{"paper_id":"3e2defd4-4512-4ee7-a873-89c77da53f84","body_text":"Benzoic acid inhibits peach root development via disrupted auxin distribution | 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 Benzoic acid inhibits peach root development via disrupted auxin distribution Qing Rong Zhang, Jilin Yao, Feiyang Ji, Zhilin Sun, Bing Bai, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6909418/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 Allelopathic effects are commonly observed in plant communities, with benzoic acid (BA) identified as a primary autotoxin contributing to peach replanting disease. However, the effects of BA on peach root growth remain unclear. In this study, we evaluated the morphological changes in peach roots and the auxin signaling responses to BA stress. Our results showed that BA treatment significantly reduced root length and inhibited lateral root emergence. Gene expression analysis revealed that several auxin-related genes, such as PpARF19 , PpAUX1 , PpYUCCA10 , were significantly reduced after BA treatment. Using transgenic peach roots with the DR5::GUS auxin reporter, generated through our optimized non-sterile root transformation protocol, we observed that BA treatment disrupted the DR5::GUS expression pattern in root tips and lateral root primordia. Hormone measurements indicated a slight reduction in auxin accumulation and a significant increase in the auxin antagonistic hormone salicylic acid. These findings suggest that BA induces autotoxicity in peach plants by impairing root growth through alterations in auxin signaling and biosynthesis pathways. This study enhances our understanding of allelopathic effects in plant interactions and provides valuable insights for mitigating challenges in peach orchards. benzoic acid Prunus persica root growth auxin root transformation system salicylic acid Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key message We established a non-sterile root transformation system in peach seedlings. Using this system, we demonstrated that BA treatment inhibits plant growth and lateral root emergence by disrupting auxin distribution. Introduction The replant problem (also known as continuous cropping obstacle) affects a wide range of plant species globally and causes significant economic losses in both the forestry and agricultural sectors (Dong et al. 2018 ). Peach ( Prunus persica ), one of the most affected species, is grown worldwide (Ricci et al., 2020 ; Zhang et al., 2023 ). Autotoxicity, a type of interspecific allelopathy, occurs when a plant species inhibits its own kind by releasing toxic chemicals into the environment. This is a major factor contributing to the peach replanting problem. Benzoic acid (BA), a key autotoxic compound in peach, strongly inhibits peach plant growth (Zhu et al. 2017 ). However, the precise impact of BA on peach root development remains unclear. Continuous cropping of peach on the same land leads to more pronounced issues compared to other Prunus species, due to their shorter life cycle and the rapid replacement of cultivars. This results in reduced growth, yield, and quality, as well as increased susceptibility to pests and diseases (Zhu et al., 2017 ; Yin et al., 2018 ; He et al., 2019 ). Replanting diseases are primarily driven by allelopathic autotoxicity, shifts in microbial communities, and soil fertility imbalances (Guo et al., 2016 ; Yim et al., 2020 ; Zhang et al., 2020). Autotoxins disrupt plant growth not only by acting as chemical signals in plant-microorganism interactions, which alter microbial community structure and indirectly affect plant growth, but also serving as stress factors that directly impair plant function and modify root secretion characteristics. This makes autotoxic substances a major contributor to replanting disease (Shen et al. 2021 ). The complex processes involved in the generation, accumulation, and detoxification of various autotoxins require a deeper exploration of the specific mechanisms through which plant roots respond to these compounds. Autotoxins are released into the surrounding environment through leaching from aboveground plant parts, volatilization, root secretion, and residue decomposition (Liu et al., 2019 ; Gallego et al., 2020 ; Zhang et al., 2020). Various autotoxic substances have been identified across numerous plant species, including phenolic compounds (such as simple phenolics, flavonoids, coumarins and quinones), terpenoids (monoterpenes, sesquiterpenes, diterpenes, triterpenes and steroids), alkaloids, nitrogen-containing chemicals (non-protein amino acids, benzoxazinoids and cyanogenic glycosides), and other chemical families (Kong et al. 2019 ). Benzoic acid (BA) is a well-known autotoxin found in root exudates and rhizosphere soils of various plant species, where it causes significant allelopathic suppression (Asaduzzaman and Asao 2012; Zhang et al. 2018 ). BA has been widely detected in peach roots and replanted soils, where it exerts a pronounced inhibitory effect on peach plant growth (Zhu et al. 2017 ). Research shows that BA stress inhibits peach seedlings growth, with higher BA concentrations leading to more severe damage to root biomass and length (Shen et al. 2021 ). However, the precise impact of BA on peach roots and the underlying molecular mechanisms remain unknown. Auxin plays a crucial role in regulating root growth and lateral root formation (Fukaki et al. 2007 ; Overvoorde et al. 2010 ). It is primarily synthesized in actively growing tissues, including shoot and root tips, young leaves, developing seeds (Pop et al. 2011 ). Auxin is transported in polarized streams via auxin transporters, a process coordinated by AUXIN RESISTANT1/LIKE AUX1 (AUX1/LAX) uptake permeases, ATP Binding Cassette subfamily B (ABCB) transporters, and PIN-FORMED (PIN) carrier proteins, driven by chemiosmotic gradients (Geisler et al., 2017 ; Zwiewka et al., 2019 ; Hammes et al., 2022 ). Although several auxin biosynthesis pathways have been identified, the IAA/YUC pathway is the predominant route for endogenous auxin biosynthesis in many plant species (Cao et al. 2019 ). This pathway involves two steps: the TAA family transaminase converts tryptophan into indole-3-pyruvate (IPA), and the flavin monooxygenase YUC family then converts IPA into indole-3-acetic acid (IAA or auxin) (Zhao 2012 ). Auxin responses are mediated by members of the Auxin Response Factor (ARF) family of transcription factors and the Aux/IAA proteins (Ulmasov et al. 1999 ). However, it remains unclear whether BA stress affects peach root development through auxin-related pathways. Investigating the mechanism of autotoxicity in peach plants by using transgenic approaches is essential (Uematsu et al., 2014 ; Zhou et al., 2015 ; Wang et al., 2019 ), although a stable transformation protocol is still lacking. Recently, a transient gene transformation method was developed for gene function analysis in peach, though its efficiency is genotype-dependent (Cheng et al. 2024 ). Additionally, the use of Agrobacterium rhizogenes strain MSU440 to generate transgenic roots in peach has also been established (Xu et al. 2020 ), but it requires a tedious tissue culture process. In this study, we first investigated the morphophysiological changes of peach plants under BA stress. The results showed that BA treatment inhibited root growth and lateral root emergence. Gene expression analysis of auxin-related genes revealed significant reductions in expression of several auxin response gene PpARFs , the biosynthesis gene PpYUC10 , and the auxin influx transporter PpAUX1 , in response to BA treatment. Next, we introduced the DR5::GUS auxin response reporter system into peach roots using a non-sterile root transformation system to generate transgenic roots. Using these transgenic roots, we observed that DR5 expression pattern in root tips and lateral root primordia was altered under BA stress. These findings were further supported by hormone measurements, which showed a reduction in auxin levels and an induction of its antagonistic hormone, salicylic acid. Overall, our data provide new insights into the auxin response mechanisms in peach roots under autotoxicity stress, and the non-sterile root transformation system will serve as a valuable tool for analyzing gene functions in peach roots. Results Benzoic acid inhibits peach root growth and lateral root emergence Benzoic acid (BA) has been identified as a major autotoxin in peach, causing stunted seedling growth or even death (Zhu et al. 2017). To evaluate the impact of BA on plant root growth, we applied 0.8 mM BA to peach seedlings (Shen et al. 2021). After two weeks, the overall size of BA treated peach seedlings was smaller than that of the control group (Figure 1A). Both stem and root lengths were significantly reduced in the BA treated seedlings compared to the control (Figure 1B). In addition, the number of lateral roots was significantly decreased upon BA treatment. Since lateral root development involves multiple steps (Péret et al. 2009), we further investigated which stage of lateral root development was affected by BA. The number of lateral root primordia in a 5 cm root fragment, including the root tip, was counted using a microscope after staining with eosin. Auxin response is a critical requirement for the development of lateral root primordium (Du and Scheres 2018). To assess the number of active lateral root primordia, we generated peach root containing the DR5::GUS reporter (see result section 3). Our results showed that the number of lateral root primordia expressing DR5::GUS was significantly lower in the BA treated roots. However, no significant difference was observed in the total number of lateral root primordia between the control and BA treated conditions. These data suggest that the reduced lateral root number following BA treatment is due to the inhibition of lateral root emergence rather than the prevention of lateral root initiation. Benzoic acid treatment downregulates the expression of auxin-related genes in peach roots Auxin plays a crucial role in plant root growth and lateral root development. To investigate the impact of BA on the expression of auxin-related genes in peach roots, we conducted RNA sequencing. To identify auxin-related genes in peach, we performed BLAST searches using Arabidopsis protein sequences for PINs, AUX1, LAXs, YUCs and ARFs (TAIR, https://www.arabidopsis.org) against the peach genome (PPGV, http://peachtree.work/home). This search identified 5 PpPIN , 3 PpLAX/AUX , 5 PpYUC and 12 PpARF gene members. An evolutionary tree was constructed based on these genes (Figure 2A). Since not all of these auxin-related genes are expressed in peach roots, subsequent analysis focused on the highly expressed genes (Supplementary Table. S1) (Shen et al. 2021). To examine the effects of exogenous BA on auxin-related gene expression in peach roots, we analyzed gene expression using digital PCR (dPCR) (Figure 2B). The primers used to amplify the fragments are listed in Supplementary Table S2. Exogenous BA treatment did not significantly affect the expression of auxin efflux transport-related genes compared to the control. In contrast, auxin uptake transport-related genes AUX/LAX showed different expression patterns. PpAUX1 was down-regulated by more than 1.8-fold, while PpLAX2 was up-regulated. No significant difference was observed for PpLAX3 compared to the control. Among the auxin response transcription factor ( ARF ) genes, PpARF4 , PpARF6 , PpARF8 , PpARF18 , and PpARF19 were down-regulated by exogenous BA, while PpARF1 and PpARF3 were up-regulated. For the auxin synthesis-related genes, PpYUC10 was down-regulated by more than 3.5-fold following exogenous BA. PpYUC2 and PpYUC6 showed no significant difference, while PpYUC8 was up-regulated. However, aside from PpYUC2 , PpYUC8 presented the lowest Cn/μL value. These findings suggest that exogenous BA treatment alters the expression of auxin-related genes in the peach root system, which may lead to disrupted auxin accumulation and, consequently, reduced root development of peach. Implementation of a simple and effective root transgenic system in peach The DR5 auxin reporter system has been successfully applied to investigate auxin distribution and response at the cellular or tissue level in plants (Benková et al. 2003; Chen et al. 2013). In this study, we aimed to explore the spatiotemporal expression patterns and signaling pathways of auxin under BA treatment using the DR5::GUS system. However, the current limitations of the gene transformation system in peach have severely restricted progress in this research. To address the challenges associated with genetic transformation of peach roots, we first optimized the infection conditions for Agrobacterium rhizogenes -mediated genetic transformation. We tested three different Agrobacterium rhizogenes infection methods: soaking infection, daubing infection, and combined infection. The results showed that soaking infection yielded the highest transformation efficiency (4.77%), followed by daubing infection (3.33%), while combined infection resulted in the lowest efficiency (0.67%) (Figure 3A-D). Therefore, soaking infection with 7 days of co-cultivation was determined to be the most suitable method for transforming peach root using this system. Next, we evaluated the impact of infection sites on transformation efficiency. Instead of infecting the hypocotyls, we infected the root meristem by excising a small portion of the tender yellow root tip (Figure 3E). This approach resulted in a significantly higher transformation efficiency of 27.11% (Figure 3F). Additionally, this root transformation method does not require a sterile working environment (Figure 3G). The optimized root transformation protocol will be further applied to generate transgenic peach roots containing DR5::GUS reporter system, enabling the investigation of spatial auxin responses following BA treatment in peach roots. Benzoic acid inhibits primary and lateral root development by disrupting auxin distribution in peach To study the spatial distribution of auxin signaling in root tip and lateral root primordia under BA stress, we introduced DR5::GUS reporter construct into peach roots using the root transformation method described above. We then analyzed the DR5::GUS expression pattern in semithin longitudinal peach root sections following two weeks of BA treatment. Examination of these semithin sections revealed reduced GUS signal expression in columella cells, stem cell niche and its surrounding cells (Figure 4A and 4B) of BA treated roots (Figure 4C and 4D). This suggests that benzoic acid treatment diminished auxin signaling in the root tips. In addition, we observed a reduction in root meristem size, root thickness, and the number of root cap cell layers following BA treatment. Next, we examined whether auxin signaling was altered in the lateral root primordia under BA stress using the DR5::GUS reporter. In the control roots, DR5 expression was detected in the lateral root primordia and its surrounding tissues, including the endodermis/cortex and vascular cells both overlying and beneath the lateral root primordium (Figure 4E and 4F). However, after BA treatment, DR5 expression persisted in the lateral root primordium but was strongly suppressed in its surrounding cells, especially in the tissues overlying and beneath the primordium (Figure 4G and 4H). Together, these results demonstrate that BA stress interferes with auxin distribution, inhibiting both primary root growth and lateral root emergence. Benzoic acid treatment interferes with auxin and salicylic acid biosynthesis To investigate the BA-mediated changes in auxin metabolism in peach roots, we measured the levels of various auxin metabolites as well as other plant hormones, including cytokinins (CK), gibberellins (GA), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA) and strigolactones (SL). A total of 51 metabolites were identified and grouped into seven categories based on their hormone classification (Supplementary Table. S3). Among the auxin-related metabolites, we observed an increase in L-tryptophan accumulation, but the levels of indole-3-carboxaldehyde and methyl indole-3-acetate were reduced, although these changes were not statistically different (Figure 5A). In contrast, the levels of salicylic acid and salicylic acid 2-O-β-glucoside were significantly increased (Figure 5B). No significant changes were detected in the levels of other hormone-related compounds. Discussion It has been reported that BA compromises peach root growth (Shen et al. 2021), and plant secondary metabolites are involved in plant growth and development, potentially influencing the regulation of plant hormones under stress by altering auxin synthesis and distribution (Vanneste & Friml, 2009; Cheng & Cheng, 2015). This study demonstrates that the peach autotoxic compound benzoic acid inhibits root growth and lateral root emergence via influencing auxin distribution in roots. BA inhibits plant root growth and lateral root emergence via auxin response Here, we showed that four parameters (stem length, root length, root weight and number of lateral roots) were significantly reduced under BA treatment (Figure 1B). These phenotypical changes are consistent with the effects of autotoxic substances observed in other species, such as cucumber (Bu et al. 2019), Medicago sativa (Wang et al., 2022), tobacco (Chen et al. 2019) and maize (Nickel et al. 1995). However, it remains unknown whether BA is also responsible for the inhibitory effects on root growth in these species. In addition to these effects, our tissue sections revealed that BA treatment significantly reduced the root meristem size, root cap cell layers and lateral root emergence. Plants respond to abiotic stresses through a series of physiological, biochemical, and metabolic modifications, including manipulating the endogenous molecular and physiological pathways. These processes are largely mediated by the involvement of the plant hormone auxin (Grunewald et al. 2009). The observed inhibition of peach root meristem and lateral root growth, along with the reduced DR5::GUS expression (Figure 4) following BA treatment, strongly supports the involvement of auxin in plant response to abiotic stress. Auxin is a key plant hormone that regulates various aspects of root development, including root cell organization, differentiation, lateral root initiation, and emergence (Yun et al. 2023). Mutants with disrupted auxin signaling or defects in the auxin transport system exhibit altered root phenotypes (Fukaki et al., 2002; Benková et al., 2003). The reduction in DR5::GUS expression in the root tip after BA treatment corresponds to the decreased meristem size and root cap cell layers (Figure 4). This is consistent with the role of auxin gradient in regulating cell differentiation from meristematic to elongation zones, as well as from distal stem cells to columella and root cap cells (Ding & Friml, 2010; Di Mambro et al., 2017; Dubreuil et al., 2018). Furthermore, the auxin response in both underlying and overlying tissues of the lateral root primordia is critical for lateral root emergence (Swarup et al., 2008; Marin et al., 2010; Porco et al., 2016; Du & Scheres, 2018). The reduced DR5::GUS expression in the cells surrounding the lateral root primordia, particularly in vascular, endodermal, and cortical cells, aligns with BA's inhibitory effect on lateral root emergence. BA affects auxin response, auxin transport and biosynthesis gene expression DR5::GUS expression results indicate auxin signaling response in peach roots is strongly inhibited under BA stress (Figure 4). Our dPCR data revealed five PpARFs ( PpARF4 , PpARF6 , PpARF8 , PpARF18 and PpARF19 ) were down-regulated, while two PpARFs ( PpARF1 and PpARF3 ) were up-regulated under BA stress. Plant auxin response genes, ARFs , are classified into three groups Class-A, B and C. Only Class-A genes act as activators, regulated by auxin through nuclear auxin pathways, while Class-B and C genes act as transcription repressors (Hernández-García et al. 2024). After BA treatment, within these reduced PpARFs, three of them ( PpARF6 , PpARF8 and PpARF19 ) belong to Class-A, primarily acting as activators, while the up-regulated PpARFs ( PpARF1 and PpARF3 ) are all from Class-B, functioning mainly as repressors. In Arabidopsis and rice, lateral root primordia outgrowth requires repression of repressor ARFs, such as ARF2 , ARF3 and ARF4 in the vascular cells (Marin et al. 2010), and upregulation of activator ARFs , such as ARF7 and ARF19 (Lee et al. 2009; Yamauchi et al. 2019). The expression profile of PpARFs , particularly the reduction in PpARF19 , helps explain the inhibition of lateral root emergence under BA treatment and directly reflects the reduced auxin response in peach roots. Polar auxin transport, which refers to the directional movement of auxin between cells, is critical for establishing plant developmental patterns. This process is mainly mediated by the asymmetric localization of auxin efflux carriers PIN-FORMED (PIN) protein family and auxin influx carriers AUXIN1/LIKE-AUX1 (AUX/LAX) protein family (Swarup & Péret, 2012). Under BA treatment, we found that the transcriptional levels of all PpPIN genes were not significantly affected by BA (Figure 2B). However, PpAUX1 expression was significantly reduced in BA treated roots compared to controls (Figure 2B). In Arabidopsis, AUX1 has been implicated in lateral root development (Swarup & Péret, 2012). Although PpLAX2 expression increased under BA treatment (Figure 2B), single mutations in LAX2 did not negatively affect lateral root formation in Arabidopsis (Da Costa et al. 2020). Collectively, auxin transporters may also contribute to inhibiting peach root system development by disrupting the auxin accumulation pattern after BA stress. Since the function of auxin transporters largely depends on their subcellular localization, especially PINs (Zhang et al., 2020), studying their protein dynamics in response to BA treatment will offer new insights into how BA interferes with polar auxin transport, ultimately leading to root growth inhibition. Additionally, we observed that PpYUC10 expression was reduced after treatment with 0.8 mM BA (Figure 2B). YUCCA flavin monooxygenases catalyze a rate-limiting step in auxin biosynthesis and play a crucial role in the formation of both embryonic and postembryonic organs (Cheng et al., 2006). Disruption of four YUC genes ( YUC1 , YUC4 , YUC10 , and YUC11 ) in Arabidopsis resulted in seedlings lacking a hypocotyl and a root meristem (Cheng et al., 2007). Our findings demonstrate that auxin signaling was significantly attenuated in the root tips and lateral root primordia under BA treatment (Figure 4). This suggests that the low expression of PpYUC10 , combined with the reduced auxin level, may mediate the peach root response to external BA, ultimately leading to slower root growth and fewer lateral roots. Accumulated SA may interfere with auxin distribution and biosynthesis during BA treatment Our data show that SA is significantly induced upon BA treatment. In the SA biosynthesis pathway, BA is thought to be catalyzed by benzoic acid hydroxylase (BA2H), converting it into SA (Leon et al. 1993). Therefore, the external application of BA leads to SA accumulation in peach roots. Additionally, the changes in auxin distribution under BA treatment may result from this SA accumulation in peach roots. For instance, protein kinase CK2 plays a crucial role in both SA and auxin pathways (Wei et al. 2021). SA influences root apical meristem patterning and lateral root formation by disturbing the auxin distribution through the regulation of auxin-related genes expression, such as auxin biosynthesis TAA1 and auxin efflux PINs, in a concentration-dependent manner (Armengot et al. 2014; Pasternak et al. 2019; Wei et al. 2021). Our results show that BA treatment causes root apical meristem patterning similar to that observed in roots treated with high levels of SA, displaying reduced meristem size and altered auxin distribution (as indicated by DR5::GUS expression). Furthermore, we found that BA treatment increases L-tryptophan levels, which may also be related to SA accumulation. It has been shown that the external application of SA for 5 days in Arabidopsis induces TAA expression (Pasternak et al. 2019). TAA is the enzyme responsible for converting L-tryptophan to indole-3-pyruvic acid (Luo and Di 2023). However, whether SA will induce L-tryptophan accumulation and the role of the increased L-tryptophan requires further investigation. Tryptophan-derived secondary metabolites are deposited in cell walls as physical defense compounds during pathogen invasion (Ishihara et al., 2008; Consonni et al., 2010). BA induced L-tryptophan is most likely related to the root defensive response rather than enhanced auxin production. After BA treatment, tryptophan-derived secondary metabolites indeed accumulated and we observed reduced auxin levels (Figure 5A) (Supplementary Table. S4) (Shen et al. 2021). In addition, during the plant defense response, it is common for SA accumulation to coincide with a reduction in auxin biosynthesis, transport and response (Zhong et al. 2021). Since BA is an important autotoxic compound, we cannot exclude the possibility that BA triggers the activation of defense factors that play an upstream role in balancing SA accumulation and auxin reduction (Wei et al. 2021). In summary, we found that BA treatment significantly inhibited plant growth, as evidenced by reduced stem length and impaired root development. Under BA stress, roots exhibited decreased auxin accumulation, accompanied by the down-regulation of auxin-responsive and auxin-synthesis genes. Using our established gene transformation system, we investigated changes in auxin expression patterns in roots under autotoxic stress. This study provides valuable insights into the molecular mechanisms by which auxin regulates peach root responses to autotoxicity. Material and methods Plant materials and growth conditions Peach ( Prunus persica ) seeds used in this study were stratified in moist sand stored at 4℃ for over 60 days. After stratification, the seeds were transplanted into vermiculite and grown under a 16 hours light cycle at 25℃ and 45% relative humidity. Benzoic acid was dissolved, prepared as a 0.8 mM stock solution, and thoroughly mixed with vermiculite to form the treatment substrate. The control substrate consisted of vermiculite mixed with water. Morphologically uniform 7-day-old seedlings and transgenic plants carrying the DR5::GUS reporter were selected and transplanted into the two substrate types for 14d. All treatments were performed with three independent biological replicates, and each replicate consisted of 3 individual plantlets. During the treatment period, the treatment groups were supplemented with 0.8 mM BA stock solution every 3 days, while the control groups received water additions to maintain consistent substrate moisture levels. All plants were cultivated in a growth chamber maintained under a 16 hours light cycle at 25℃. After 14 days culture, the seedlings were collected from both BA treatment and control groups for comprehensive analyses, including physiological parameter measurements, digital PCR assays, metabolite profiling and semithin sections preparation. Vector construction To generate DR5::GUS construct, seven tandem direct repeats of 11 bp, including the auxin responsive factor gene-binding site (TGTCTC), were amplified by PCR using high fidelity Phusion polymerase (Thermo-Fisher). The PCR product was then cloned into the pENTR-D/-TOPO (Invitrogen). After sequence verification, the DR5 promoter region was recombined into pKGW243 using LR clonase II (Invitrogen), resulting in the DR5::GUS construct. RNA extraction and Digital PCRs analysis Total RNA was extracted from peach roots using the E.Z.N.A. Plant RNA Kit, following the manufacturer’s protocol. RNA quality was assessed via agarose gel electrophoresis. One microgram of RNA was used for cDNA synthesis using the RevertAid RT kit (Thermo Scientific). Gene expression of auxin-related genes in the roots was quantified using Sniper DQ24 Digital PCR Platform (Sniper, Suzhou, China). The cDNA was diluted 100-fold and used for dPCR analysis with the 2×dPCR EvaGreen Master mix (Rox) (Sniper, Suzhou, China) and gene-specific primers on the Sniper DQ24 Digital System. The 22 μL EvaGreen dPCR mixture contained 11 μL of 2×dPCR EvaGreen Master Mix (Rox) (Sniper, Suzhou, China), 5 μL of template suspension, 1 μL of each forward and reverse primers (10 μmol·L −1 ), and 4 μL of sterile ultrapure water. The EvaGreen dPCR program was set as follows: droplet generation at 60 ℃ for 5 minutes, pre-denaturation at 95 ℃ for 15 minutes, followed by 40 cycles of denaturation at 95 ℃ for 20 s and annealing/elongation at 58 ℃ for 30 s. Data acquisition and analysis were performed by SightPro software (Sniper). Non-sterile Agrobacterium rhizogenes -mediated root transformation The A. rhizogenes MSU440 strain carrying the DR5::GUS plasmids was plated on solid LB medium containing 100 mg/L spectinomycin and incubated at 28℃ for 2-3 days. Single colonies were selected and cultured overnight in LB liquid medium (28℃, 200 RPM). Aliquots of the overnight culture were further propagated with LB solid and liquid medium for either 2 days or overnight. The liquid culture was grown until the OD600 reached 0.8-1.2. Freshly grown bacteria were then centrifuged at 3500 rpm for 15 minutes and resuspended in a suspension buffer (Woody Plant regeneration Medium, 9.75 g/L MES, 0.1% sucrose, 0.5g/L CaCl 2 , 0.8mg IBA, 100 mM acetosyringone and 0.05% Silwet L-77; pH 5.7). The bacterial suspensions, along with bacteria harvested from solid media, were used to directly infect explants. Peach seedlings with 1-2 cm of emerged radicles, germinated in a non-sterile environment, were used for transformation. The root tips were excised either below hypocotyl swelling or from the meristematic zone. Multiple small wounds were gently made on the remaining hypocotyl or root using a sterile scalpel. The wounded seedlings underwent one of three infection methods: 1) soaking infection: seedlings were immersed in an A.rhizogenes strain MSU440 suspension containing the target plasmid at room temperature for 40 minutes; 2) daubing infection: seedlings were immersed in distilled water containing 100 mM acetosyringone for 20 minutes at 25℃ in the dark, then the wounded region was coated with A. rhizogenes collected from solid medium; or 3) combined infection: seedlings were immersed in the A.rhizogenes strain MSU440 suspension and then dipped with A. rhizogenes from a solid medium. After infection, seedlings were transplanted onto co-cultivation medium (solidified with 0.7% agar) and incubated in a growth chamber for 7 days under dark conditions. They were then returned to normal growth conditions, at 25℃ under a 16-hour light cycle. Adventitious roots, which developed from the hypocotyl after 2-3 weeks, were selected for the identification of positive transformants by detecting DsRED using a fluorescence stereomicroscope equipped with a digital camera (SMZ25, Nikon). Fluorescence signals were observed using a 400 nm excitation filter and 600 nm emission filter. Root staining, embedding and sectioning Transgenic roots containing DR5::GUS constructs were stained with GUS buffer (Biotopped, China) and then incubated at 37℃ for 15 minutes to 3 hours as described previously (Na et al. 2011). After staining, the root segments were placed in a fixation buffer (5% glutaraldehyde in 100 mM phosphate buffer, pH 7.2) and incubated at 4℃ overnight. Roots were then dehydrated through an ethanol series (10%, 30%, 50%, 70%, 90% and 100% ethanol for 30 minutes each), followed by infiltration and embedding in Technovit 7100 (Hereus-Kulzer, Germany). The embedded roots were sectioned into 5 μm longitudinal slices using a microtome (Leica 2035) and stained in 0.1% Ruthenium Red for 15 minutes. To analyze the total number of lateral root primordia, freshly collected roots were first fixed in 70% ethanol for 12 hours, and then immersed in a 1% Eosin B solution dissolved in ethanol for 2 minutes. Statistical analysis All treatments in this study were biologically replicated at least three times. Data are presented as mean values ± SE of the replicates. Statistical analyses were performed using the Statistical Product and Service Solutions (SPSS) software (IBM Co., Armonk, NY, USA). The significance of differences between groups was determined using the least significant difference (LSD) test at a significance level of P < 0.05. All experimental data were also analyzed using Student’s t-test. Graphs and figures were generated using GraphPad Prism 9.0.0 software (GraphPad, San Diego, CA, USA). Declarations Acknowledgements This work was supported by the 2024 Classification Development Quota Project-Talent Introduction Start-up Fund of Beijing University of Agriculture (5066516006/005) and the 2023 Classification Development Quota Project - Science and Technology Innovation Capacity Enhancement Plan - Mechanisms of Plant-Microbe Interactions Mediated by Peach Secondary Metabolites in Continuous Cropping Soil of Beijing University of Agriculture (5076016183/069). Author contributions QRZ, JY, QZ and TTX designed the experiments. QRZ and JY performed the experiments with occasional help from ZS, FJ, BB, JZ HL and QC. QRZ, JY and FJ analyzed the data. QRZ and TTX wrote the manuscript. All authors read and approved the final manuscript. Conflict of interest Authors declare that they have no competing interests. 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(A) Morphological changes of peach plants following 2 weeks of BA treatment. (B) Quantitative analysis of growth parameters, including stem length, root length, root weight, number of lateral roots, number of lateral root primordia with \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e expression and total number of lateral root primordia after 2 weeks of BA treatment.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6909418/v1/275491eba76c3e970639cff6.png\"},{\"id\":88812948,\"identity\":\"be7ae9e6-bdb4-41cb-88a3-e8444579a0e8\",\"added_by\":\"auto\",\"created_at\":\"2025-08-11 15:52:45\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":7553426,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePhylogenetic analysis and expression profiling of auxin-related genes in peach roots following exogenous BA treatment.\\u003cstrong\\u003e \\u003c/strong\\u003e(A) Phylogenetic analysis of \\u003cem\\u003ePp\\u003c/em\\u003eARF, \\u003cem\\u003ePp\\u003c/em\\u003eYUC, \\u003cem\\u003ePp\\u003c/em\\u003eLAX/\\u003cem\\u003ePp\\u003c/em\\u003eAUXand \\u003cem\\u003ePp\\u003c/em\\u003ePIN proteins in peach. (B) Expression levels of \\u003cem\\u003ePpARFs\\u003c/em\\u003e, \\u003cem\\u003ePpYUCs\\u003c/em\\u003e, \\u003cem\\u003ePpPINs\\u003c/em\\u003e, \\u003cem\\u003ePpAUX1\\u003c/em\\u003e and \\u003cem\\u003ePpLAXs\\u003c/em\\u003e in peach roots in response to 2 weeks exogenous BA treatment. Error bars represent mean ± standard deviation (SD) from three biological replicates. An asterisk (*) indicates a significant difference at \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6909418/v1/1cc496f5b1c3805ef171f12a.png\"},{\"id\":88810933,\"identity\":\"0a3ae0ad-7cf7-412a-9c96-66639ff8bce3\",\"added_by\":\"auto\",\"created_at\":\"2025-08-11 15:36:45\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":18321617,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eNon-sterile \\u003cem\\u003eAgrobacterium rhizogenes\\u003c/em\\u003e-mediated root transformation system in peach. (A, B and C) Workflow of the different \\u003cem\\u003eA. rhizogenes \\u003c/em\\u003einfection methods. (D) Transformation efficiency of various infection methods. (E) Workflow of the meristem infection using \\u003cem\\u003eA. rhizogenes\\u003c/em\\u003e. (F) Transformation efficiency with different infection sites and co-culture times. Transformation frequency was calculated as the average (number of plants expressing the target gene/total number of injected plants) × 100%. (G) Workflow of the non-sterile \\u003cem\\u003eAgrobacterium rhizogenes\\u003c/em\\u003e-mediated roots transformation system for peach seedlings. (H) A peach plant with transgenic root generated with the workflow described in (G). Transgenic roots expressing \\u003cem\\u003eDsRED\\u003c/em\\u003e marker gene, bright light (I) and fluorescent light (J). (K) Transgenic roots containing \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e reporter system, showing GUS\\u003cem\\u003e \\u003c/em\\u003eexpression in the root tips and lateral root primordia. Close-up image of (K) showing lateral roots (L) and root tip (M).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6909418/v1/e8054a557d40cb407133387f.png\"},{\"id\":88809944,\"identity\":\"601c8329-94c2-4d75-b07d-abc784efeee8\",\"added_by\":\"auto\",\"created_at\":\"2025-08-11 15:28:45\",\"extension\":\"png\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":16943320,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cem\\u003eDR5::GUS\\u003c/em\\u003e distribution pattern in peach roots is altered after BA treatment. (A-D) \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e expression in root tips. (A) \\u003cem\\u003eDR5\\u003c/em\\u003e activity is observed in the stem cell niche and its surrounding area, columella, and vasculature cells under control conditions. (C) In BA-treated roots, \\u003cem\\u003eDR5\\u003c/em\\u003e expression is reduced, being present only in stem cell niche and columella cells. Additionally, the meristem size and root cap cell layer were also reduced. (B and D) Magnified details of (A) and (C), respectively. (E-H) \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e expression in lateral root primordia. (E) \\u003cem\\u003eDR5\\u003c/em\\u003eis expressed in the lateral root primordium and its surrounding tissues, including both overlying and underlying tissues under normal conditions. (G) In BA-treated roots, \\u003cem\\u003eDR5 \\u003c/em\\u003eactivity is detected only in the lateral root primordium. (F and H) Magnified details of (E) and (G), respectively. Bar, 50 μm.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6909418/v1/3c471e7eea692c1cf50af626.png\"},{\"id\":88810931,\"identity\":\"fa09be3e-91c2-4ba4-9819-c22d53fe4fba\",\"added_by\":\"auto\",\"created_at\":\"2025-08-11 15:36:45\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":4158444,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSchematic diagram of auxin and salicylic acid biosynthesis pathways in peach roots in response to BA stress. (A) In the auxin biosynthesis pathway, BA treatment reduces the accumulation of IAA and its metabolite IAId. (B) In salicylic acid biosynthesis pathway, BA treatment significantly increases the accumulation of SA. Error bars indicate means ± standard deviation (SD) from three biological replicates.An asterisk (*) indicates a significant difference at \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6909418/v1/aa8058d2d256d2663b83685d.png\"},{\"id\":90914683,\"identity\":\"45865fa8-bd65-4fe9-bb58-62e0949f8216\",\"added_by\":\"auto\",\"created_at\":\"2025-09-09 14:05:25\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":61030236,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6909418/v1/97185efa-9d1f-4f0e-ae64-e31a20852138.pdf\"},{\"id\":88809936,\"identity\":\"1a08fdb7-6837-4a51-bbf0-d43b0da84fae\",\"added_by\":\"auto\",\"created_at\":\"2025-08-11 15:28:45\",\"extension\":\"xlsx\",\"order_by\":5,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":21644,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Supplymentaltable.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6909418/v1/bd415e56eab05c61fe8dfdfc.xlsx\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Benzoic acid inhibits peach root development via disrupted auxin distribution\",\"fulltext\":[{\"header\":\"Key message\",\"content\":\"\\u003cp\\u003eWe established a non-sterile root transformation system in peach seedlings. Using this system, we demonstrated that BA treatment inhibits plant growth and lateral root emergence by disrupting auxin distribution.\\u003c/p\\u003e\"},{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eThe replant problem (also known as continuous cropping obstacle) affects a wide range of plant species globally and causes significant economic losses in both the forestry and agricultural sectors (Dong et al. \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). Peach (\\u003cem\\u003ePrunus persica\\u003c/em\\u003e), one of the most affected species, is grown worldwide (Ricci et al., \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Zhang et al., \\u003cspan citationid=\\\"CR57\\\" class=\\\"CitationRef\\\"\\u003e2023\\u003c/span\\u003e). Autotoxicity, a type of interspecific allelopathy, occurs when a plant species inhibits its own kind by releasing toxic chemicals into the environment. This is a major factor contributing to the peach replanting problem. Benzoic acid (BA), a key autotoxic compound in peach, strongly inhibits peach plant growth (Zhu et al. \\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). However, the precise impact of BA on peach root development remains unclear.\\u003c/p\\u003e\\u003cp\\u003eContinuous cropping of peach on the same land leads to more pronounced issues compared to other \\u003cem\\u003ePrunus\\u003c/em\\u003e species, due to their shorter life cycle and the rapid replacement of cultivars. This results in reduced growth, yield, and quality, as well as increased susceptibility to pests and diseases (Zhu et al., \\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Yin et al., \\u003cspan citationid=\\\"CR55\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; He et al., \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Replanting diseases are primarily driven by allelopathic autotoxicity, shifts in microbial communities, and soil fertility imbalances (Guo et al., \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e; Yim et al., \\u003cspan citationid=\\\"CR54\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Zhang et al., 2020). Autotoxins disrupt plant growth not only by acting as chemical signals in plant-microorganism interactions, which alter microbial community structure and indirectly affect plant growth, but also serving as stress factors that directly impair plant function and modify root secretion characteristics. This makes autotoxic substances a major contributor to replanting disease (Shen et al. \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). The complex processes involved in the generation, accumulation, and detoxification of various autotoxins require a deeper exploration of the specific mechanisms through which plant roots respond to these compounds.\\u003c/p\\u003e\\u003cp\\u003eAutotoxins are released into the surrounding environment through leaching from aboveground plant parts, volatilization, root secretion, and residue decomposition (Liu et al., \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e; Gallego et al., \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Zhang et al., 2020). Various autotoxic substances have been identified across numerous plant species, including phenolic compounds (such as simple phenolics, flavonoids, coumarins and quinones), terpenoids (monoterpenes, sesquiterpenes, diterpenes, triterpenes and steroids), alkaloids, nitrogen-containing chemicals (non-protein amino acids, benzoxazinoids and cyanogenic glycosides), and other chemical families (Kong et al. \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Benzoic acid (BA) is a well-known autotoxin found in root exudates and rhizosphere soils of various plant species, where it causes significant allelopathic suppression (Asaduzzaman and Asao 2012; Zhang et al. \\u003cspan citationid=\\\"CR58\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). BA has been widely detected in peach roots and replanted soils, where it exerts a pronounced inhibitory effect on peach plant growth (Zhu et al. \\u003cspan citationid=\\\"CR64\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). Research shows that BA stress inhibits peach seedlings growth, with higher BA concentrations leading to more severe damage to root biomass and length (Shen et al. \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). However, the precise impact of BA on peach roots and the underlying molecular mechanisms remain unknown.\\u003c/p\\u003e\\u003cp\\u003eAuxin plays a crucial role in regulating root growth and lateral root formation (Fukaki et al. \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2007\\u003c/span\\u003e; Overvoorde et al. \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2010\\u003c/span\\u003e). It is primarily synthesized in actively growing tissues, including shoot and root tips, young leaves, developing seeds (Pop et al. \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). Auxin is transported in polarized streams via auxin transporters, a process coordinated by AUXIN RESISTANT1/LIKE AUX1 (AUX1/LAX) uptake permeases, ATP Binding Cassette subfamily B (ABCB) transporters, and PIN-FORMED (PIN) carrier proteins, driven by chemiosmotic gradients (Geisler et al., \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e; Zwiewka et al., \\u003cspan citationid=\\\"CR65\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e; Hammes et al., \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Although several auxin biosynthesis pathways have been identified, the IAA/YUC pathway is the predominant route for endogenous auxin biosynthesis in many plant species (Cao et al. \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). This pathway involves two steps: the TAA family transaminase converts tryptophan into indole-3-pyruvate (IPA), and the flavin monooxygenase YUC family then converts IPA into indole-3-acetic acid (IAA or auxin) (Zhao \\u003cspan citationid=\\\"CR61\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e). Auxin responses are mediated by members of the Auxin Response Factor (ARF) family of transcription factors and the Aux/IAA proteins (Ulmasov et al. \\u003cspan citationid=\\\"CR47\\\" class=\\\"CitationRef\\\"\\u003e1999\\u003c/span\\u003e). However, it remains unclear whether BA stress affects peach root development through auxin-related pathways.\\u003c/p\\u003e\\u003cp\\u003eInvestigating the mechanism of autotoxicity in peach plants by using transgenic approaches is essential (Uematsu et al., \\u003cspan citationid=\\\"CR46\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e; Zhou et al., \\u003cspan citationid=\\\"CR63\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Wang et al., \\u003cspan citationid=\\\"CR50\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e), although a stable transformation protocol is still lacking. Recently, a transient gene transformation method was developed for gene function analysis in peach, though its efficiency is genotype-dependent (Cheng et al. \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). Additionally, the use of \\u003cem\\u003eAgrobacterium rhizogenes\\u003c/em\\u003e strain MSU440 to generate transgenic roots in peach has also been established (Xu et al. \\u003cspan citationid=\\\"CR52\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e), but it requires a tedious tissue culture process.\\u003c/p\\u003e\\u003cp\\u003eIn this study, we first investigated the morphophysiological changes of peach plants under BA stress. The results showed that BA treatment inhibited root growth and lateral root emergence. Gene expression analysis of auxin-related genes revealed significant reductions in expression of several auxin response gene \\u003cem\\u003ePpARFs\\u003c/em\\u003e, the biosynthesis gene \\u003cem\\u003ePpYUC10\\u003c/em\\u003e, and the auxin influx transporter \\u003cem\\u003ePpAUX1\\u003c/em\\u003e, in response to BA treatment. Next, we introduced the \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e auxin response reporter system into peach roots using a non-sterile root transformation system to generate transgenic roots. Using these transgenic roots, we observed that \\u003cem\\u003eDR5\\u003c/em\\u003e expression pattern in root tips and lateral root primordia was altered under BA stress. These findings were further supported by hormone measurements, which showed a reduction in auxin levels and an induction of its antagonistic hormone, salicylic acid. Overall, our data provide new insights into the auxin response mechanisms in peach roots under autotoxicity stress, and the non-sterile root transformation system will serve as a valuable tool for analyzing gene functions in peach roots.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eBenzoic acid inhibits peach root growth and lateral root emergence\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eBenzoic acid (BA) has been identified as a major autotoxin in peach, causing stunted seedling growth or even death\\u0026nbsp;(Zhu et al. 2017). To evaluate the impact of BA on plant root growth, we applied 0.8 mM BA to peach seedlings\\u0026nbsp;(Shen et al. 2021). After two weeks, the overall size of BA treated peach seedlings was smaller than that of the control group (Figure 1A). Both stem and root lengths were significantly reduced in the BA treated seedlings compared to the control (Figure 1B). In addition, the number of lateral roots was significantly decreased upon BA treatment.\\u003c/p\\u003e\\n\\u003cp\\u003eSince lateral root development involves multiple steps (P\\u0026eacute;ret et al. 2009), we further investigated which stage of lateral root development was affected by BA. The number of lateral root primordia in a 5 cm root fragment, including the root tip, was counted using a microscope after staining with eosin. Auxin response is a critical requirement for the development of lateral root primordium (Du and Scheres 2018). To assess the number of active lateral root primordia, we generated peach root containing the \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e reporter (see result section 3). Our results showed that the number of lateral root primordia expressing \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e was significantly lower in the BA treated roots. However, no significant difference was observed in the total number of lateral root primordia between the control and BA treated conditions. These data suggest that the reduced lateral root number following BA treatment is due to the inhibition of lateral root emergence rather than the prevention of lateral root initiation.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eBenzoic acid treatment downregulates the expression of auxin-related genes in peach roots\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAuxin plays a crucial role in plant root growth and lateral root development. To investigate the impact of BA on the expression of auxin-related genes in peach roots, we conducted RNA sequencing. To identify auxin-related genes in peach, we performed BLAST searches using Arabidopsis protein sequences for PINs, AUX1, LAXs, YUCs and ARFs\\u003cem\\u003e\\u0026nbsp;\\u003c/em\\u003e(TAIR, https://www.arabidopsis.org) against the peach genome (PPGV, http://peachtree.work/home). This search identified 5\\u003cem\\u003e\\u0026nbsp;PpPIN\\u003c/em\\u003e, 3 \\u003cem\\u003ePpLAX/AUX\\u003c/em\\u003e, 5 \\u003cem\\u003ePpYUC\\u003c/em\\u003e and 12 \\u003cem\\u003ePpARF\\u003c/em\\u003e gene members. An evolutionary tree was constructed based on these genes (Figure 2A). Since not all of these auxin-related genes are expressed in peach roots, subsequent analysis focused on the highly expressed genes (Supplementary Table. S1)\\u0026nbsp;(Shen et al. 2021).\\u003c/p\\u003e\\n\\u003cp\\u003eTo examine the effects of exogenous BA on auxin-related gene expression in peach roots, we analyzed gene expression using digital PCR (dPCR) (Figure 2B). The primers used to amplify the fragments are listed in Supplementary Table S2. Exogenous BA treatment did not significantly affect the expression of auxin efflux transport-related genes compared to the control. In contrast, auxin uptake transport-related genes \\u003cem\\u003eAUX/LAX\\u0026nbsp;\\u003c/em\\u003eshowed different expression patterns. \\u003cem\\u003ePpAUX1\\u003c/em\\u003e was down-regulated by more than 1.8-fold, while \\u003cem\\u003ePpLAX2\\u003c/em\\u003e was up-regulated. No significant difference was observed for \\u003cem\\u003ePpLAX3\\u003c/em\\u003e compared to the control. Among the auxin response transcription factor (\\u003cem\\u003eARF\\u003c/em\\u003e) genes, \\u003cem\\u003ePpARF4\\u003c/em\\u003e, \\u003cem\\u003ePpARF6\\u003c/em\\u003e, \\u003cem\\u003ePpARF8\\u003c/em\\u003e, \\u003cem\\u003ePpARF18\\u003c/em\\u003e, and \\u003cem\\u003ePpARF19\\u003c/em\\u003e were down-regulated by exogenous BA, while \\u003cem\\u003ePpARF1\\u003c/em\\u003e and \\u003cem\\u003ePpARF3\\u003c/em\\u003e were up-regulated. For the auxin synthesis-related genes, \\u003cem\\u003ePpYUC10\\u0026nbsp;\\u003c/em\\u003ewas down-regulated by more than 3.5-fold following exogenous BA.\\u003cem\\u003e\\u0026nbsp;PpYUC2\\u0026nbsp;\\u003c/em\\u003eand\\u003cem\\u003e\\u0026nbsp;PpYUC6\\u0026nbsp;\\u003c/em\\u003eshowed no significant difference, while \\u003cem\\u003ePpYUC8\\u003c/em\\u003e was up-regulated. However, aside from \\u003cem\\u003ePpYUC2\\u003c/em\\u003e, \\u003cem\\u003ePpYUC8\\u003c/em\\u003e presented the lowest Cn/\\u0026mu;L value.\\u003c/p\\u003e\\n\\u003cp\\u003eThese findings suggest that exogenous BA treatment alters the expression of auxin-related genes in the peach root system, which may lead to disrupted auxin accumulation and, consequently, reduced root development of peach.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eImplementation of a simple and effective root transgenic system in peach\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe\\u003cem\\u003e\\u0026nbsp;DR5\\u0026nbsp;\\u003c/em\\u003eauxin reporter system has been successfully applied to investigate auxin distribution and response at the cellular or tissue level in plants (Benkov\\u0026aacute; et al. 2003; Chen et al. 2013). In this study, we aimed to explore the spatiotemporal expression patterns and signaling pathways of auxin under BA treatment using the \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e system. However, the current limitations of the gene transformation system in peach have severely restricted progress in this research.\\u003c/p\\u003e\\n\\u003cp\\u003eTo address the challenges associated with genetic transformation of peach roots, we first optimized the infection conditions for \\u003cem\\u003eAgrobacterium rhizogenes\\u003c/em\\u003e-mediated genetic transformation. We tested three different \\u003cem\\u003eAgrobacterium rhizogenes\\u003c/em\\u003e infection methods: soaking infection, daubing infection, and combined infection. The results showed that soaking infection yielded the highest transformation efficiency (4.77%), followed by daubing infection (3.33%), while combined infection resulted in the lowest efficiency (0.67%) (Figure 3A-D). Therefore, soaking infection with 7 days of co-cultivation was determined to be the most suitable method for transforming peach root using this system. Next, we evaluated the impact of infection sites on transformation efficiency. Instead of infecting the hypocotyls, we infected the root meristem by excising a small portion of the tender yellow root tip (Figure 3E). This approach resulted in a significantly higher transformation efficiency of 27.11% (Figure 3F). Additionally, this root transformation method does not require a sterile working environment (Figure 3G).\\u003c/p\\u003e\\n\\u003cp\\u003eThe optimized root transformation protocol will be further applied to generate transgenic peach roots containing \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e reporter system, enabling the investigation of spatial auxin responses following BA treatment in peach roots.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eBenzoic acid inhibits primary and lateral root development by disrupting auxin distribution in peach\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo study the spatial distribution of auxin signaling in root tip and lateral root primordia under BA stress, we introduced \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e reporter construct into peach roots using the root transformation method described above. We then analyzed the \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e expression pattern in semithin longitudinal peach root sections following two weeks of BA treatment.\\u003c/p\\u003e\\n\\u003cp\\u003eExamination of these semithin sections revealed reduced GUS signal expression in columella cells, stem cell niche and its surrounding cells (Figure 4A and 4B) of BA treated roots (Figure 4C and 4D). This suggests that benzoic acid treatment diminished auxin signaling in the root tips. In addition, we observed a reduction in root meristem size, root thickness, and the number of root cap cell layers following BA treatment. Next, we examined whether auxin signaling was altered in the lateral root primordia under BA stress using the \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e reporter. In the control roots, \\u003cem\\u003eDR5\\u003c/em\\u003e expression was detected in the lateral root primordia and its surrounding tissues, including the endodermis/cortex and vascular cells both overlying and beneath the lateral root primordium (Figure 4E and 4F). However, after BA treatment, \\u003cem\\u003eDR5\\u003c/em\\u003e expression persisted in the lateral root primordium but was strongly suppressed in its surrounding cells, especially in the tissues overlying and beneath the primordium (Figure 4G and 4H).\\u003c/p\\u003e\\n\\u003cp\\u003eTogether, these results demonstrate that BA stress interferes with auxin distribution, inhibiting both primary root growth and lateral root emergence.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eBenzoic acid treatment interferes with auxin and salicylic acid biosynthesis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo investigate the BA-mediated changes in auxin metabolism in peach roots, we measured the levels of various auxin metabolites as well as other plant hormones, including cytokinins (CK), gibberellins (GA), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA) and strigolactones (SL). A total of 51 metabolites were identified and grouped into seven categories based on their hormone classification (Supplementary Table. S3).\\u003c/p\\u003e\\n\\u003cp\\u003eAmong the auxin-related metabolites, we observed an increase in L-tryptophan accumulation, but the levels of indole-3-carboxaldehyde and methyl indole-3-acetate were reduced, although these changes were not statistically different (Figure 5A). In contrast, the levels of salicylic acid and salicylic acid 2-O-\\u0026beta;-glucoside were significantly increased (Figure 5B). No significant changes were detected in the levels of other hormone-related compounds.\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eIt has been reported that BA compromises peach root growth (Shen et al. 2021), and plant secondary metabolites are involved in plant growth and development, potentially influencing the regulation of plant hormones under stress by altering auxin synthesis and distribution (Vanneste \\u0026amp; Friml, 2009; Cheng \\u0026amp; Cheng, 2015). This study demonstrates that the peach autotoxic compound benzoic acid inhibits root growth and lateral root emergence via influencing auxin distribution in roots.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eBA inhibits plant root growth and lateral root emergence via auxin response\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eHere, we showed that four parameters (stem length, root length, root weight and number of lateral roots) were significantly reduced under BA treatment (Figure 1B). These phenotypical changes are consistent with the effects of autotoxic substances observed in other species, such as cucumber\\u0026nbsp;(Bu et al. 2019), \\u003cem\\u003eMedicago sativa\\u0026nbsp;\\u003c/em\\u003e(Wang et al., 2022), tobacco\\u0026nbsp;(Chen et al. 2019)\\u0026nbsp;and maize\\u0026nbsp;(Nickel et al. 1995). However, it remains unknown whether BA is also responsible for the inhibitory effects on root growth in these species.\\u003c/p\\u003e\\n\\u003cp\\u003eIn addition to these effects, our tissue sections revealed that BA treatment significantly reduced the root meristem size, root cap cell layers and lateral root emergence. Plants respond to abiotic stresses through a series of physiological, biochemical, and metabolic modifications, including manipulating the endogenous molecular and physiological pathways. These processes are largely mediated by the involvement of the plant hormone auxin\\u0026nbsp;(Grunewald et al. 2009). The observed inhibition of peach root meristem and lateral root growth, along with the reduced\\u003cem\\u003e\\u0026nbsp;DR5::GUS\\u0026nbsp;\\u003c/em\\u003eexpression (Figure 4) following BA treatment, strongly supports the involvement of auxin in plant response to abiotic stress.\\u003c/p\\u003e\\n\\u003cp\\u003eAuxin is a key plant hormone that regulates various aspects of root development, including root cell organization, differentiation, lateral root initiation, and emergence (Yun et al. 2023). Mutants with disrupted auxin signaling or defects in the auxin transport system exhibit altered root phenotypes (Fukaki et al., 2002; Benkov\\u0026aacute; et al., 2003). The reduction in \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e expression in the root tip after BA treatment corresponds to the decreased meristem size and root cap cell layers (Figure 4). This is consistent with the role of auxin gradient in regulating cell differentiation from meristematic to elongation zones, as well as from distal stem cells to columella and root cap cells (Ding \\u0026amp; Friml, 2010; Di Mambro et al., 2017; Dubreuil et al., 2018). Furthermore, the auxin response in both underlying and overlying tissues of the lateral root primordia is critical for lateral root emergence (Swarup et al., 2008; Marin et al., 2010; Porco et al., 2016; Du \\u0026amp; Scheres, 2018). The reduced \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e expression in the cells surrounding the lateral root primordia, particularly in vascular, endodermal, and cortical cells, aligns with BA\\u0026apos;s inhibitory effect on lateral root emergence.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eBA affects auxin response, auxin transport and biosynthesis gene expression\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cem\\u003eDR5::GUS\\u003c/em\\u003e expression results indicate auxin signaling response in peach roots is strongly inhibited under BA stress (Figure 4). Our dPCR data revealed five \\u003cem\\u003ePpARFs\\u003c/em\\u003e (\\u003cem\\u003ePpARF4\\u003c/em\\u003e, \\u003cem\\u003ePpARF6\\u003c/em\\u003e, \\u003cem\\u003ePpARF8\\u003c/em\\u003e, \\u003cem\\u003ePpARF18\\u0026nbsp;\\u003c/em\\u003eand \\u003cem\\u003ePpARF19\\u003c/em\\u003e) were down-regulated, while two \\u003cem\\u003ePpARFs\\u0026nbsp;\\u003c/em\\u003e(\\u003cem\\u003ePpARF1\\u003c/em\\u003e and \\u003cem\\u003ePpARF3\\u003c/em\\u003e) were up-regulated under BA stress. Plant auxin response genes,\\u003cem\\u003e\\u0026nbsp;ARFs\\u003c/em\\u003e, are classified into three groups Class-A, B and C. Only Class-A genes act as activators, regulated by auxin through nuclear auxin pathways, while Class-B and C genes act as transcription repressors\\u0026nbsp;(Hern\\u0026aacute;ndez-Garc\\u0026iacute;a et al. 2024). After BA treatment, within these reduced \\u003cem\\u003ePpARFs,\\u003c/em\\u003e three of them (\\u003cem\\u003ePpARF6\\u003c/em\\u003e, \\u003cem\\u003ePpARF8\\u003c/em\\u003e and \\u003cem\\u003ePpARF19\\u003c/em\\u003e) belong to Class-A, primarily acting as activators, while the up-regulated\\u003cem\\u003e\\u0026nbsp;PpARFs\\u0026nbsp;\\u003c/em\\u003e(\\u003cem\\u003ePpARF1\\u003c/em\\u003e and \\u003cem\\u003ePpARF3\\u003c/em\\u003e) are all from Class-B, functioning mainly as repressors. In Arabidopsis and rice, lateral root primordia outgrowth requires repression of repressor \\u003cem\\u003eARFs,\\u003c/em\\u003e such as \\u003cem\\u003eARF2\\u003c/em\\u003e, \\u003cem\\u003eARF3\\u003c/em\\u003e and\\u003cem\\u003e\\u0026nbsp;ARF4\\u0026nbsp;\\u003c/em\\u003ein the vascular cells\\u0026nbsp;(Marin et al. 2010), and upregulation of activator \\u003cem\\u003eARFs\\u003c/em\\u003e, such as\\u003cem\\u003e\\u0026nbsp;ARF7\\u003c/em\\u003e and \\u003cem\\u003eARF19\\u003c/em\\u003e (Lee et al. 2009; Yamauchi et al. 2019). The expression profile of\\u003cem\\u003e\\u0026nbsp;PpARFs\\u003c/em\\u003e, particularly the reduction in \\u003cem\\u003ePpARF19\\u003c/em\\u003e, helps explain the inhibition of lateral root emergence under BA treatment and directly reflects the reduced auxin response in peach roots.\\u003c/p\\u003e\\n\\u003cp\\u003ePolar auxin transport, which refers to the directional movement of auxin between cells, is critical for establishing plant developmental patterns. This process is mainly mediated by the asymmetric localization of auxin efflux carriers PIN-FORMED (PIN) protein family and auxin influx carriers AUXIN1/LIKE-AUX1 (AUX/LAX) protein family (Swarup \\u0026amp; P\\u0026eacute;ret, 2012). Under BA treatment, we found that the transcriptional levels of all \\u003cem\\u003ePpPIN\\u003c/em\\u003e genes were not significantly affected by BA (Figure 2B). However, \\u003cem\\u003ePpAUX1\\u003c/em\\u003e expression was significantly reduced in BA treated roots compared to controls (Figure 2B). In Arabidopsis,\\u003cem\\u003e\\u0026nbsp;AUX1\\u003c/em\\u003e has been implicated in lateral root development (Swarup \\u0026amp; P\\u0026eacute;ret, 2012). Although \\u003cem\\u003ePpLAX2\\u003c/em\\u003e expression increased under BA treatment (Figure 2B), single mutations in \\u003cem\\u003eLAX2\\u003c/em\\u003e did not negatively affect lateral root formation in Arabidopsis (Da Costa et al. 2020). Collectively, auxin transporters may also contribute to inhibiting peach root system development by disrupting the auxin accumulation pattern after BA stress. Since the function of auxin transporters largely depends on their subcellular localization, especially PINs (Zhang et al., 2020), studying their protein dynamics in response to BA treatment will offer new insights into how BA interferes with polar auxin transport, ultimately leading to root growth inhibition.\\u003c/p\\u003e\\n\\u003cp\\u003eAdditionally, we observed that \\u003cem\\u003ePpYUC10\\u0026nbsp;\\u003c/em\\u003eexpression was reduced after treatment with 0.8 mM BA (Figure 2B). YUCCA flavin monooxygenases catalyze a rate-limiting step in auxin biosynthesis and play a crucial role in the formation of both embryonic and postembryonic organs (Cheng et al., 2006). Disruption of four \\u003cem\\u003eYUC\\u003c/em\\u003e genes (\\u003cem\\u003eYUC1\\u003c/em\\u003e, \\u003cem\\u003eYUC4\\u003c/em\\u003e, \\u003cem\\u003eYUC10\\u003c/em\\u003e, and \\u003cem\\u003eYUC11\\u003c/em\\u003e) in Arabidopsis resulted in seedlings lacking a hypocotyl and a root meristem (Cheng et al., 2007). Our findings demonstrate that auxin signaling was significantly attenuated in the root tips and lateral root primordia under BA treatment (Figure 4). This suggests that the low expression of \\u003cem\\u003ePpYUC10\\u003c/em\\u003e, combined with the reduced auxin level, may mediate the peach root response to external BA, ultimately leading to slower root growth and fewer lateral roots.\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAccumulated SA may interfere with auxin distribution and biosynthesis during BA treatment\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eOur data show that SA is significantly induced upon BA treatment. In the SA biosynthesis pathway, BA is thought to be catalyzed by benzoic acid hydroxylase (BA2H), converting it into SA\\u0026nbsp;(Leon et al. 1993). Therefore, the external application of BA leads to SA accumulation in peach roots. Additionally, the changes in auxin distribution under BA treatment may result from this SA accumulation in peach roots. For instance, protein kinase CK2 plays a crucial role in both SA and auxin pathways\\u0026nbsp;(Wei et al. 2021).\\u003c/p\\u003e\\n\\u003cp\\u003eSA influences root apical meristem patterning and lateral root formation by disturbing the auxin distribution through the regulation of auxin-related genes expression, such as auxin biosynthesis TAA1 and auxin efflux PINs, in a concentration-dependent manner\\u0026nbsp;(Armengot et al. 2014; Pasternak et al. 2019; Wei et al. 2021). Our results show that BA treatment causes root apical meristem patterning similar to that observed in roots treated with high levels of SA, displaying reduced meristem size and altered auxin distribution (as indicated by \\u003cem\\u003eDR5::GUS\\u0026nbsp;\\u003c/em\\u003eexpression).\\u003c/p\\u003e\\n\\u003cp\\u003eFurthermore, we found that BA treatment increases L-tryptophan levels, which may also be related to SA accumulation. It has been shown that the external application of SA for 5 days in Arabidopsis induces TAA expression\\u0026nbsp;(Pasternak et al. 2019). TAA is the enzyme responsible for converting L-tryptophan to indole-3-pyruvic acid\\u0026nbsp;(Luo and Di 2023). However, whether SA will induce L-tryptophan accumulation and the role of the increased L-tryptophan requires further investigation. Tryptophan-derived secondary metabolites are deposited in cell walls as physical defense compounds during pathogen invasion (Ishihara et al., 2008; Consonni et al., 2010). BA induced L-tryptophan is most likely related to the root defensive response rather than enhanced auxin production. After BA treatment, tryptophan-derived secondary metabolites indeed accumulated and we observed reduced auxin levels (Figure 5A) (Supplementary Table. S4)\\u0026nbsp;(Shen et al. 2021).\\u003c/p\\u003e\\n\\u003cp\\u003eIn addition, during the plant defense response, it is common for SA accumulation to coincide with a reduction in auxin biosynthesis, transport and response (Zhong et al. 2021). Since BA is an important autotoxic compound, we cannot exclude the possibility that BA triggers the activation of defense factors that play an upstream role in balancing SA accumulation and auxin reduction (Wei et al. 2021).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eIn summary, we found that BA treatment significantly inhibited plant growth, as evidenced by reduced stem length and impaired root development. Under BA stress, roots exhibited decreased auxin accumulation, accompanied by the down-regulation of auxin-responsive and auxin-synthesis genes. Using our established gene transformation system, we investigated changes in auxin expression patterns in roots under autotoxic stress. This study provides valuable insights into the molecular mechanisms by which auxin regulates peach root responses to autotoxicity.\\u003c/p\\u003e\"},{\"header\":\"Material and methods\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003ePlant materials and growth conditions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ePeach (\\u003cem\\u003ePrunus persica\\u003c/em\\u003e) seeds used in this study were stratified in moist sand stored at 4℃ for over 60 days. After stratification, the seeds were transplanted into vermiculite and grown under a 16 hours light cycle at 25℃ and 45% relative humidity. Benzoic acid was dissolved, prepared as a 0.8 mM stock solution, and thoroughly mixed with vermiculite to form the treatment substrate. The control substrate consisted of vermiculite mixed with water. Morphologically uniform 7-day-old seedlings and transgenic plants carrying the \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e reporter were selected and transplanted into the two substrate types for 14d. All treatments were performed with three independent biological replicates, and each replicate consisted of 3 individual plantlets. During the treatment period, the treatment groups were supplemented with 0.8 mM BA stock solution every 3 days, while the control groups received water additions to maintain consistent substrate moisture levels. All plants were cultivated in a growth chamber maintained under a 16 hours light cycle at 25℃. After 14 days culture, the seedlings were collected from both BA treatment and control groups for comprehensive analyses, including physiological parameter measurements, digital PCR assays, metabolite profiling and semithin sections preparation.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eVector construction\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo generate \\u003cem\\u003eDR5::GUS\\u0026nbsp;\\u003c/em\\u003econstruct, seven tandem direct repeats of 11 bp, including the auxin responsive factor gene-binding site (TGTCTC), were amplified by PCR using high fidelity Phusion polymerase (Thermo-Fisher). The PCR product was then cloned into the pENTR-D/-TOPO (Invitrogen). After sequence verification, the DR5 promoter region was recombined into pKGW243 using LR clonase II (Invitrogen), resulting in the \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e construct.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eRNA extraction and Digital PCRs analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTotal RNA was extracted from peach roots using the E.Z.N.A. Plant RNA Kit, following the manufacturer\\u0026rsquo;s protocol. RNA quality was assessed via agarose gel electrophoresis. One microgram of RNA was used for cDNA synthesis using the RevertAid RT kit (Thermo Scientific).\\u003c/p\\u003e\\n\\u003cp\\u003eGene expression of auxin-related genes in the roots was quantified using Sniper DQ24 Digital PCR Platform (Sniper, Suzhou, China). The cDNA was diluted 100-fold and used for dPCR analysis with the 2\\u0026times;dPCR EvaGreen Master mix (Rox) (Sniper, Suzhou, China) and gene-specific primers on the Sniper DQ24 Digital System. The 22 \\u0026mu;L EvaGreen dPCR mixture contained 11 \\u0026mu;L of 2\\u0026times;dPCR EvaGreen Master Mix (Rox) (Sniper, Suzhou, China), 5 \\u0026mu;L of template suspension, 1 \\u0026mu;L of each forward and reverse primers (10 \\u0026mu;mol\\u0026middot;L\\u003csup\\u003e\\u0026minus;1\\u003c/sup\\u003e), and 4 \\u0026mu;L of sterile ultrapure water. The EvaGreen dPCR program was set as follows: droplet generation at 60 ℃ for 5 minutes, pre-denaturation at 95 ℃ for 15 minutes, followed by 40 cycles of denaturation at 95 ℃ for 20 s and annealing/elongation at 58 ℃ for 30 s. Data acquisition and analysis were performed by SightPro software (Sniper).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eNon-sterile\\u003cem\\u003e\\u0026nbsp;Agrobacterium rhizogenes\\u003c/em\\u003e-mediated root transformation\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe \\u003cem\\u003eA. rhizogenes\\u003c/em\\u003e MSU440 strain carrying the \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e plasmids was plated on solid LB medium containing 100 mg/L spectinomycin and incubated at 28℃ for 2-3 days. Single colonies were selected and cultured overnight in LB liquid medium (28℃, 200 RPM). Aliquots of the overnight culture were further propagated with LB solid and liquid medium for either 2 days or overnight. The liquid culture was grown until the OD600 reached 0.8-1.2. Freshly grown bacteria were then centrifuged at 3500 rpm for 15 minutes and resuspended in a suspension buffer (Woody Plant regeneration Medium, 9.75 g/L MES, 0.1% sucrose, 0.5g/L CaCl\\u003csub\\u003e2\\u003c/sub\\u003e, 0.8mg IBA, 100 mM acetosyringone and 0.05% Silwet L-77; pH 5.7). The bacterial suspensions, along with bacteria harvested from solid media, were used to directly infect explants.\\u003c/p\\u003e\\n\\u003cp\\u003ePeach seedlings with 1-2 cm of emerged radicles, germinated in a non-sterile environment, were used for transformation. The root tips were excised either below hypocotyl swelling or from the meristematic zone. Multiple small wounds were gently made on the remaining hypocotyl or root using a sterile scalpel. The wounded seedlings underwent one of three infection methods: 1) soaking infection: seedlings were immersed in an \\u003cem\\u003eA.rhizogenes\\u003c/em\\u003e strain MSU440 suspension containing the target plasmid at room temperature for 40 minutes; 2) daubing infection: seedlings were immersed in distilled water containing 100 mM acetosyringone for 20 minutes at 25℃ in the dark, then the wounded region was coated with \\u003cem\\u003eA. rhizogenes\\u003c/em\\u003e collected from solid medium; or 3) combined infection: seedlings were immersed in the \\u003cem\\u003eA.rhizogenes\\u003c/em\\u003e strain MSU440 suspension and then dipped with \\u003cem\\u003eA. rhizogenes\\u003c/em\\u003e from a solid medium.\\u003c/p\\u003e\\n\\u003cp\\u003eAfter infection, seedlings were transplanted onto co-cultivation medium (solidified with 0.7% agar) and incubated in a growth chamber for 7 days under dark conditions. They were then returned to normal growth conditions, at 25℃ under a 16-hour light cycle. Adventitious roots, which developed from the hypocotyl after 2-3 weeks, were selected for the identification of positive transformants by detecting \\u003cem\\u003eDsRED\\u003c/em\\u003e using a fluorescence stereomicroscope equipped with a digital camera (SMZ25, Nikon). Fluorescence signals were observed using a 400 nm excitation filter and 600 nm emission filter.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eRoot staining, embedding and sectioning\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTransgenic roots containing \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e constructs were stained with GUS buffer (Biotopped, China) and then incubated at 37℃ for 15 minutes to 3 hours as described previously (Na et al. 2011). After staining, the root segments were placed in a fixation buffer (5% glutaraldehyde in 100 mM phosphate buffer, pH 7.2) and incubated at 4℃ overnight. Roots were then dehydrated through an ethanol series (10%, 30%, 50%, 70%, 90% and 100% ethanol for 30 minutes each), followed by infiltration and embedding in Technovit 7100 (Hereus-Kulzer, Germany). The embedded roots were sectioned into 5 \\u0026mu;m longitudinal slices using a microtome (Leica 2035) and stained in 0.1% Ruthenium Red for 15 minutes. To analyze the total number of lateral root primordia, freshly collected roots were first fixed in 70% ethanol for 12 hours, and then immersed in a 1% Eosin B solution dissolved in ethanol for 2 minutes.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eStatistical analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll treatments in this study were biologically replicated at least three times. Data are presented as mean values \\u0026plusmn; SE of the replicates. Statistical analyses were performed using the Statistical Product and Service Solutions (SPSS) software (IBM Co., Armonk, NY, USA). The significance of differences between groups was determined using the least significant difference (LSD) test at a significance level of \\u003cem\\u003eP\\u003c/em\\u003e \\u0026lt; 0.05. All experimental data were also analyzed using Student\\u0026rsquo;s t-test. Graphs and figures were generated using GraphPad Prism 9.0.0 software (GraphPad, San Diego, CA, USA).\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was supported by the 2024 Classification Development Quota Project-Talent Introduction Start-up Fund of Beijing University of Agriculture (5066516006/005) and the 2023 Classification Development Quota Project - Science and Technology Innovation Capacity Enhancement Plan - Mechanisms of Plant-Microbe Interactions Mediated by Peach Secondary Metabolites in Continuous Cropping Soil of Beijing University of Agriculture (5076016183/069).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eQRZ, JY, QZ and TTX designed the experiments. QRZ and JY performed the experiments with occasional help from ZS, FJ, BB, JZ HL and QC. QRZ, JY and FJ analyzed the data. QRZ and TTX wrote the manuscript. All authors read and approved the final manuscript.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflict of interest\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAuthors declare that they have no competing interests.\\u003cstrong\\u003e\\u003cbr\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n \\u003cli\\u003eArmengot L, Marqu\\u0026egrave;s‐Bueno MM, Soria‐Garcia A, et al (2014) Functional interplay between protein kinase CK 2 and salicylic acid sustains \\u003cem\\u003e\\u0026nbsp;PIN\\u0026nbsp;\\u003c/em\\u003e transcriptional expression and root development. The Plant Journal 78:411\\u0026ndash;423. https://doi.org/10.1111/tpj.12481\\u003c/li\\u003e\\n \\u003cli\\u003eAsaduzzaman Md, Asao T (2012) Autotoxicity in beans and their allelochemicals. Scientia Horticulturae 134:26\\u0026ndash;31. https://doi.org/10.1016/j.scienta.2011.11.035\\u003c/li\\u003e\\n \\u003cli\\u003eBenkov\\u0026aacute; E, Michniewicz M, Sauer M, et al (2003) Local, Efflux-Dependent Auxin Gradients as a Common Module for Plant Organ Formation. Cell 115:591\\u0026ndash;602. https://doi.org/10.1016/S0092-8674(03)00924-3\\u003c/li\\u003e\\n \\u003cli\\u003eBu R, Wang R, Wei Q, et al (2019) Silencing of glycerol-3-phosphate acyltransferase 6 (GPAT6) gene using a newly established virus induced gene silencing (VIGS) system in cucumber alleviates autotoxicity mimicked by cinnamic acid (CA). Plant Soil 438:329\\u0026ndash;346. https://doi.org/10.1007/s11104-019-03996-0\\u003c/li\\u003e\\n \\u003cli\\u003eCao X, Yang H, Shang C, et al (2019) The Roles of Auxin Biosynthesis YUCCA Gene Family in Plants. IJMS 20:6343. https://doi.org/10.3390/ijms20246343\\u003c/li\\u003e\\n \\u003cli\\u003eChen Y, Chen W, Lan Y, et al (2019) Determination of 18 phenolic acids in tobacco and rhizosphere soil by ultra high performance liquid chromatography combined with triple quadrupole mass spectrometry. J of Separation Science 42:816\\u0026ndash;825. https://doi.org/10.1002/jssc.201800819\\u003c/li\\u003e\\n \\u003cli\\u003eChen Y, Yordanov YS, Ma C, et al (2013) DR5 as a reporter system to study auxin response in Populus. Plant Cell Rep 32:453\\u0026ndash;463. https://doi.org/10.1007/s00299-012-1378-x\\u003c/li\\u003e\\n \\u003cli\\u003eCheng F, Cheng Z (2015) Research Progress on the use of Plant Allelopathy in Agriculture and the Physiological and Ecological Mechanisms of Allelopathy. Front Plant Sci 6:. https://doi.org/10.3389/fpls.2015.01020\\u003c/li\\u003e\\n \\u003cli\\u003eCheng J, Shao Y, Hu X, et al (2024) A simple and efficient gene functional analysis method for studying the growth and development of peach seedlings. Horticulture Research 11:uhae155. https://doi.org/10.1093/hr/uhae155\\u003c/li\\u003e\\n \\u003cli\\u003eCheng Y, Dai X, Zhao Y (2006) Auxin biosynthesis by the YUCCA flavin monooxygenases controls the formation of floral organs and vascular tissues in \\u003cem\\u003eArabidopsis\\u003c/em\\u003e. Genes Dev 20:1790\\u0026ndash;1799. https://doi.org/10.1101/gad.1415106\\u003c/li\\u003e\\n \\u003cli\\u003eCheng Y, Dai X, Zhao Y (2007) Auxin Synthesized by the YUCCA Flavin Monooxygenases Is Essential for Embryogenesis and Leaf Formation in \\u003cem\\u003eArabidopsis\\u003c/em\\u003e. 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Front Plant Sci 10:985. https://doi.org/10.3389/fpls.2019.00985\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"benzoic acid, Prunus persica, root growth, auxin, root transformation system, salicylic acid\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6909418/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6909418/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eAllelopathic effects are commonly observed in plant communities, with benzoic acid (BA) identified as a primary autotoxin contributing to peach replanting disease. However, the effects of BA on peach root growth remain unclear. In this study, we evaluated the morphological changes in peach roots and the auxin signaling responses to BA stress. Our results showed that BA treatment significantly reduced root length and inhibited lateral root emergence. Gene expression analysis revealed that several auxin-related genes, such as \\u003cem\\u003ePpARF19\\u003c/em\\u003e, \\u003cem\\u003ePpAUX1\\u003c/em\\u003e, \\u003cem\\u003ePpYUCCA10\\u003c/em\\u003e, were significantly reduced after BA treatment. Using transgenic peach roots with the \\u003cem\\u003eDR5::GUS\\u003c/em\\u003e auxin reporter, generated through our optimized non-sterile root transformation protocol, we observed that BA treatment disrupted the \\u003cem\\u003eDR5::GUS\\u003c/em\\u003eexpression pattern in root tips and lateral root primordia. Hormone measurements indicated a slight reduction in auxin accumulation and a significant increase in the auxin antagonistic hormone salicylic acid. These findings suggest that BA induces autotoxicity in peach plants by impairing root growth through alterations in auxin signaling and biosynthesis pathways. This study enhances our understanding of allelopathic effects in plant interactions and provides valuable insights for mitigating challenges in peach orchards.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Benzoic acid inhibits peach root development via disrupted auxin distribution\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-08-11 15:28:40\",\"doi\":\"10.21203/rs.3.rs-6909418/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"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\":\"3a183a9e-1ae3-4ad7-a900-609443fe351a\",\"owner\":[],\"postedDate\":\"August 11th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-09-09T13:56:46+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-08-11 15:28:40\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6909418\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6909418\",\"identity\":\"rs-6909418\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}