1f24ff24e79bb2.html"},{"id":92857989,"identity":"2d501c26-fbee-447a-9ff9-4f68b90bde37","added_by":"auto","created_at":"2025-10-06 11:49:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1247503,"visible":true,"origin":"","legend":"\u003cp\u003eMorphological changes in\u003cem\u003e R. glutinosa\u003c/em\u003e mother and adventitious roots. 20-day-old seedlings were treated with ABA (40 μM) or GA (150 μM) for 14 days. CK: the control group, ABA: the ABA-treated group, GA: the GA-treated group (GA).\u003c/p\u003e","description":"","filename":"Figure.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7635435/v1/6c9f5138e43a6810f747be74.png"},{"id":92857995,"identity":"558a1c39-8550-4aed-8d16-f718e8001dd8","added_by":"auto","created_at":"2025-10-06 11:49:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2420738,"visible":true,"origin":"","legend":"\u003cp\u003eQuality control of sequencing data. (A) Principal component analysis (PCA) of RNA-seq data. PC1: the first principal component, PC2: the second principal component. The value is the contribution of each principal component. Different classification criteria are distinguished by colour and shape. (B) Heatmap of sample correlation based on gene expression. The closer the coefficient of correlation r^2 is to 1, the stronger the correlation between the two samples. Abbreviations: AM: the mother root ABA treatment group; AR: the adventitious root ABA treatment group; CM: the mother root control; CR: the adventitious root control; GM: the mother root GA treatment group; GR: the adventitious root GA treatment group.\u003c/p\u003e","description":"","filename":"Figure.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7635435/v1/c82bc70db91d2a955303f227.png"},{"id":92858859,"identity":"2bbf0f0d-840b-4638-a4ac-7b7958bbba80","added_by":"auto","created_at":"2025-10-06 11:57:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4855259,"visible":true,"origin":"","legend":"\u003cp\u003eExpression patterns of genes related to ABA and GA metabolism and signal transduction in \u003cem\u003eR. glutinosa\u003c/em\u003e under ABA or GA treatment. (A) The number of DEGs. (B) Heatmap showing the expression profiles of DEGs related to ABA. (C) Heatmap showing the expression profiles of DEGs related to GA. Abbreviations: ZEP: zeaxanthin epoxidase. NCED: 9′-cis-epoxycarynthase. ABA2: Xanthoxin dehydrogenase. CYP707A: abscisic acid 8'-hydroxylase. XER: E3 ubiquitin-protein ligase XERICO. PP2C: protein phosphatases type 2C. PYL: PYL receptor. ABF: abscisic acid-insensitive. SnRK2.6: SNF1-related protein kinase 2.6. CPS: copalyl diphosphate synthase. KS: ent-Kaurenes. KO: ent-Kaurene oxidase. KAO: ent-Kaurenoic acid oxidase. GA20ox: gibberellin 20-oxidases. GA3ox: gibberellin 3-oxidase. GA2oX: gibberellin 2-oxidase. GID1: gibberellin-insensitive dwarf 1. The value was normalized as Z-scores using FPKM.\u003c/p\u003e","description":"","filename":"Figure.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7635435/v1/6bdb2644e22a40945f8b6e2d.png"},{"id":92857991,"identity":"e73993b0-5d1b-4224-a08d-6f680e49aa00","added_by":"auto","created_at":"2025-10-06 11:49:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2988977,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional enrichment of DEGs. (A) KEGG pathway enrichment analysis of DEGs from AR vs. CR. (B) KEGG pathway enrichment analysis of DEGs from GR vs. CR. The dot plots of (A) and (B) show the top 10 most significant KEGG pathways. The dots in (A) and (B) are colored by \u003cem\u003eq \u003c/em\u003evalue and sized by the number of genes involved in the pathway. (C) and (D) Gene set enrichment analysis (GSEA) plots for the starch and sucrose metabolism pathway set using the DEGs from AR vs. CR (C) and GR vs. CR (D). The enrichment plots show the enrichment scores and NES values. Negative NES values indicate down-regulation of pathway expression, while positive values indicate up-regulation of that. The black line in the GSEA enrichment plots represents the set of genes examined.\u003c/p\u003e","description":"","filename":"Figure.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7635435/v1/3fc6286faf56d032a158bb81.png"},{"id":92859464,"identity":"7eb04fb2-b96f-456d-9efa-a4ac09ed7b35","added_by":"auto","created_at":"2025-10-06 12:05:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2076332,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram showing the expression heatmaps of key enzymes involved in starch and sucrose metabolism. Abbreviations: INV: beta-fructofuranosidase [EC:3.2.1.26]. SUS: sucrose synthase [EC:2.4.1.13]. ENPP1_3: ectonucleotide pyrophosphatase/phosphodiesterase family member 1/3 [EC:3.1.4.1 3.6.1.9]. GBE1: 1,4-alpha-glucan branching enzyme [EC:2.4.1.18]. glgC: glucose-1-phosphate adenylyltransferase [EC:2.7.7.27]. glgA: starch synthase [EC:2.4.1.21]. WAXY: granule-bound starch synthase [EC:2.4.1.242]. ISA: isoamylase [EC:3.2.1.68]. The value was normalized as Z-scores using FPKM.\u003c/p\u003e","description":"","filename":"Figure.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7635435/v1/44a006c6f03e4535faf075c2.png"},{"id":92858863,"identity":"9dcbaec1-f684-4aae-aabe-431ed59954ff","added_by":"auto","created_at":"2025-10-06 11:57:47","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":17254754,"visible":true,"origin":"","legend":"\u003cp\u003eGene set enrichment analysis (GSEA) analysis of cell wall biosynthesis. (A) and (B) GSEA plots for the cell wall organization and cell wall biological processes set using the DEGs from AR vs. CR (A) and GR vs. CR (B). The enrichment plots show the enrichment scores and NES values. Negative NES values indicate down-regulation of pathway expression, while positive values indicate up-regulation of that. The black line in the GSEA enrichment plots represents the set of genes examined. (C) Heatmap showing the expression profiles of DEGs related to cell wall. Abbreviations: CESA： cellulose synthase A. CSL: cellulose synthase-like protein. XTH: xyloglucan endotransglucosylase/hydrolase protein. IRX: probable beta-1,4-xylosyltransferase. PME: pectinesterase. PAE: pectin acetylesterase. GAUT: galacturonosyltransferase. PG: polygalacturonase. EXP: expansin. WAK: wall-associated receptor kinase. The value was normalized as Z-scores using FPKM.\u003c/p\u003e","description":"","filename":"Figure.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7635435/v1/c85baafb0aa767ab15ec6838.png"},{"id":92857998,"identity":"8520e967-aa3e-4330-baa1-bc59f5e49284","added_by":"auto","created_at":"2025-10-06 11:49:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":4580554,"visible":true,"origin":"","legend":"\u003cp\u003eWeighted gene co-expression network analysis (WGCNA). (A) Co-expression module clustering dendrogram. Based on the genetic system cluster tree drawn by TOM, different colors represent different modules. (B) The heatmap showed the correlations between modules and traits. The upper number in each cell refers to the correlation coefficient of each module in the trait, and the lower number is the corresponding P-value. Red represents high adjacency (positive correlation) and blue represents low adjacency (negative correlation). Visualization of key co-expression networks in darkred modules (C) and turquoise (D) modules. The color and size of the circles represent the degree of connectivity, and the yellow circles represent genes with antagonistic expression levels.\u003c/p\u003e","description":"","filename":"Figure.7.png","url":"https://assets-eu.researchsquare.com/files/rs-7635435/v1/f2e78599f14951b3e72c56a9.png"},{"id":92859466,"identity":"5a776e94-0f29-4e43-9eb1-98886c6c4566","added_by":"auto","created_at":"2025-10-06 12:05:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2818416,"visible":true,"origin":"","legend":"\u003cp\u003eThe qRT-PCR profiles of 12 genes were randomly selected. GAPDH was also used as the internal reference primer. The expression level was detected by 2 −ΔΔCt. With the expression level of the adventitious root control (CR) as the reference state, set as 1. The error bar is the standard error (SEM) of the average value, and * above indicates that there are significant differences in \u003cem\u003eR. glutinosa\u003c/em\u003e under different treatments (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Abbreviations: CYP707A: abscisic acid 8'-hydroxylase. EXP: expansin. PAE: pectin acetylesterase. XTH: xyloglucan endotransglucosylase/hydrolase protein. CSL: cellulose synthase-like protein. PER10: peroxidase 10. ERF017: ethylene-responsive transcription factor ERF017. ERF025: ethylene-responsive transcription factor ERF025. DREB1B: dehydration-responsive element-binding protein 1B. DREB1C: dehydration-responsive element-binding protein 1C. DREB1D: dehydration-responsive element-binding protein 1D.\u003c/p\u003e","description":"","filename":"Figure.8.png","url":"https://assets-eu.researchsquare.com/files/rs-7635435/v1/872526301433317e3dc1912d.png"},{"id":92858014,"identity":"1d4e9fb1-ed62-4e17-b04d-2221a4d57782","added_by":"auto","created_at":"2025-10-06 11:49:47","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2100859,"visible":true,"origin":"","legend":"\u003cp\u003eThe identified key regulators in ABA/GA antagonism are involved in ABA and GA metabolism. (A) Expression differences of DREBs, ERFs, and PERs genes between adventitious roots and fibrous roots. (B) Changes in the expression of ABA and GA related metabolic and responsive genes in tobacco leaves 48 hours after transient transformation with the RgERF017 and RgPER10 genes. The error bar is the SEM of the average value, and * above indicates that there are significant differences in \u003cem\u003eR. glutinosa\u003c/em\u003e under different treatments (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). Abbreviations: PER: peroxidase. ERF: ethylene-responsive transcription factor. DREB: dehydration-responsive element binding protein. CYP707A: abscisic acid 8'-hydroxylase. RD22: BURP domain protein RD22. GA20ox: gibberellin 20-oxidases. GA3ox: gibberellin 3-oxidase. GA2ox: gibberellin 2-oxidase.\u003c/p\u003e","description":"","filename":"Figure.9.png","url":"https://assets-eu.researchsquare.com/files/rs-7635435/v1/cda228fe1a9abc778813c8a4.png"},{"id":92858866,"identity":"c5b5ee6a-2434-4e64-98ba-418284b798c2","added_by":"auto","created_at":"2025-10-06 11:57:47","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":4872368,"visible":true,"origin":"","legend":"\u003cp\u003eConceptual model for the potential mechanism of ABA/GA antagonism in the initiation of \u003cem\u003eR. glutinosa\u003c/em\u003e tuberous roots. The arrows simply represent the directional flow of the process, without implying positive or negative effects.\u003c/p\u003e","description":"","filename":"Figure.10.png","url":"https://assets-eu.researchsquare.com/files/rs-7635435/v1/b61e610ce8d29abf7bda6ed5.png"},{"id":93259535,"identity":"598182ac-6915-43c1-93f0-d9acbc908a15","added_by":"auto","created_at":"2025-10-10 17:44:59","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":40046155,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7635435/v1/ac28d564-b21f-400f-a501-e247ffdd2f93.pdf"},{"id":92858857,"identity":"918980fa-ee8c-4362-82b6-240072a6a6c2","added_by":"auto","created_at":"2025-10-06 11:57:46","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":8461896,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Figure 1.\u003c/strong\u003e DEGs in the adventitious roots and mother roots of \u003cem\u003eR. glutinosa\u003c/em\u003e treated with ABA or GA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Figure 2.\u003c/strong\u003e Venn diagram of DEGs in AM vs. CM and AR vs. CR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Figure 3.\u003c/strong\u003e Venn diagram of DEGs in GM vs. CM and GR vs. CR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Figure 4.\u003c/strong\u003e Venn diagram of DEGs in ARM and GRM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Figure 5. \u003c/strong\u003e160 DEGs with opposite expression patterns between ARM and GRM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Figure 6. \u003c/strong\u003ePCR verification of RgERF017 and RgPER10 expression in tobacco leaves infiltrated with recombinant plasmids.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Figure 7. \u003c/strong\u003eNBT staining was performed to detect reactive oxygen species (ROS) accumulation in tobacco leaves.\u003c/p\u003e","description":"","filename":"SupplementaryTables.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7635435/v1/4832938f8221e3e69055f6c6.xlsx"},{"id":92858855,"identity":"7a7d5e93-1bc9-4479-b2ae-4b18564a5814","added_by":"auto","created_at":"2025-10-06 11:57:46","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1306504,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table 1\u003c/strong\u003e. Quality control of RNA-Seq data.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table 2\u003c/strong\u003e. Unigenes annotated statistical table.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table 3\u003c/strong\u003e. 7,416 DEGs in AR vs. CR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table 4\u003c/strong\u003e. 9,034 DEGs in GR vs. CR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table 5\u003c/strong\u003e. KEGG pathway analysis of DEGs from AR vs. CR and GR vs. CR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table 6\u003c/strong\u003e. GSEA analysis of KEGG pathways from AR vs. CR and GR vs. CR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table 7\u003c/strong\u003e. Unigenes involved in the starch and sucrose metabolism pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table 8\u003c/strong\u003e. GSEA analysis of Biological Processes from AR vs. CR and GR vs. CR.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table 9\u003c/strong\u003e. 9897 DEGs in ARM and GRM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table 10\u003c/strong\u003e. Unigenes from different modules in WGCNA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table 11\u003c/strong\u003e. Unigenes information of the darkred and turquoise modules in the STRING database.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table 12\u003c/strong\u003e. Expression and annotation of 160 genes in transcriptome analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplemental Table 13\u003c/strong\u003e. The primers used in this study.\u003c/p\u003e","description":"","filename":"SupplementalFigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-7635435/v1/b7b1edd7b1b59a02ddee24e6.docx"}],"financialInterests":"","formattedTitle":"Transcriptome profiling of Rehmannia glutinosa uncovers ABA/GA antagonism mechanisms in tuberous root initiation","fulltext":[{"header":"Key Message","content":"\u003cp\u003eDownstream responses to ABA/GA antagonism and novel key regulators in its network were identified, among which RgERF017 and RgPER10 induce ABA/GA balance shift through regulating ABA/GA metabolism.\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003e\u003cem\u003eRehmannia glutinosa\u003c/em\u003e is a perennial plant with significant medicinal and dietary properties. Its tuberous roots are widely used in traditional Chinese medicine due to richness of bioactive compounds. These bioactive compounds accumulate gradually along with the development of \u003cem\u003eR. glutinosa\u003c/em\u003e roots (Zhang et al., \u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Huang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). Therefore, \u003cem\u003eR. glutinosa\u003c/em\u003e tuberous root development decides the values of \u003cem\u003eR. glutinosa\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cem\u003eR. glutinosa\u003c/em\u003e tuberous roots originate from adventitious roots, undergoing five developmental stages: adventitious roots, fibrous roots, initiated tuberous roots, medium tuberous roots, and late tuberous roots (Li et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). \u003cem\u003eR. glutinosa\u003c/em\u003e is usually propagated vegetatively using the enlarged roots in the later developmental stage, which we call mother roots. Adventitious roots stem from mother roots. And the transition from adventitious roots to fibrous roots is prerequisite for tuberous root formation and is intricately regulated by plant hormones (Sun et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). Abscisic acid (ABA) and gibberellins (GA) are two essential plant hormones that play pivotal roles in plant growth and development (Liu \u0026amp; Hou \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Shu et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Zhang et al., \u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Numerous studies have demonstrated their antagonistic interactions in regulating tuberous root development (Liu et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Lin et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Generally, GA inhibits the initiation of tuberous root by promoting cell elongation and lignification (Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e), while ABA stimulates tuberous root formation through enhancing starch biosynthesis, cell division, and expansion (Li et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e) and thus antagonizes the effect of GA (Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). The antagonistic regulation of ABA and GA in tuberous root development depends on the dynamic change of the content ratio of ABA/GA. When the ratio of ABA/GA increases, it was beneficial to the initiation of tuberous roots (Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). The determination of endogenous hormones level in \u003cem\u003eR. glutinosa\u003c/em\u003e showed the increased ratio of ABA/GA in the early developmental stage, during which adventitious roots had initiated expansion and been transformed into fibrous roots (Sun et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). This demonstrated the antagonism of ABA/GA might be involved in the early stage of \u003cem\u003eR. glutinosa\u003c/em\u003e tuberous roots development.\u003c/p\u003e\u003cp\u003eThe antagonistic crosstalk between ABA and GA mainly involves two layers (Liu \u0026amp; Hou \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Firstly, their antagonistic effects are evident in the reciprocal regulation of the metabolism pathways (Liu \u0026amp; Hou \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Key enzymes involved in ABA metabolism include 9-\u003cem\u003ecis\u003c/em\u003e-epoxycarotenoid dioxygenase (NCED) and the abscisic acid 8'-hydroxylase (CYP707A). Gibberellin 20-oxidase (GA20ox), gibberellin 3-oxidase (GA3ox), and gibberellin 2-oxidase (GA2ox) are the classical enzymes of GA metabolism. The trade-off of the expression of these key enzymes was observed in diverse plant biological processes antagonistically mediated by ABA and GA (Oh et al., \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Oh et al., \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Seo et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Oh et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Gubler et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kashiwakura et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). The modulation triggers the shift of ratio of endogenous ABA/GA. The higher level of GA detected in ABA-deficient mutants (Seo et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) and the enhancement of \u003cem\u003eNCEDs\u003c/em\u003e expression and repression of \u003cem\u003eCYP707A\u003c/em\u003es observed in GA-deficient mutants (Oh et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) are two typical examples. Secondly, the crosstalk between ABA and GA signal transduction pathways further underpins the antagonistic effects of the two hormones. ABA and GA signaling intersect at different transcription factors (TFs), which are involved in both ABA and GA signaling and metabolism. Abscisic acid-insensitive 5 (ABI5) is a basic leucine zipper TF, which is viewed as the central factor in ABA/GA antagonism during seed germination (Li et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). It can control the expression of many ABA and GA signaling and metabolism genes (Li et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). The expression of \u003cem\u003eABI5\u003c/em\u003e can be regulated by an AP2-domain-containing TF, abscisic acid-insensitive 4 (ABI4) (Finkelstein et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Mu\u0026ntilde;iz Garc\u0026iacute;a et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), which plays a critical role in ABA/GA antagonism as well (Li et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e; Xian et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). It not only interacts with and stabilizes DELLA, the master negative regulator in GA signaling pathway, but also controls the expression of \u003cem\u003eDELLA\u003c/em\u003e, \u003cem\u003eGA2ox7\u003c/em\u003e, and \u003cem\u003eNCED6\u003c/em\u003e (Xian et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Overexpression of \u003cem\u003eABI4\u003c/em\u003e will result in a higher ratio of ABA/GA (Shu et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn \u003cem\u003eR. glutinosa\u003c/em\u003e, the growing ratio of ABA/GA during the transition from adventitious roots to fibrous and initiated tuberous roots is also mainly attributed to the improved expression of ABA biosynthetic and signaling genes and the reduced expression of \u003cem\u003eCYP707A\u003c/em\u003es (Li et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, the specific mechanisms underlying the dynamic balance of ABA/GA and ultimately the initiation of tuberous roots are still totally obscure so far in \u003cem\u003eR. glutinosa\u003c/em\u003e. To explore the mechanisms, we performed transcriptomic analysis of \u003cem\u003eR. glutinosa\u003c/em\u003e roots exposed to ABA and GA to elucidate the antagonistic regulatory network of ABA and GA in tuber development. Exogenous ABA or GA disrupted the original ABA/GA homeostasis. The one with a higher concentration activated its corresponding signaling pathway, while the other signaling was triggered soon as well due to the crosstalk of ABA/GA signaling. Some DREBs (dehydration-responsive element binding proteins), ERFs (ethylene-responsive transcription factors) and PERs (peroxidases) were identified as the key hub elements in the ABA/GA signaling crosstalk, which orchestrated ABA/GA antagonism by integrating ABA/GA metabolism. And according to the analysis, the expansion of adventitious roots arose from the promotion of starch and sucrose metabolism, cell wall biogenesis and modification, cell division and expansion. Inversely, the suppression of these biological processes plus the induction of lignification led to the inhibition of root enlargement. Taken together, these findings not only offer novel entry points and candidate molecules for in-depth investigations of the antagonism of ABA/GA, but aid in uncovering the fundamental principles of tuber development in \u003cem\u003eR. glutinosa\u003c/em\u003e.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePlant materials\u003c/h2\u003e\u003cp\u003e\u003cem\u003eR. glutinosa\u003c/em\u003e (cultivar Jinjiu) was grown in 11cm\u0026times;11cm pots filled with a mixture of vermiculite and humus soil (1:1.5, V: V) in climate chambers at 28\u0026deg;C, 70% relative humidity, with a photoperiod of 16 hours of light and 8 hours of darkness. \u003cem\u003eR. glutinosa\u003c/em\u003e 20-day-old seedlings were divided into three groups: control group (CK), ABA-treated group (ABA), and GA-treated group (GA). The seedlings in the three groups were irrigated continuously with distilled water, 40 \u0026micro;M ABA, and 150 \u0026micro;M GA for two weeks respectively. Then, both newly grown and mother roots were collected for transcriptome sequencing. All samples were frozen in liquid nitrogen immediately after collection and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for subsequent experiments.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eTranscriptome sequencing and data analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from samples using an RNA extraction kit. One \u0026micro;g of RNA per sample was utilized for sequencing library construction. The sequencing libraries were generated using the NEBNext\u0026reg; Ultra\u0026trade; RNA Library Preparation Kit (NEB, USA). Paired-end sequencing was then performed on the Illumina Hiseq 2000 platform. Q20, Q30, GC content, and sequence repetition levels were calculated and the clean reads with high quality were screened from raw reads through in-house perl scripts. Transcriptome assembly was accomplished using Trinity (Grabherr et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The data obtained were further processed with the BMKCloud online platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.biocloud.net\" target=\"_blank\"\u003ewww.biocloud.net\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.biocloud.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Based on all expressed genes, principal component analysis (PCA) was performed to elucidate the correlation between the samples. Pearson's Correlation Coefficient was employed to evaluate the correlation between the samples.\u003c/p\u003e\u003cp\u003eNR, Pfam, KOG/COG/eggNOG, Swiss-Prot, and KEGG were used to annotate the unigenes. Differential expression analysis was performed using the DESeq R package. A fold change (FC)\u0026thinsp;\u0026ge;\u0026thinsp;2 and a false discovery rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were used to identify differentially expressed genes (DEGs). Gene Ontology (GO) enrichment analysis of the DEGs was implemented by the topGO R packages based Kolmogorov\u0026ndash;Smirnov test. KOBAS software was used to detect the statistical enrichment of DEGs in the KEGG pathway (Kanehisa et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Xie et al., \u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Gene Set Enrichment Analysis (GSEA) analysis of all genes for their role in GO and KEGG-related biological processes are carried out using the BMKCloud web platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.biocloud.net\" target=\"_blank\"\u003ewww.biocloud.net\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.biocloud.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), with a \u003cem\u003ep\u003c/em\u003e-value less than 0.01.\u003c/p\u003e\n\u003ch3\u003eWeighted gene co-expression network analysis (WGCNA)\u003c/h3\u003e\n\u003cp\u003eWGCNA was conducted using the DEGs contained in ARM or GRM (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, S3). The threshold criteria included an average FPKM value of 1 for gene expression, an inter-module similarity threshold of 0.5, and a minimum of 30 genes per module. The protein-protein interaction (PPI) network of genes in key modules, derived from the WGCNA analysis, were analyzed using the STRING website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cn.string-db.org/\u003c/span\u003e\u003cspan address=\"https://cn.string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and visualized using Cytoscape (version 3.8.2) software for visualization.\u003c/p\u003e\n\u003ch3\u003eRT-qPCR analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from \u003cem\u003eR. glutinosa\u003c/em\u003e roots or tobacco leaves using RNAprep Pure Plant Kit (Vazyme Biotech, Nanjing, China). The first strand cDNA was synthesized with 1 \u0026micro;g total RNA using the first strand cDNA synthesis kit (Vazyme Biotech, Nanjing, China). And qRT-PCR was performed on the LightCycler\u0026reg;96 instrument system (Roche, Switzerland) using SYBR qPCR Mix (Vazyme Biotech, Nanjing, China). Specific primers were designed using Primer Premier 5.0, with \u003cem\u003eRgGAPDH\u003c/em\u003e or \u003cem\u003eNtActin\u003c/em\u003e as the internal reference primer. The expression level was analyzed by 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt. And the specific primers were shown in supplementary file 14.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTransient transformation of\u003c/b\u003e \u003cb\u003eRgERF017\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003eRgPER10\u003c/b\u003e \u003cb\u003ein tobacco\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe sequences of \u003cem\u003eRgERF017\u003c/em\u003e (\u003cem\u003eUnigene_033855\u003c/em\u003e) and \u003cem\u003eRgPER10\u003c/em\u003e (\u003cem\u003eUnigene_155852\u003c/em\u003e) were cloned into the pMD19-T vector (TaKaRa) and verified by colony PCR using M13-F/R primers followed by sequencing. Correctly sequenced CDS fragments were cloned into the Super1300-GFP expression vector using the ClonExpress II One Step Cloning Kit (Vazyme). The recombinant constructs and the empty Super1300-GFP vector were subsequently transformed into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e strain GV3101 competent cells. One-month-old tobacco was used for agroinfiltration, with the left side of each leaf infiltrated with Agrobacterium harboring the empty Super1300-GFP vector and the right side infiltrated with Agrobacterium containing the recombinant construct. After 48 h of dark incubation, infiltrated leaf tissues were collected for other analyses.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eMorphological responses of\u003c/b\u003e \u003cb\u003eR. glutinosa\u003c/b\u003e \u003cb\u003eadventitious roots to exogenous ABA and GA\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe development of \u003cem\u003eR. glutinosa\u003c/em\u003e tuberous root begins with the swelling of adventitious roots. During this process, the antagonistic regulation of ABA and GA plays a critical role. To assess the effects of ABA and GA on the morphological changes of \u003cem\u003eR. glutinosa\u003c/em\u003e adventitious roots, 20-day-old seedlings were continuously irrigated with distilled water, 40 \u0026micro;M ABA, and 150 \u0026micro;M GA respectively for two weeks. In the control group, adventitious roots showed no swelling. However, ABA treatment led to a significant enlargement of these roots. In contrast, GA treatment did not induce swelling but resulted in a reduction in root length and a significant decrease in the number of adventitious roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). No significant differences were observed in the mother roots across the three treatments, nor were any noted before or after treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). These results suggest that ABA promotes the development of \u003cem\u003eR. glutinosa\u003c/em\u003e adventitious roots, whereas GA exerts an inhibitory effect. The two hormones exhibit antagonism in regulating the early transition of adventitious roots, but no effect on the morphology of mother roots.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eQuality control of RNA-Seq data and function annotations\u003c/h2\u003e\u003cp\u003eTo investigate the mechanisms underlying the antagonistic effects of ABA and GA on \u003cem\u003eR. glutinosa\u003c/em\u003e tuberous root initiation, RNA-seq technology was employed to analyze both adventitious roots (CR: control roots, AR: ABA-treated roots and GR: GA-treated roots) and mother roots (CM: control mother roots, AM: ABA-treated mother roots and GM: GA-treated mother roots). Twelve cDNA libraries were constructed for Illumina sequencing, and the effects of exogenous ABA and GA on the development of \u003cem\u003eR. glutinosa\u003c/em\u003e adventitious root were comprehensively analyzed at the transcriptional level. As a result, a total of 258,732,013 clean reads were obtained. The average content of GC was 44.74%. The average values of Q20 and Q30 were 98.14 and 94.60, respectively (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The PCA results reveal distinct differences among the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). And the roots treated with GA were notably farther from the CK groups than those treated with ABA, especially for adventitious roots. This suggests that the GA treatment groups exhibit greater variability, and the endogenous ABA level should increase in the early development stage under normal conditions. The inference agrees with the result of Sun et al. (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), who determined the level of endogenous hormones during \u003cem\u003eR. glutinosa\u003c/em\u003e tuberous root development. The Pearson correlation coefficient among adventitious root replicates ranged from 0.914 to 0.991, while those across various treatments varied from 0.332 to 0.620. The correlations among mother root replicates ranged from 0.844 to 0.955, whereas those among different treatments ranged from 0.592 to 0.870. The higher values of different treatments in mother root groups indicated that mother root was insensitive to plant hormones (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eBased on the NR, Swiss-Prot, COG, KOG, eggNOG, GO, KEGG, and Pfam databases, 30,432 unigenes were annotated totally. And 28,332 (93.10%), 21,203 (69.67%), 9,451 (31.06%), 17,034 (56.00%), 24,881 (81.76%), 24,432 (80.29%), 20,435 (67.15%), and 23,137 (76.03%) unigenes were annotated across these eight databases, respectively. Furthermore, 6,033 (19.82%) unigenes were found to be homologous across all eight databases (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDEGs involved in ABA or GA metabolism and signal transduction\u003c/h3\u003e\n\u003cp\u003eFC\u0026thinsp;\u0026ge;\u0026thinsp;2 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were used to identify DEGs. A total of 12608 DEGs were identified from adventitious roots in response to exogenous ABA and GA. The number of DEGs both in AR vs. CR and GR vs. CR was 7,416 (3,293 up-regulated and 4,123 down-regulated) (Table S3; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), and 9,034 (3,492 up-regulated and 5,542 down-regulated) (Table S4; Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). The results indicated a higher number of DEGs in response to GA treatment, with a greater proportion of genes being down-regulated under ABA or GA treatments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAmong these genes, 55 DEGs related to ABA metabolism and signal transduction pathways and 24 DEGs involved in GA metabolism and signal transduction pathways were chosen for further analysis (Figure. 3B C). The expression levels of ABA biosynthesis genes \u003cem\u003ezeaxanthin epoxidase\u003c/em\u003es (\u003cem\u003eZEPs\u003c/em\u003e) and \u003cem\u003eNCEDs\u003c/em\u003e were increased following ABA treatment. However, the expression of another ABA biosynthesis gene \u003cem\u003eabscisic acid deficient 2\u003c/em\u003e (\u003cem\u003eABA2\u003c/em\u003e) was reduced after ABA treatment, but up-regulated by exogenous GA. Moreover, an overwhelmingly majority of ABA catabolism-associated genes \u003cem\u003eCYP707A\u003c/em\u003es were up-regulated post-ABA treatment, while down-regulated under GA treatment. XERICOs (XER\u003cem\u003es\u003c/em\u003e) act as inducers of ABA biosynthesis (Ko et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). By and large, both ABA and GA stimulated the expression of \u003cem\u003eXERs\u003c/em\u003e, especially the latter. The expression of ABA signaling negative regulators \u003cem\u003ePP2Cs\u003c/em\u003e (clade A protein phosphatases of type 2Cs) were elevated remarkably in AR group. Under GA treatment, only a small subset of \u003cem\u003ePP2C\u003c/em\u003es were up-regulated. In contrast, most \u003cem\u003epyrabactin resistance-like protein\u003c/em\u003es (\u003cem\u003ePYL\u003c/em\u003es), which encode ABA receptors and inhibit the effect of \u003cem\u003ePP2Cs\u003c/em\u003e (Zhao et al., \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), were down-regulated under ABA treatment. And a subset of \u003cem\u003ePYL\u003c/em\u003es was significantly up-regulated under GA treatment. Both ABFs and SnRK2 play positive roles in ABA signaling process (Zhao et al., \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Collin et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Ali et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). They also exhibited significantly up-regulated expressions under both ABA and GA treatment. Copalyl diphosphate synthase (CPS) is involved in GA synthesis (Bour\u0026eacute; \u0026amp; Arnaud \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). It was down-regulated following GA treatment, but up-regulated under ABA treatment. Ent-Kaurenes (KS), ent-Kaurene oxidase (KO) and ent-Kaurenoic acid oxidase (KAO) are entailed in the production of the common GA precursor GA\u003csub\u003e12\u003c/sub\u003e (Bour\u0026eacute; \u0026amp; Arnaud \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). However, they showed different expression patterns. \u003cem\u003eKSs\u003c/em\u003e increased after GA treatment but decreased under ABA treatment. \u003cem\u003eKO\u003c/em\u003e showed minimal changes following GA treatment but was up-regulated under ABA treatment. \u003cem\u003eKAO\u003c/em\u003es expression was up-regulated under both GA and ABA treatments, with a more pronounced increase observed under ABA treatment. The biosynthesis of bioactive GAs requires \u003cem\u003eGA20ox\u003c/em\u003es and \u003cem\u003eGA3ox\u003c/em\u003es (Shani et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), which were down-regulated after GA treatment (Figure. 3C). \u003cem\u003eGA2oxs\u003c/em\u003e, as GA-deactivating enzymes, exhibited a significant down-regulation following GA treatment as well (Figure. 3C). \u003cem\u003eDELLA\u003c/em\u003e/\u003cem\u003eGAI\u003c/em\u003e, the key negative regulator in the GA signaling pathway, was predominantly up-regulated following GA treatment, while it was down-regulated under ABA treatment. And the expression of GA receptor \u003cem\u003eGibberellin-Insensitive Dwarf 1\u003c/em\u003e (\u003cem\u003eGID1\u003c/em\u003e) was suppressed by both ABA and GA. Although these genes displayed differential expression patterns in the presence of exogenous ABA and GA, it is still noticeable that the exogenous ABA or GA prevented the biosynthesis and signaling of itself and facilitated that of antagonist. Consequently, the antagonistic regulation of ABA/GA balance was actuated by excess ABA or GA. And this antagonism may depend more on ABA metabolism and signal transduction pathways according to Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and C.\u003c/p\u003e\n\u003ch3\u003eKEGG pathway enrichment analysis\u003c/h3\u003e\n\u003cp\u003eThe KEGG enrichment analysis of DEGs revealed the top 10 pathways based on their \u003cem\u003eq\u003c/em\u003e values (Table S5). The metabolic pathways enriched in both AR and GR included the \"Plant MAPK signaling pathway\" (Ko04016), \"Plant hormone signal transduction\" (Ko04075), \"Phenylpropanoid biosynthesis\" (Ko00940), \"Plant-pathogen interaction\" (Ko04626), and \"Isoquinoline alkaloid biosynthesis\" (Ko00950) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B; Table S5). Notably, among the top five pathways, \"Starch and sucrose metabolism\" (Ko00500) was significantly enriched in AR but not in GR, with a total of 140 DEGs involved in this pathway. To further examine the modulation of the starch and sucrose metabolism pathway under ABA or GA treatment, a GSEA was conducted. The GSEA result for AR indicated a normalized enrichment score (NES) of 1.413, with 122 core genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC; Table S6). In contrast, GSEA for GR revealed an NES of -1.291, with 82 core genes and a relatively higher \u003cem\u003ep\u003c/em\u003e-value (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD; Table S6). The result suggested that the starch and sucrose metabolism pathway was regulated antagonistically by ABA and GA. It was up-regulated significantly in AR and down-regulated slightly in GR.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eStarch and sucrose metabolism response to ABA and GA\u003c/h2\u003e\u003cp\u003eTo further elucidate the regulatory roles of ABA and GA in starch and sucrose metabolism, we examined the expression profiles of relevant DEGs within this pathway. It was evident that the metabolism pathway from sucrose to starch was up-regulated by ABA, as all enzymes involved were virtually accumulated transcriptionally under ABA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e; Table S7). These enzymes included sucrose synthase (SUS), ectonucleotide pyrophosphatase/phosphodiesterase family member 1/3 (ENPP1_3), glucose-1-phosphate adenylyltransferase (glgC), granule-bound starch synthase (WAXY), starch synthase (glgA), 1,4-alpha-glucan branching enzyme (GBE1), and isoamylase (ISA). However, GA did not negatively regulate these enzymes. \u003cem\u003eGBE1\u003c/em\u003e, \u003cem\u003eWAXY\u003c/em\u003e, and \u003cem\u003eglgC\u003c/em\u003e showed minimal changes in GR. And others were up-regulated by GR. This demonstrated that the antagonism of ABA/GA was not reflected in starch biosynthesis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eCell wall biogenesis and organization\u003c/h2\u003e\u003cp\u003eGO analysis was performed using GSEA with a \u003cem\u003ep\u003c/em\u003e-value threshold of 0.01. The result showed that the top 5 biological processes ranked by \u003cem\u003ep\u003c/em\u003e-value in AR were activated (Table S8). However, the top 5 biological processes in GR were repressed, including carbohydrate metabolic process, defense response, response to biotic stimulus, lipid metabolic process, and cell wall organization (Table S8). And given the aforesaid analysis on cellulose metabolism, we paid more attention to the biological processes associated with cell wall. It was found that the GO terms associated with the cell wall exhibited contrasting patterns under GA and ABA treatments. In AR, significant enrichment was observed for the gene sets of \"cell wall biogenesis\" (GO:0042546) and \"cell wall organization\" (GO:0071555). The NES for \"cell wall biogenesis\" and \"cell wall organization\" in AR were 1.562 and 1.678, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA; Table S8). The two biological processes were primarily up-regulated in AR. More biological processes related to cell wall were enriched in GR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB; Table S8), and they were all suppressed. In sum, ABA and GA showed antagonism in the biological processes of \"cell wall biogenesis\" and \"cell wall organization\".\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further explore the effects of ABA and GA on cell wall-related genes, a heatmap of the DEGs was plotted (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Among the DEGs, \u003cem\u003ecellulose synthase\u003c/em\u003es (\u003cem\u003eCESA\u003c/em\u003es) involved in cellulose synthesis were significantly down-regulated by GA, while their expression in AR showed minimal change. Another cellulose-related gene \u003cem\u003ecellulose synthase-like protein\u003c/em\u003es (\u003cem\u003eCSLs\u003c/em\u003e) showed the different expression profile in the presence of ABA and GA, most elevated in AR but reduced in GR. Xyloglucan endotransglucosylase/hydrolase proteins (XTHs) and robable beta-1,4-xylosyltransferase (IRX) are associated with hemicellulose synthesis. \u003cem\u003eXTHs\u003c/em\u003e were antagonistically regulated by ABA and GA, whereas probable \u003cem\u003ebeta-1,4-xylosyltransferase\u003c/em\u003e (\u003cem\u003eIRX\u003c/em\u003e) genes were up-regulated in response to both ABA and GA. Genes related to pectin synthesis and modification, including \u003cem\u003epolygalacturonase\u003c/em\u003es (\u003cem\u003ePG\u003c/em\u003es), \u003cem\u003epectin methylesterases\u003c/em\u003e (\u003cem\u003ePME\u003c/em\u003es), \u003cem\u003epectin acetylesterase\u003c/em\u003es (\u003cem\u003ePAE\u003c/em\u003es), and \u003cem\u003egalacturonosyltransferase\u003c/em\u003es (\u003cem\u003eGAUT\u003c/em\u003es), were predominantly up-regulated in AR and down-regulated in GR. Additionally, the expression patterns of another two important genes affecting the cell wall structure were also depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Expansins (\u003cem\u003eEXPs\u003c/em\u003e) were up-regulated in AR but showed little change in GR. Wall-associated receptor kinases (WAKs) belong to receptor-like protein family. They are cross-linked pectins and involved in cell wall integrity signaling pathway. The heatmap showed that ABA predominantly down-regulated \u003cem\u003eWAKs\u003c/em\u003e and most \u003cem\u003eWAK\u003c/em\u003es in GR showed minimal change with only a few being down-regulated. Overall, the DEGs directly implicated in cell wall biosynthesis, cell wall remodeling and modification were up-regulated in AR and down-regulated in GR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003e\u003cb\u003eKey genes for tuberous roots initiation antagonistically regulated by ABA/GA identified using WGCNA and PPI network\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDespite the antagonistic regulation of ABA and GA on the initiation of \u003cem\u003eR. glutinosa\u003c/em\u003e tuberous root, neither ABA nor GA induced any morphological alterations in mother roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The insensitivity of \u003cem\u003eR. glutinosa\u003c/em\u003e mother roots to ABA and GA were also manifested in Pearson correlation coefficient as well (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). This implied that the variation at transcriptomics level in mother roots was inadequate for the root enlargement initiated by ABA/GA, and the DEGs in AM vs. CM or GM vs. CM did not contain the key genes for the induction of root swelling regulated by ABA/GA. Therefore, the DEGs in the overlapping regions of AM vs. CM and AR vs. CR were excluded and the rest of DEGs in AR vs. CR were referred to as ARM (Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The DEGs in GR vs. CR were processed similarly and designated as GRM (Figure S3).\u003c/p\u003e\u003cp\u003eTo investigate the regulatory effects of ABA and GA on tuberous root initiation and identify the related key genes, we audaciously performed WGCNA on the 9,897 DEGs in ARM or GRM (Table S9; Figure S4). This analysis resulted in the identification of 11 distinct modules (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA; Table S10). The module-sample correlation analysis was then conducted to identify specific modules that strongly correlate with ABA and GA treatment. The darkred module containing 348 genes and the turquoise module containing 1055 genes were finally highlighted in bright red (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB; Table S10). In the darkred module, \u003cem\u003eNCED3\u003c/em\u003e was identified as a hub gene in the PPI network, along with other highly connected genes such as \u003cem\u003eDREB1B\u003c/em\u003e, \u003cem\u003ePP2CA\u003c/em\u003e, \u003cem\u003eDREB1C\u003c/em\u003e, \u003cem\u003eRD22\u003c/em\u003e, \u003cem\u003eprotein phosphatase 2C 7\u003c/em\u003e (\u003cem\u003eHAB2\u003c/em\u003e), and \u003cem\u003eXTH22\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Similarly, the turquoise module PPI network highlighted \u003cem\u003eflavone 3'-O-methyltransferase 1\u003c/em\u003e (\u003cem\u003eOMT1\u003c/em\u003e) as a hub gene, with other highly connected genes, including \u003cem\u003ecinnamyl alcohol dehydrogenase 5\u003c/em\u003e (\u003cem\u003eCAD5\u003c/em\u003e), \u003cem\u003ealdehyde dehydrogenase family 2 member C4\u003c/em\u003e (\u003cem\u003eALDH2C4\u003c/em\u003e), probable \u003cem\u003enicotianamine synthase 4\u003c/em\u003e (\u003cem\u003eNAS4\u003c/em\u003e), \u003cem\u003eglycerol kinase\u003c/em\u003e (\u003cem\u003eGLPK\u003c/em\u003e), \u003cem\u003eprotein PHR1-LIKE 1\u003c/em\u003e (\u003cem\u003ePHL1\u003c/em\u003e), and \u003cem\u003eserine-glyoxylate aminotransferase\u003c/em\u003e (\u003cem\u003eAGT1\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD; Table S11). Based on FPKM values, 160 DEGs with opposite expression patterns in ABA and GA treatments were selected from GRM and ARM (Table S12; Figure S5). Among these, 95 DEGs showed up-regulated expression in AR but down-regulated expression in GR, while 65 DEGs exhibited the opposite pattern. DEGs with contrasting expression patterns are denoted in yellow in the PPI interaction map. In the PPI network analysis, seven genes including \u003cem\u003eDREB1B\u003c/em\u003e, \u003cem\u003eDREB1C\u003c/em\u003e, \u003cem\u003eDREB1D\u003c/em\u003e, \u003cem\u003eERF025\u003c/em\u003e, \u003cem\u003eERF017\u003c/em\u003e, \u003cem\u003eROH1\u003c/em\u003e (\u003cem\u003eF6A14.15\u003c/em\u003e), and \u003cem\u003eprotein phosphatase 2C 63\u003c/em\u003e (\u003cem\u003eF17I5.110\u003c/em\u003e) displayed reduced expression patterns in GR (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Conversely, in the PPI network of GR, 11 genes, including probable \u003cem\u003eubiquitin-conjugating enzyme E2 24\u003c/em\u003e (\u003cem\u003eUBC24\u003c/em\u003e), \u003cem\u003ePER47\u003c/em\u003e, \u003cem\u003ePER10\u003c/em\u003e, \u003cem\u003ePER59\u003c/em\u003e, \u003cem\u003eNAS4\u003c/em\u003e, probable \u003cem\u003eserine/threonine-protein kinase PBL15\u003c/em\u003e (\u003cem\u003ePBL15\u003c/em\u003e), \u003cem\u003etype IV inositol polyphosphate 5-phosphatase 9\u003c/em\u003e (\u003cem\u003eIP5P9\u003c/em\u003e), \u003cem\u003ephosphatidylinositol/phosphatidylcholine transfer protein SFH2\u003c/em\u003e (\u003cem\u003eSFH2\u003c/em\u003e), \u003cem\u003eWAT1-related protein At1g43650\u003c/em\u003e (\u003cem\u003eUMAMIT22\u003c/em\u003e), and \u003cem\u003eWAT1-related protein At5g07050\u003c/em\u003e (\u003cem\u003eUMAMIT9\u003c/em\u003e), exhibited decreased expression patterns in AR (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). These genes labeled yellow may play crucial roles in the antagonism of ABA/GA and affect the expression of the forementioned hub genes potentially associated with tuberous root initiation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eThe gene expression pattern was verified by qRT-PCR\u003c/h2\u003e\u003cp\u003eTo confirm the RNA-Seq precision, 12 genes were selected for qRT-PCR assessment. The results revealed a resemblance between the expression patterns of these 12 genes tested by qRT-PCR and those detected by RNA-Seq, thus substantiating the reliability of RNA-Seq (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e; Table S13).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTransient expression\u003c/b\u003e \u003cb\u003eof RgERF017\u003c/b\u003e \u003cb\u003eor\u003c/b\u003e \u003cb\u003eRgPER10\u003c/b\u003e \u003cb\u003ein tobacco affected the expression of ABA and GA metabolism genes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo initially explore their roles in ABA/GA antagonism, the identified hub genes, \u003cem\u003eDREB\u003c/em\u003es, \u003cem\u003eERF\u003c/em\u003es and \u003cem\u003ePER\u003c/em\u003es, were selected. We examined the expression of these genes during \u003cem\u003eR. glutinosa\u003c/em\u003e early development stage. The elevated expression was detected in fibrous roots, with \u003cem\u003eERFs and PERs\u003c/em\u003e showing particularly notable increase (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). Two representative candidates with significant changes, \u003cem\u003eRgERF017\u003c/em\u003e and \u003cem\u003eRgPER10\u003c/em\u003e, were then cloned and used for the transient transformation analysis (Figure S6). The significant transcript accumulation of key ABA biosynthetic genes NCEDs and ABA-responsive genes RD22s and the downregulation of ABA catabolic genes CYP707As were detected in the tobacco leaves transiently expressing \u003cem\u003eRgERF017\u003c/em\u003e or \u003cem\u003eRgPER10\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). This revealed an elevation in endogenous ABA levels in tobacco leaves. GA biosynthetic genes GA20oxs and GA3ox were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB), but GA catabolic genes GA2oxs showed different responses. The transient expression of \u003cem\u003eRgPER10\u003c/em\u003e caused the upregulation of \u003cem\u003eGA2ox\u003c/em\u003es, suggesting a reduction in GA levels. However, \u003cem\u003eRgERF017\u003c/em\u003e led to the reduced accumulation of \u003cem\u003eGA2ox\u003c/em\u003es transcripts. These results suggested that \u003cem\u003eRgERF017\u003c/em\u003e and \u003cem\u003eRgPER10\u003c/em\u003e are involved in ABA/GA antagonism through the regulation of ABA and GA metabolism genes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cb\u003eABA and GA antagonistically regulate the initiation of\u003c/b\u003e \u003cb\u003eR. glutinosa\u003c/b\u003e \u003cb\u003eroot expansion\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe antagonistic actions of ABA and GA in tuberization were first found in potato (Vreugdenhil et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The ABA-deficient potato lines can recover tuberization with the inhibition of GA biosynthesis (Vreugdenhil et al., \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). Endogenous GA level was negatively correlated with tuber formation and exogenous ABA stimulated tuberization in potato (Xu et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). It was thus deemed that the positive regulatory effect of ABA on potato tuberization was achieved by counteracting GA (Xu et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). Afterwards, the roles of ABA or GA were identified in various stem/root tubers, including yam stem tuber and bulbil (Kim et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), carrot, turnip and radish taproot (Wang et al., \u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Meng et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), \u003cem\u003eGladiolus hybridus\u003c/em\u003e corm (Li et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e) and \u003cem\u003ePseudostellaria heterophylla\u003c/em\u003e tuberous root (Wang et al., \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e2024a\u003c/span\u003e). Except for yam tuber and bulbil, ABA stimulates, and GA represses the initiation of tuberization in general. To ascertain the roles of ABA and GA in \u003cem\u003eR. glutinosa\u003c/em\u003e tuberization, we treated \u003cem\u003eR. glutinosa\u003c/em\u003e seedlings with ABA and GA respectively. Phenotypically, exogenous ABA significantly promoted root expansion, whereas GA treatment inhibited tuberization and root growth, resulting in reduced root length and quantity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Therefore, it is evident that ABA and GA antagonistically regulate the initiation of \u003cem\u003eR. glutinosa\u003c/em\u003e root expansion, with ABA acting as a positive regulator.\u003c/p\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eThe feedback regulation on metabolism involves the antagonistic action of ABA and GA\u003c/h2\u003e\u003cp\u003eExcess phytohormones tips the balance between ABA and GA. It actuates the mediation of ABA/GA homeostasis, which involves ABA and GA metabolism and signaling pathways. The endogenous ABA levels are directly associated with both ABA synthesis and catabolism. The ABA biosynthetic genes, \u003cem\u003eZEP\u003c/em\u003es and \u003cem\u003eNCED\u003c/em\u003es, were up-regulated following ABA treatment, while \u003cem\u003eABA2\u003c/em\u003e was repressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). ABA2 functions in plastids after ZEP and NCED (Finkelstein, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The suppression of \u003cem\u003eABA2\u003c/em\u003e prevented ABA biosynthesis. However, ABA catabolism was promoted, because most catabolic genes \u003cem\u003eCYP707A\u003c/em\u003es were upregulated by exogenous ABA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). And in terms of ABA accumulation, catabolism outweighs biosynthesis (Finkelstein, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), so the expression pattens of ABA metabolic genes demonstrated that the content of ABA may decrease after the sharp increase caused by ABA treatment. ABA metabolism is tightly regulated through the feedback signaling mechanisms (Finkelstein, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), in which ABFs play a vital role (Wang et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Collin et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). ABFs are activated by the canonical ABA signaling pathway at both transcriptional and protein level in the presence of ABA (Lopez-Molina et al., \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The active ABFs then induce the expression of ABA-responsive genes, including \u003cem\u003eNCED\u003c/em\u003es, and ABA signaling negative regulator \u003cem\u003ePP2C\u003c/em\u003es (Zong et al., \u003cspan citationid=\"CR105\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Collin et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The elevated expression of \u003cem\u003eABF\u003c/em\u003es, \u003cem\u003eNCED\u003c/em\u003es and \u003cem\u003ePP2C\u003c/em\u003es was detected in our study and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB. PYLs usually count as ABA receptors. They are also the targets of ABFs (Zhao et al., \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), but ABFs may mainly contribute to the maintenance of \u003cem\u003ePYL\u003c/em\u003es expression (Zhao et al., \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). PYLs possess unique functions in various biological processes, which implies their diverse expression patterns (Zhao et al., \u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Santiago et al. (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) and Zhao et al. (\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) observed that some \u003cem\u003ePYL\u003c/em\u003es were up-regulated by exogenous ABA but some were depressed or exhibited no obvious response. In \u003cem\u003eR. glutinosa\u003c/em\u003e, most \u003cem\u003ePYL\u003c/em\u003es showed decreased expression but some kept no change in post-ABA situation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The expression of \u003cem\u003eABF\u003c/em\u003es and the other two core ABA signaling components, \u003cem\u003ePP2C\u003c/em\u003es and \u003cem\u003ePYL\u003c/em\u003es, gave a hint that the feedback regulation of ABA pathway was already activated and the core ABA signaling was suppressed.\u003c/p\u003e\u003cp\u003eLike ABA, GA synthesis and catabolism directly dictate endogenous GA levels, which are controlled by feedback loop (Bour\u0026eacute; \u0026amp; Arnaud \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). CPS, KS KO, and KAO are the essential enzymes for the biosynthesis of bioactive GA precursor GA\u003csub\u003e12\u003c/sub\u003e (Shani et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). After GA treatment, these enzymes other than KS were all repressed at transcriptional level in \u003cem\u003eR. glutinosa\u003c/em\u003e roots (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Although some of the genes responded similarly to GA application in other plants and were thought to be involved in the feedback regulation of GA (Zhang et al., \u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Guan et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), the unchanged bioactive GA in Arabidopsis overexpressing \u003cem\u003eAtCPS\u003c/em\u003e and \u003cem\u003eAtKS\u003c/em\u003e (Fleet et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2003\u003c/span\u003e) indicated that the enzymes may be incapable of influencing bioactive GA content straightly. The key enzymes remarkably affecting the biologically active GAs are 2-oxoglutarate-dependent dioxygenases (2-ODDs), namely the biosynthetic GA20ox, GA3ox, and catabolic GA2ox (Bour\u0026eacute; \u0026amp; Arnaud \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shani et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). There are numerous reports of repressed \u003cem\u003eGA20ox\u003c/em\u003e and \u003cem\u003eGA3ox\u003c/em\u003e and enhanced \u003cem\u003eGA2ox\u003c/em\u003e by exogenous GA (Hern\u0026aacute;ndez-Garc\u0026iacute;a et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Bour\u0026eacute; \u0026amp; Arnaud \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Shani et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). However, not only the biosynthetic \u003cem\u003eGA20ox\u003c/em\u003es and \u003cem\u003eGA3ox\u003c/em\u003e but the majority of \u003cem\u003eGA2ox\u003c/em\u003es as well were downregulated by GA in \u003cem\u003eR. glutinosa\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The inhibited expression of \u003cem\u003eGA2ox\u003c/em\u003es was also reported in GA-treated yam, which actually took place 30 days after GA treatment (Zhou et al., \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). So, it is speculated that the feedback regulation started by GA treatment may move into the later stage. The feedback responses on GA metabolism modulated by GA signaling, in which DELLA proteins function as critical repressors (Shani et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). DELLA proteins are degraded after GA binding to its receptor GID1(Shani et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The GA biosynthetic genes \u003cem\u003eGA20ox2\u003c/em\u003e, \u003cem\u003eGA3ox1\u003c/em\u003e and GA receptor \u003cem\u003eGID1\u003c/em\u003e are early DELLA-responsive genes (Zentella et al., \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Their transcript levels are reduced after GA treatment, which resulted from the reduced accumulation of DELLA proteins (Silverstone et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Griffiths et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Zentella et al., \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Weston et al., \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The expression patterns of these genes in \u003cem\u003eR. glutinosa\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC) were consistent with the finding and implied the decreased DELLA at protein level. Nevertheless, long-term treatment of GA led to the increased expression of \u003cem\u003eDELLA\u003c/em\u003e at transcriptional level in tubers (Zhou et al., \u003cspan citationid=\"CR104\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Meng et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). This result was also detected in \u003cem\u003eR. glutinosa\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). The up-regulated \u003cem\u003eDELLA\u003c/em\u003e is not indicative of the accumulation of DELLA proteins. The accumulated transcript of \u003cem\u003eDELLA\u003c/em\u003e may serve as a reservoir for the timely up-regulation of DELLA proteins, which indicated that GA signaling would be repressed.\u003c/p\u003e\u003cp\u003eABA/GA homeostasis is not merely regulated by individual ABA or GA signaling pathways, but by the interplay between the two signaling pathways (Bour\u0026eacute; \u0026amp; Arnaud \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). DELLAs and ABFs are their respective key factors and serve as the crosstalk interface as well. The ABA/GA antagonism in seed germination and potato tuberization correlates with DELLAs and ABFs (Mu\u0026ntilde;iz Garc\u0026iacute;a et al., \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). However, \u003cem\u003eDELLA\u003c/em\u003es were antagonistically regulated by exogenous ABA and GA, while both ABA and GA promoted the expression of \u003cem\u003eABF\u003c/em\u003es in \u003cem\u003eR. glutinosa\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, C). It is notable that the degree of up-regulated expression of \u003cem\u003eABF\u003c/em\u003es varies among individuals following ABA or GA treatment. This may lead to an antagonistic effect on the expression of ABF targets, \u003cem\u003eNCED\u003c/em\u003es and \u003cem\u003ePYL\u003c/em\u003es (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). ABI5, a member of ABFs, is recruited and activated by DELLA (Lim et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). It also negatively controls the expression of \u003cem\u003eGA3ox\u003c/em\u003e (Lim et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), to which the observation in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC conformed. Furthermore, XER is another crosstalk point. It is conducive to the accumulation of ABA through transcriptional upregulation of \u003cem\u003eNCED\u003c/em\u003e (Ko et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). And it was also identified as one DELLA-responsive gene (Zentella et al., \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The expression of \u003cem\u003eXER\u003c/em\u003e kept in line with \u003cem\u003eDELLA\u003c/em\u003es, but \u003cem\u003eNCED\u003c/em\u003es displayed the opposite expression patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). It is because NCEDs are modulated by multiple factors, such as ABI5 (Lim et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and ABI4 (Shu et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), besides XER. In Arabidopsis, RGL2 (a DELLA protein) stimulates ABI5 activity and ABA formation through the XER protein (Piskurewicz et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Therefore, the expression of \u003cem\u003eDELLA\u003c/em\u003es, \u003cem\u003eABF\u003c/em\u003es and \u003cem\u003eXER\u003c/em\u003es after GA treatment suggested that ABA levels would be elevated. This hypothesis about ABA levels can be reinforced by the suppressed expression of \u003cem\u003eCYP707A\u003c/em\u003es under GA treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003eOverall, most genes presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and C produced antagonistic responses to ABA and GA treatment. And the expression of these genes denotes the declined ABA levels post-ABA treatment and elevated ABA levels after GA application according to the discussion above. Compared to ABA, the trend of GA metabolism is not evident following exposure to the two hormones. But as mentioned above, GA signaling would be suppressed following GA treatment. Shu et al. (\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) proposed a model of the antagonism of ABA and GA, which suggests that ABA and GA tend to exhibit a negative correlation (Shu et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This view is drawn from the endogenous concentrations of ABA and GA, while the exogenous ABA and GA appear to elicit different responses in our study. The persistent excessive exogenous ABA or GA suppressed respective biosynthesis and signaling and stimulated reciprocally through feedback regulation and crosstalk. These responses reset ABA/GA homeostasis, which remains true to the core tenets of the model. Moreover, given more antagonistic expression patterns present in key genes related to ABA metabolism and signaling, it is potential that the ABA/GA balance mainly depends on ABA metabolism. Alternatively, it is also plausible that feedback responses implicating ABA levels persists for a longer duration, or that the feedback associated with GA metabolism is more sensitive to exogenous phytohormones. In sum, ABA/GA homeostasis depends on the antagonistic action of ABA and GA on their metabolism and signaling crosstalk.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThe potential key regulators in the antagonistic action of ABA and GA during\u003c/b\u003e \u003cb\u003eR. glutinosa\u003c/b\u003e \u003cb\u003etuberous root initiation\u003c/b\u003e\u003c/p\u003e\u003cp\u003eExposure to ABA or GA leads to thousands of gene expression changes (Table S9), among which there are key regulators involved in the regulatory antagonism of ABA and GA. After removing mother root DEGs not relevant to this biological process, those from ARM or GRM were subjected to WGCNA for key regulator identification (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). To date, as per the existing studies the regulators involving ABA/GA balance primarily encompass TFs (Yaish et al., \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Lee et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tang et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Jin et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Xian et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Xie et al., \u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), reactive oxygen species (ROS) (Chen et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e), and proteins related to ubiquitin and ubiquitin-like modification (Miura et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Lin et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). According to the WGCNA and PPI visualized in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and D, the hub TFs antagonistically regulated by ABA and GA were only shown in the distinctive module with a robust correlation to ABA. These hub TFs are DREB1C, DREB1B, DREB1D, ERF017 and ERF025. They are all induced by ABA but suppressed by GA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). And under normal growing conditions the significant accumulation of \u003cem\u003eDREB1C, ERF017\u003c/em\u003e and \u003cem\u003eERF025\u003c/em\u003e transcripts occurred during the tuberous root initiation (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). Both DREBs and ERFs belong to AP2/ERF super-family. AP2/ERF super-family is well-recognized for their regulatory functions in hormonal signaling pathways and various aspects of plant development (Feng et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Shu et al. (\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) proposed that the research on AP2-domain-containing TFs may be helpful to disclose the interplay of ABA and GA and understand the mechanisms underlying ABA/GA antagonism. And a series of investigations have confirmed this opinion, among which ABI4 has been the most thoroughly studied (Yaish et al., \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Shu et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Shu et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Xian et al., \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Although the hub TFs identified in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e are from different subfamilies, DREB1C, DREB1B, and DREB1D falling into DREB subfamily, ERF017 and ERF025 belonging to ERF subfamily, some DREBs and ERFs still exhibit an association with ABA or GA metabolism or signaling (Tang et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR103\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Ma et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Vonapartis et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We investigated the expression of ABA and GA metabolism genes in the tobacco levels transiently expressing \u003cem\u003eRgERF017.\u003c/em\u003e The expression levels of both ABA and GA metabolism genes were significantly affected, which also resulted in the accumulated endogenous ABA. Therefore, these TFs are the candidates participating in the modulation of ABA/GA antagonism despite their obscure role in ABA/GA antagonism crosstalk (Shu et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition to AP2/ERF TFs, some ROS-related PERs were also identified as hub genes in the turquoise module (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Thereinto, \u003cem\u003ePER10, PER47\u003c/em\u003e, and \u003cem\u003ePER59\u003c/em\u003e inversely responded to exogenous ABA and GA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). And their transcripts were also significantly accumulated in \u003cem\u003eR. glutinosa\u003c/em\u003e early development stage under normal conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). ROS are the inevitable by-products of metabolism (Wang et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). The accumulation of ROS is toxic to organisms, but low-level ROS still serve as key signaling molecules, implicating in plant development (Wang et al., \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e2024b\u003c/span\u003e). In Arabidopsis, ROS mediate seed dormancy and germination through regulating the balance of ABA/GA (Liu et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Li et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). ABA catabolic gene \u003cem\u003eCYP707A\u003c/em\u003es and GA biosynthesis are stimulated by ROS (Liu et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Hence the genes for peroxidases capable of eliminating ROS appear to affect ABA/GA homeostasis. AtPER1 is such a gene that has been experimentally demonstrated to be involved in the trade-off between ABA and GA (Chen et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In our study, the transient expression of \u003cem\u003eRgPER10\u003c/em\u003e in tobacco also induced the change of ratio of ABA/GA through regulating ABA and GA metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). But interestingly its ROS-scavenging capacity appears to be less potent than that of \u003cem\u003eRgEFR017\u003c/em\u003e (Figure S7). The results demonstrated that the involvement of \u003cem\u003eRgERF017\u003c/em\u003e in ABA/GA antagonism may also be associated with but not limited to the scavenging of ROS. Furthermore, the PERs were predicted to interact with CADs, MEE, OMT etc. in PPI analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). These proteins, including PERs, are closely associated with lignin biosynthesis (Liu et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Martin et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). And their expression was all elevated by GA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). It is reported that exogenous GA promotes lignin biosynthesis through positively regulating these lignin biosynthetic genes (Wang et al., \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). And the increased lignin content in roots can arrest root growth (Wang et al., \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Taken together, our results on \u003cem\u003ePER\u003c/em\u003es suggest that on the one hand \u003cem\u003ePER\u003c/em\u003es in \u003cem\u003eR. glutinosa\u003c/em\u003e may be involved in ROS-mediated feedback loop of ABA/GA homeostasis, and on the other hand \u003cem\u003ePER\u003c/em\u003es also exert direct influence on root development through mediating lignin biosynthesis.\u003c/p\u003e\u003cp\u003eApart from TFs and ROS, other proteins, including UBC24 and NAS4, were also identified as hub genes antagonistically regulated by ABA and GA in our WGCNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). UBC24, a ubiquitin-conjugating E2 enzyme, and its interacting proteins are more closely related to phosphate homeostasis (Liu et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). NAS4 and its predicted interacting proteins predominantly participate in the transport of metal ions (Wang et al., \u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yang et al., \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). So far, no studies have shown that they have a crucial function in the biological processes regulated by ABA/GA antagonism, and the relationship between them and ABA/GA antagonism as well as \u003cem\u003eR. glutinosa\u003c/em\u003e root development calls for further investigation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eThe downstream responses to ABA/GA antagonism\u003c/h2\u003e\u003cp\u003eDownstream responses and plant development are controlled by the ratio of ABA/GA. The prevailing one with higher content dominates the expression of downstream key genes and thus determines the direction of development.\u003c/p\u003e\u003cp\u003eStarch and sucrose metabolism are closely linked to tuber development, as these carbohydrates serve as vital sources of energy and structural materials for tuber growth and expansion (Sojikul et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Pan et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Cai et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Dynamic transcriptional profiling of \u003cem\u003eR. glutinosa\u003c/em\u003e tuberous root showed that starch and sucrose metabolism is the most overrepresented group in carbohydrate metabolism during root development (Sun et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In analogy with \u003cem\u003eR. glutinosa\u003c/em\u003e roots under normal developmental conditions, starch and sucrose metabolism was also enriched in the swelling fibrous roots induced by the addition of ABA, and this pathway was upregulated in its entirety, especially the biosynthetic process from sucrose to starch (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The upregulation of genes encoding starch synthases, including \u003cem\u003eGBE1\u003c/em\u003es, \u003cem\u003eglgA\u003c/em\u003es and \u003cem\u003eWAXY\u003c/em\u003es (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), demonstrated that ABA actuated the accumulation of starch. Starch biosynthesis and metabolism are regulated by sucrose (Yoon et al., \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). It can provide energy, processed materials and even hexose-based sugar signals for sinks development after its cleavage (Yoon et al., \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Sucrose cleavage can be catalyzed by INVs or SUSs. These two enzymes channel sucrose into two distinct metabolic pathways, with SUSs starting the pathways for starch synthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The silencing of \u003cem\u003eSUS\u003c/em\u003es in tuber plants, potato (Herbers \u0026amp; Sonnewald \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) and \u003cem\u003eGladiolus hybridus\u003c/em\u003e (Li et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e), gave rise to the decreased starch content and tuber yield. In \u003cem\u003eR. glutinosa\u003c/em\u003e, the remarkably elevated expression of \u003cem\u003eSUS\u003c/em\u003es was observed in the swelling roots treated by ABA, and meanwhile all the \u003cem\u003eINV\u003c/em\u003es showed significantly lower expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). This may be one reason for the accumulation of starch in \u003cem\u003eR. glutinosa\u003c/em\u003e roots. Thus, it is reasonable to conjecture that \u003cem\u003eSUS\u003c/em\u003e and \u003cem\u003eINV\u003c/em\u003e may be key genes for the downstream responses. However, SUSs and INVs were not obviously regulated antagonistically by ABA and GA. GA also induced the expression of most \u003cem\u003eSUS\u003c/em\u003es, but to a lower degree than ABA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Only \u003cem\u003eunigene_0941071\u003c/em\u003e was repressed by exogenous GA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). As for \u003cem\u003eINV\u003c/em\u003es, the declined expression was also detected in most homologous unigenes post-GA treatment, except for \u003cem\u003eunigene_154406\u003c/em\u003e and \u003cem\u003eunigene_131992\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Probably, only certain genes, those subject to antagonistic regulation, are the pivotal nodes in the ABA/GA antagonistic network, determining the direction of sucrose cleavage and eventually even the root development. Alternatively, \u003cem\u003eSUS\u003c/em\u003es and even starch biosynthesis are more sensitive to ABA than to GA, as the regulatory effect of GA on starch biosynthesis was weaker overall.\u003c/p\u003e\u003cp\u003eCell wall biosynthesis and modification underpin the tuber expansion and shaping (Jarvis, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Liu et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). During tuberous root development, the metabolic process related to cell wall metabolism was enriched (Li et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Cai et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). And in \u003cem\u003eR. glutinosa\u003c/em\u003e, those DEGs associated with cell wall metabolism were mainly identified in the initiation stage of fibrous roots expansion (Li et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Similarly, Our GSEA analysis also detected up-regulated cell wall-related biological processes during the tuberous root initiation induced by ABA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e; Table S8). By contrast, all biological processes related to cell wall were significantly downregulated under GA treatment. Cell walls are tied to cell division and expansion (Delmer et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Plus, the GESA analysis also revealed that the biological processes related to cell cycle were only significantly enriched in AR group, rather than in GR group (Table S8), so cell proliferation and expansion may occur extensively during the initiation of tuberous root in AR group. Cell walls are mainly composed of cellulose, hemicellulose, pectin, wall-associated proteins, etc. (Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Delmer et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Cell wall biogenesis and expansion growth cannot proceed without the involvement of cell wall polymers and their synthesis enzymes, wall-modifying enzymes and other important wall-associated proteins (Chen et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Delmer et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). It is obvious that the enzymes responsible for the synthesis of cellulose and pectin, CESAs and GAUTs (Anderson \u0026amp; Kieber \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Delmer et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), maintained high expression levels in both CR and AR groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In contrast, they are significantly suppressed by GA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). This indicated that the synthesis of cellulose and pectin remains highly active during the normal developmental stage, likely priming cells for division and expansion. And ABA sustains this synthetic activity, while GA suppresses it. Moreover, important wall-modifying enzymes, XTHs and PMEs, hemicellulose synthase CSLs and cell wall signal transducers (WAKs) (Chen et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e) were antagonistically regulated by ABA and GA (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). XTHs cleave or reorganize the backbone of xyloglucan, the most abundant hemicellulose in the primary walls (Delmer et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). PMEs demethylesterify pectic backbones of homogalacturonan, which is initially generated in a highly methyl-esterified form by GAUTs (Delmer et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The higher expression of these enzymes suggests enhanced wall remodeling for cell expansion takes place in the presence of ABA. And this conclusion can be supported by the expression pattern of EXPs as well. The overwhelming majority of EXPs exhibited significantly upregulated expressions exclusively in the AR group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). EXPs facilitate cell wall-loosening through disrupting the noncovalent interactions between laterally aligned polysaccharides and thus help to cell enlargement (Cosgrove, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Consequently, the specific expression of wall-associated enzymes and proteins further disclosed that exogenous ABA promotes cell expansion to a greater extent compared with normal conditions, and GA arrests tuberous root initiation by retarding cell division and expansion. Previous studies (Xu et al., \u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Carrera et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Bou-Torrent et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Cheng et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Roumeliotis et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Davi\u0026egrave;re \u0026amp; Achard \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e) have shown that the inhibitory effect of GA on tuber formation is associated with enhanced cell elongation, manifested as stimulation of stolon elongation or promotion of aerial growth. The elongated overground stem observed after GA treatment (not shown) supports the latter part of the conclusion, but as we argued above, GA directly suppresses cell division and expansion in \u003cem\u003eR. glutinosa\u003c/em\u003e roots. Furthermore, the stimulation of lignification in roots by GA also contributes to the inhibition of tuber expansion (Chen et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). The increased lignification induced by exogenous GA, as identified through WGCNA, supports this opinion.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we elucidated the molecular mechanisms underlying the antagonistic regulation of adventitious root swelling in \u003cem\u003eR. glutinosa\u003c/em\u003e by ABA and GA through metabolic, signaling pathway, and transcription factor network analyses, complemented by phenotypic observations and gene expression profiling. Exogenous ABA or GA disrupts ABA/GA homeostasis and the one with a higher concentration will then activate its corresponding signaling pathway. The other signaling will be triggered soon due to the crosstalk of ABA/GA signaling. The key hub elements in ABA/GA signaling crosstalk, such as ABFs, DELLAs, DERBs and ERFs, orchestrate ABA/GA antagonism by integrating ABA/GA metabolism and downstream responses. In general, higher ratio of ABA/GA results in the promotion of starch and sucrose metabolism, cell wall biogenesis and modification, cell division and expansion, while lower ratio of ABA/GA suppresses these biological processes and induces lignification. The outputs of these downstream responses, like ROS, also function as feedback regulators of ABA/GA metabolism. Both ABA/GA signaling and the regulators from downstream responses modulate ABA/GA metabolism mainly through regulating the expression of key metabolic genes. The modulation of ABA/GA metabolism finally contributes to the trade-off between ABA and GA. Meanwhile, the downstream responses triggers or inhibits the initiation of \u003cem\u003eR. glutinosa\u003c/em\u003e tuberous roots. This proposed model was depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFundings:\u003c/h2\u003e\u003cp\u003eThis work was supported by Natural Science Foundation of Henan Province (No. 242300421579 and No. 252300421280), Key Research Development and Promotion Project (in Science and Technology) of Henan Province (No. 232102110028), and Program for Innovative Research Team (in Science and Technology) in University of Henan Province (No. 23IRTSTHN022).\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003eRuixue Yang and Peilei Chen conceived and designed the experiments. Ruixue Yang, Yiying Du, Zhenyang Fang and Heyang Wang performed the experiments and analyzed the data. Ruixue Yang and Peilei Chen wrote the manuscript. Qingxiang Yang complemented the manuscript. Peilei Chen and Hongying Duan supervised the work and corrected the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eThe authors acknowledge financial support from the Natural Science Foundation of China [grant numbers 32372746], and the support of Henan Province University\u0026rsquo;s Engineering and Technology Center of Conservation and Utilization for Genuine Chineses Medicinal Herbs, and Xinxiang Engineering and Technology Center of Conservation and Utilization for Chinese Medicinal Herbs.\u003c/p\u003e\u003ch2\u003eData availability statement\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the manuscript and within its supplementary materials published online.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAli, F., Qanmber, G., Li, F. \u0026amp; Wang, Z. 2022. \u0026quot;Updated role of ABA in seed maturation, dormancy, and germination.\u0026quot; \u003cem\u003eJournal of Advanced Research,\u003c/em\u003e 35\u003cstrong\u003e:\u003c/strong\u003e 199-214. https://doi.org/10.1016/j.jare.2021.03.011.\u003c/li\u003e\n\u003cli\u003eAnderson, C. 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The swelling of its adventitious roots is a prerequisite for tuberous roots initiation, which is antagonistically regulated by abscisic acid (ABA) and gibberellins (GA). However, the antagonistic mechanisms between ABA and GA remain poorly understood. In this study, exogenous ABA significantly promoted adventitious roots swelling, whereas GA exerted an inhibitory effect. To investigate the potential mechanisms, transcriptome analysis was carried out on \u003cem\u003eR. glutinosa\u003c/em\u003e roots treated with ABA or GA. Compared to control samples, 7,416 differentially expressed genes (DEGs) (ABA) and 9,034 DEGs (GA) were identified in adventitious roots. The expression patterns of DEGs involved in ABA or GA metabolism and signal transduction revealed the dynamic regulation of ABA/GA equilibrium. Kyoto Encyclopedia of Genes and Genomes and Gene set enrichment analysis showed enhanced starch accumulation and cell wall biosynthesis and modification in ABA-treated adventitious roots. The lignification induced by GA and key regulators in the antagonistic action of ABA and GA, including DREBs, ERFs and PERs, were identified using weighted gene co-expression network analysis and protein-protein interaction network analysis. And the transient expression analysis of \u003cem\u003eRgERF017\u003c/em\u003e and \u003cem\u003eRgPER10\u003c/em\u003e in tobacco further demonstrated that these two genes are involved in ABA/GA antagonism through the control of ABA and GA metabolism. This study provides a comprehensive analysis of the antagonistic regulatory network between ABA and GA during \u003cem\u003eR. glutinosa\u003c/em\u003e tuberous roots initiation, deepening our understanding of the molecular mechanisms underlying tuberous root formation.\u003c/p\u003e","manuscriptTitle":"Transcriptome profiling of Rehmannia glutinosa uncovers ABA/GA antagonism mechanisms in tuberous root initiation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-06 11:49:41","doi":"10.21203/rs.3.rs-7635435/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":"01d9e095-e3bd-4bee-a2fa-11543673ca60","owner":[],"postedDate":"October 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-10T17:36:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-06 11:49:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7635435","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7635435","identity":"rs-7635435","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}