A semi-hydroponic cultivation system designed for collecting root exudates from maize in symbiosis with arbuscular mycorrhiza fungi

A semi-hydroponic cultivation system designed for collecting root exudates from maize in symbiosis with arbuscular mycorrhiza fungi | 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 A semi-hydroponic cultivation system designed for collecting root exudates from maize in symbiosis with arbuscular mycorrhiza fungi Xinhao Luo, Xiaowan Geng, Jing Zhou, Jin Chen, Beijiu Cheng, Xiaoyu Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7506078/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Apr, 2026 Read the published version in Journal of Plant Research → Version 1 posted 5 You are reading this latest preprint version Abstract The collection of root exudates, particularly those from plants symbiotically associated with arbuscular mycorrhizal fungi (AMF), were notably challenging. A semi-hydroponic cultivation system (SHCS) was designed to collect rhizosphere exudates from maize in symbiosis with AMF. This system utilizes perlite as a solid support to simulate soil barriers, combined with drip irrigation to facilitate symbiosis and the collection of maize root exudates. SHCS consists of a culture bottle, a solution bottle providing nutrients, a peristaltic pump for powering the system, silicone tubes connecting all components, a flat-jaw pinchcock for operation, and a device shelf for placing all items. Then it was used to collect root exudates from maize-wild type B73 and AMF-inoculated B73, followed by metabolomics analysis using LC-MS/MS. Through comparative analysis, we identified significant differences in metabolite levels between B73 and RiB73. Briefly, a total of 54 metabolites exhibited AMF-related characteristics, and these metabolites were enriched in 15 metabolic pathways. Key metabolites include lumichrome, riboflavin, indolelactic acid, abscisic acid, gibberellin a116, and l-histidinol phosphate. Among them, l-histidinol phosphate significantly decreased after AMF inoculation, while the other metabolites showed a notable increase in content. Semi-hydroponic root exudates maize arbuscular mycorrhizal fungi Metabolomics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Plants continuously release various compounds into their surrounding environment through a process known as exudation. This procedure plays a pivotal role in mediating diverse interactions within the rhizosphere, contributing to the cycling of carbon and nitrogen. (Chai and Schachtman 2022 ; Panchal et al. 2022 ) These compounds, known as root exudates, encompass a diverse array of high- and low-molecular-weight substances spanning various chemical classes, including amino acids, organic acids, alcohols, polypeptides, sugars, phenolics, enzymes, proteins, and hormones. (Baetz and Martinoia 2014 ) And there is still uncertainty regarding whether these compounds secreted by the roots or absorbed from the soil. Under specific conditions, root exudates are harnessed to boost crop yields and enhance tolerance to environmental stresses. Therefore, obtaining pure, uncontaminated root exudates for research is of paramount importance. According to existing research, the methods for collecting root exudates primarily include hydroponics and soil substrate cultivation. Hydroponics typically utilizes pure water or nutrient solutions for cultivation, facilitating the detection of root exudates, as it avoids mechanical damage caused by removing roots from solid substrates. (Strehmel et al. 2014 ) However, this approach cannot emulate the natural mechanical constraints of soil on root development, and the substantial volume of nutrient solution complicates the collection of root exudates. Soil substrate cultivation offers greater versatility and serves effectively as a preliminary screening tool, as it is applied to plants grown in field conditions. (Canarini et al. 2016 ) Whereas, the microbial communities in soil not only affect root exudates, but also secrete their own compounds, which can easily mix with the root exudates, leading to impure samples and interference in subsequent analysis. (Eichmann et al. 2021 ) Additionally, during the washing process, some degree of root damage is inevitable. (Oburger et al. 2013 ) Therefore, to simulate the natural mechanical constraints of soil, avoid root damage, and eliminate the confounding influence of soil microbes, the use of hydroponic systems or supported hydroponics with substrates such as gels, glass beads, or vermiculite presents a superior approach for studying root exudates. (Vranova et al. 2013 ) The symbiotic relationship between plants and arbuscular mycorrhizal fungi (AMF) is a widespread and mutually beneficial interaction, present in over two-thirds of terrestrial plant species. At the ecosystem level, AMF play a crucial role in nutrient cycling and regulate the ecosystem's response to environmental fluctuations, highlighting their irreplaceable ecological significance. (Luo et al. 2024 ; Xu et al. 2022 ; Xu et al. 2024 ) Symbiosis with AMF offers multifaceted benefits to maize, primarily including enhanced nutrient uptake (Cheng et al. 2023 ), improved stress resistance (Colombo et al. 2017 ), better root development (Han et al. 2023 ), strengthened soil health and increased yield and quality (Wang et al. 2019 ). Metabolomics approaches offer a comprehensive view of chemical profiles and have been widely applied in medicine, plant sciences, and food research. (Li et al. 2022 ; Li et al. 2021a ) Nowadays, many studies employ metabolomics to analyze various plant root exudates, aiming to identify all metabolites present in the rhizosphere. Kaur et al has reported that AMF symbiosis can significantly influence the levels of key metabolites such as sugars, organic acids, and amino acids, thereby enhancing plant resistance. (Kaur and Suseela 2020 ) Zhang et al found that AMF can influence the levels of phenolic acids in cotton root exudates, thereby reducing the incidence of Fusarium wilt in cotton. (Zhang et al. 2012 ) Meanwhile, Shukla et al discovered that after AMF colonization, the release of root exudates can be altered to restrict nematode movement, thereby reducing nematode invasion in tomatoes. (Shukla et al. 2015 ) Based on previous research, we designed a semi-hydroponic system for collecting root exudates from maize or other plants, utilizing perlite as a solid support to simulate soil separation. SHCS is integrated with drip irrigation to support symbiosis and facilitate the collection of maize root exudates. At the same time, non-targeted metabolomics analysis of the extracted root exudates was conducted to preliminarily investigate the differences in the presence or absence of AMF symbiosis, thereby validating the applicability of SHCS we designed 2. Materials and methods 2.1 Experimental materials and planting treatments The study material consisted of maize wild-type B73, which were provided by the National Engineering Laboratory of Crop Stress Resistance at Anhui Agricultural University. And the AMF species used was Rhizophagus irregularis (Ri) DAOM 197198, provided by Sun Yat-Sen University, Guangzhou, China. Mycorrhizal colonization and arbuscule size analyses were performed as previously described. (Xue et al. 2015 ) 2.2 Semi-hydroponic The experiment was conducted in a greenhouse to prevent airborne contamination. All assembly equipment was sterilized in an autoclave prior to use. The components of the system all consist of common items, including a 1 L culture bottle, several silicone tubes, an angle iron assembly for the device shelf, a flatjaw pinchcock to control circulation, a 500 mL solution bottle, and a multi-channel peristaltic pump. The growth substrate is perlite, which is sieved through a 60-mesh screen and then sterilized at 121°C for 60 minutes. After cooling for 2 days, it is ready for use. (Fig. 1 ) Select B73 maize seedlings with similar growth, and secure them using perlite. First, place the filter membrane into the culture vessel, followed by a 1–2 cm layer of sterile perlite. Position the seedlings in an appropriate location and add more perlite to secure them. To maintain sterile conditions, the culture vessel should be thoroughly cleaned and then wiped with 75% ethanol before use. Rinse the vessel filled with perlite twice with sterile full-strength Hoagland nutrient solution, then add 350 mL of sterile nutrient solution for cultivation. The vessel should be wrapped with aluminum foil and black opaque tape to prevent algal growth. Finally, cover the bottom of the culture vessel with aluminum foil to reserve space for the plants to protrude. The high-phosphorus Hoagland nutrient solution consists of 5 mmol/L KNO₃, 5 mmol/L Ca(NO₃)₂·4H₂O, 2 mmol/L MgSO₄·7H₂O, 20 µmol/L FeEDTA, 1 mmol/L KH₂PO₄, 1 µmol/L H₃BO₃, and trace elements including 2 µmol/L MnCl₂·4H₂O, 2 µmol/L ZnSO₄, 0.2 µmol/L CuSO₄·5H₂O, and 0.2 µmol/L (NH₄)₆Mo₇O₂₄. A constant nutrient solution flow rate of 350 mL/h was maintained. For each AMF maize plant, a mixture of 40 g of spore-sand (200 spores) was added. Wrap the solution bottle with aluminum foil and black light-blocking tape to simulate the darkness of the soil environment as closely as possible. After planting, keep the apparatus in a greenhouse with a 16-hour light/26°C and 8-hour dark/18°C cycle, and a relative humidity of approximately 60%. (Fig. 2 ) 2.3 Trypan Blue Staining Trypan blue staining of mycorrhizal roots from maize was performed according to established protocols. (Sportes et al. 2021 ; Xu et al. 2022 ) Root segments of maize were initially fixed in FAA (formalin acetic acid alcohol) for 4 hours. Subsequently, the segments were incubated at high temperature with 10% KOH, acidified, and clarified. Finally, the segments were stained with a 0.05% trypan blue solution. The AM fungal structures within the root segments were subsequently visualized using a DM5000B microscope (Leica, Wetzlar, Germany). The mycorrhizal infection rate and infection intensity of maize inoculated with Ri for 30 and 60 days were assessed following published methods. (Wang et al. 2022 ) 2.4 Sample preparation On the 60 days after planting, fill the culture bottles with 300 mL of ultrapure water to collect maize root exudates. After 12 hours, transfer the liquid to labeled sealed bags and store at -80°C for 24 hours. Next, freeze-dry the samples for 72 hours to obtain the lyophilized root exudate powder. Store the powder in centrifuge tubes for subsequent metabolomic sequencing. 50 mg solid sample was added to a 2 mL centrifuge tube along with a 6 mm diameter grinding bead. Metabolite extraction was performed using 400 µL of extraction solution (methanol: water = 4:1 (v:v)) containing 0.02 mg/mL of internal standard (L-2-chlorophenylalanine). The samples were ground using the Wonbio-96c frozen tissue grinder (Shanghai Wanbo Biotechnology Co., LTD) for 6 minutes (-10°C, 50 Hz), followed by low-temperature ultrasonic extraction for 30 minutes (5°C, 40 kHz). The samples were then placed at -20°C for 30 minutes, centrifuged for 15 minutes (4°C, 13000 g), and the supernatant was transferred to the injection vial for LC-MS/MS analysis. 2.5 Metabolomics analysis of UPLC-Orbitrap-MS/MS The LC-MS/MS analysis of the sample was conducted on a Thermo UHPLC-Q Exactive system equipped with an ACQUITY HSS T3 column (100 mm × 2.1 mm i.d., 1.8 µm; Waters, USA). The mobile phases consisted of 0.1% formic acid in water (95:5, v/v) (solvent A) and 0.1% formic acid in acetonitrile: isopropanol (47.5:47.5:5, v/v) (solvent B). Positive ion mode separation gradient: 0–3 min (20% B), 3-4.5 min (35% B), 4.5-5 min (100% B), 5-6.3 min (maintained at 100% B), 6.3–6.4 min (0% B), 6.4-8 min (maintained at 0% B); Negative ion mode separation gradient: 0-1.5 min (5% B), 1.5-2 min (10% B), 2-4.5 min (30% B), 4.5-5 min (100% B), 5-6.3 min (maintained at 100% B), 6.3–6.4 min (0% B), 6.4-8 min (maintained at 0% B). The flow rate was 0.40 mL/min and the column temperature was 40℃. 2.6 Statistical analysis Raw data were obtained from UPLC-Orbitrap-MS/MS. The preprocessed datasets, encompassing both positive and negative modes, were analyzed using unsupervised principal component analysis (PCA), supervised orthogonal partial least squares discriminant analysis (OPLS-DA) with SIMCA-P software (version 14.1) (Umetrics, Umeå, Sweden). Potential differential metabolites among B73 and RiB73 were identified based on VIP > 1.0, p-value 1.5. VIP is a metric used in multivariate statistical methods like PLS-DA. It quantifies the contribution of each variable (e.g., metabolites, genes, or features) to the model, and fold change measures the ratio of change between two conditions, typically expressed as a magnitude of up- or down-regulation. It is commonly used in biological and omics studies. (Li et al. 2022 ; Li et al. 2021b ) A heat map was generated using OECloud tools at https://cloud.oebiotech.com . Metabolite peaks were annotated using accurate mass measurements through online metabolite databases such as HMDB ( https://hmdb.ca/ ) and PubChem ( https://pubchem.ncbi.nlm.nih.gov/ ). All results were analyzed as mean ± S.E.M. T-tests were employed to determine significant differences among different groups or between two specific groups, respectively, using GraphPad Prism 9 (GraphPad Software, USA). Statistical significance was established at p < 0.05. 3. Results 3.1 Maize grows normally in a SHCS To assess the growth of wild-type maize B73 under semi-hydroponic conditions, we measured several basic physiological parameters, including plant height, root length, aboveground biomass, underground biomass, blade length, and total chlorophyll level. Under semi-hydroponic conditions, maize showed a significant increase in plant height, root length, shoot biomass, root biomass, and leaf length at 30 days compared to 15 days. Additionally, total chlorophyll level remained stable, with no significant difference observed between 15 and 30 days. (Fig. S1 ) These results demonstrate that maize can grow normally in the semi-hydroponic system we established. 3.2 AMF promoted maize growth The colonization of AM fungi can significantly enhance plant growth. At 60 days, RiB73 (wild-type maize B73 colonize Rhizophagus irregularis ) showed a significant increase in plant height, root length, shoot biomass, root biomass, and leaf length at 60 days compared to B73. Meanwhile, total chlorophyll level show no significant difference between B73 and RiB73. (Fig. 2 ) The inoculation effectiveness was assessed through trypan blue staining. No fungal structures of AMF were observed under a standard microscope after trypan blue staining in the untreated B73 group. Following AMF inoculation, structures such as arbuscules, vesicles, and hyphaws were observed in the maize. (Fig. 3 h-j) Simultaneously, we assessed the mycorrhizal infection rate and infection intensity in maize inoculated with Ri to ensure successful colonization. (Fig.S4) 3.3 Significant different chemical profiles among B73 and RiB73 by UPLC-Orbitrap-MS/MS Chemical profiles of B73 and RiB73 were detected by UPLC-Orbitrap-MS/MS system. A total of 3313 ion features in positive ion mode and 3687 ion features negative ion mode were detected, respectively. (Fig. S1 ) Firstly, the PCA was applied to evaluate intra- and inter-group differences and to reduce background noises. (Fig. 4 a, b) Secondly, the OPLS-DA statistical analysis methods were further employed to evaluate the deference of chemical profiles between B73 and RiB73. Furthermore, R2X, R2Y and Q2 were calculated as 0.695, 0.999 and 0.953 in positive and 0.911, 0.999, 0.992 in negative, respectively which indicated that the models were established successfully without over-fitting B73 and RiB73 exhibited significant separation, indicating distinct chemical profiles. (Fig. 4 c, d) And R2Y and Q2 of permutation were calculated as 0.998 and 0.509 in positive and 0.998 and 0.738 in negative. (Fig. 4 e, f) 3.4 Characteristic metabolites of B73 and RiB73 Variables with VIP > 1, p-value 1.5 were identified as potential differential metabolites. These metabolites were characterized by comparing their retention time (RT) and m/z values with relevant data published in the literature or available in databases. A total of 54 metabolites were tentatively identified, comprising 34 in positive mode and 20 in negative mode. The heat map based on the 54 differentially abundant metabolites offered a comprehensive overview of the differences in metabolite content between B73 and RiB73. (Fig. 5 a, b) In the plot, red and blue colors represent values higher and lower than the mean, respectively. 3.5 Metabolic pathway analysis between B73 and RiB73 The analysis of metabolic pathways based on differential metabolites helps us understand how metabolic pathways were enriched between B73 and RiB73. It presented an enrichment analysis of metabolic pathways in B73 and RiB73. (Fig. 6 a) The data were visualized with the logarithm of the significance level (p-value) on the y-axis and the pathway impact on the x-axis. Through enrichment analysis and topology, we identified 15 metabolic pathways, including phenylalanine, tyrosine, and tryptophan biosynthesis, biosynthesis of various plant secondary metabolites, phenylpropanoid biosynthesis, linoleic acid metabolism, arachidonic acid metabolism, riboflavin metabolism, histidine metabolism, biotin metabolism, pentose phosphate pathway, carbon fixation in photosynthetic organisms, sphingolipid metabolism, α-linolenic acid metabolism, diterpenoid biosynthesis, glycerophospholipid metabolism, and cysteine and methionine metabolism. In these enriched pathways, we focused on metabolites of interest. For instance, riboflavin and lumichrome in the riboflavin metabolism pathway, L-histidinol phosphate in the histidine metabolism pathway, gibberellin A116 in the diterpenoid biosynthesis pathway, as well as key plant hormones like indolelactic acid (iaa) and abscisic acid (aba). (Fig. 6 b-g) except for L-histidinol phosphate, the levels of all other metabolites significantly increased after AMF inoculation. 4. Discussion The collection of root exudates poses a significant challenge in studying AMF metabolism. Hydroponic systems lack the natural mechanical barriers provided by soil, while soil cultivation cannot avoid microbial influences and mechanical damage during sampling. (Barros et al. 2020 ; Yang et al. 2023 ) To address these challenges, a SHCS was designed to collect rhizosphere exudates from maize in symbiosis with AMF. This system utilizes perlite as a solid support to simulate soil barriers, combined with drip irrigation to facilitate symbiosis and the collection of maize root exudates. Then it was used to collect root exudates from B73 and AMF-inoculated B73, followed by metabolomics analysis using LC-MS/MS. Through comparative analysis, we identified significant differences in metabolite levels between B73 and RiB73. Our recirculating semi-hydroponic system effectively supports the healthy growth of maize plants and is well-suited for the collection of root exudates. The advantages of this system are comprehensive. Firstly, the nutrient solution supplied to the plants throughout the experiment remains sterile, as the system uses a peristaltic pump to distribute the sterilized nutrient solution directly to the plants through silicone tubing. Additionally, a 240-mesh filter membrane is placed over the top of the plant culture bottle, creating a barrier to prevent the loss of the substrate. The system is placed in a greenhouse to ensure optimal plant growth conditions, including controlled humidity, as fluctuations in humidity may affect both plant growth and the distribution of root exudates. Furthermore, this controlled environment allows for standardized collection procedures, including specific plant secretion collection times, to eliminate the influence of confounding factors on root exudates. A similar system used for tomato plants has been successful in maintaining sterility in the rhizosphere. (Kuijken et al. 2015 ) Secondly, this system allows for continuous collection of root exudates without disturbing the roots. Like the non-invasive root box method, it benefits the study of plant root exudates by minimizing the risk of root damage and artificial interference with exudate secretion, as seen in sampling systems under soil or sand cultivation conditions. (Tiziani et al. 2020 ) Lastly, the use of a recirculating semi-hydroponic system better simulates soil conditions compared to hydroponic or aeroponic systems. Currently, the collection devices for plant root exudates are still not fully optimized. In some studies, pure hydroponic systems with nutrient solutions have been employed. the root exudates in the nutrient solution were collected. (Yang et al. 2023 ; Zhao et al. 2021 ) But the primary shortcomings of this method are its inability to replicate the natural mechanical barriers experienced by roots in soil, and the large volume of nutrient solution, which makes the collection of root exudates challenging. Additionally, some studies have attempted to collect exudates in situ from field soil or greenhouse potted plants. (Barros et al. 2020 ; Phillips et al. 2008 ) In our study, root exudates are metabolized by the soil microbial community, which, along with the compounds secreted by microorganisms into the rhizosphere, complicates the analysis of root exudates in soil. Therefore, there is an urgent need for a new root exudate collection device to address the issues present in existing technologies. In metabolomic studies, a total of 54 differentiated metabolites were putatively identified between B73 and RiB73. Among them, a few of the differential metabolites have been reported in previous studies. Riboflavin, also known as vitamin B2, is an essential nutrient for humans and animals and can be synthesized by plants and microorganisms. (You et al. 2021 ) In a study on tomatoes, vitamins such as riboflavin were found to be related to defense mechanisms in AMF. (Sanchez-Bel et al. 2016 ) Although our study did not impose stress treatments, we similarly observed higher levels of riboflavin in RiB73, this could be an interesting finding. The role of lumichrome is often associated with riboflavin, as the latter can be easily converted enzymatically or photochemically into lumichrome. (Dakora et al. 2015 ) They are considered a novel molecule that stimulates plant development, particularly in the relationship between leguminous plants and rhizobia. (Matiru and Dakora 2005 ; Sanchez-Bel et al. 2016 ) In our study, lumichrome has been reported for the first time in AMF, and it has the potential to serve as a differential metabolite distinguishing between AMF-treated and untreated samples. Two common plant hormones, indoleacetic acid and abscisic acid, along with other substances related to hormone signaling, such as gibberellin A116, was found to be present at higher levels in RiB73. In tomato roots, AMF induced an increase in endogenous IAA levels. (Wang et al. 2021 ) In Funneliformis mosseae, mycorrhiza stimulates root hair growth as well as IAA synthesis and transport in trifoliate orange. (Liu et al. 2018 ) In mycorrhizal roots, the ABA content was consistently higher compared to that in the roots of nonmycorrhizal control plants. This has been confirmed in plants such as tomatoes, maize, and soybeans. (Charpentier et al. 2014 ; Danneberg et al. 1993 ; Herrera-Medina et al. 2007 ) Dihydrophaseic acid were identified as metabolites of ABA. (Baek et al. 2020 ) Its content follows the same trend as ABA. Gibberellin A116 is also known as GA12. In Arabidopsis, the root-to-shoot translocation of GA12 allows for a flexible growth response to changes in ambient temperature, (Camut et al. 2019 ) may have implications for controlling developmental phase transitions and adapting to adverse environments. (Regnault et al. 2015 ) 5. Conclusions Overall, a new plant growth system was established, combining perlite-supported semi-hydroponics and a semi-automated drip irrigation system for the repetitive collection of plant root exudates. This growth system offers several advantages over other systems, as it simulates certain aspects of mechanical barrier rooting in soil. It also allows for the collection of root exudates without disturbing or damaging the roots. Maize can grow normally in the semi-hydroponic system established. Meanwhile, the growth-promoting effect of AMF inoculation on maize was most pronounced at 60 days. This study analyzed and compared the chemical profiles between B73 and RiB73, identifying a total of 54 differentially abundant metabolites. Furthermore, a total of 54 metabolites exhibited AMF-related characteristics, enriched across 15 metabolic pathways. Key metabolites include lumichrome, riboflavin, indolelactic acid, abscisic acid, gibberellin A116, and L-histidinol phosphate. These metabolites have the potential to serve as biomarkers for studying maize root exudates following AMF inoculation. Declarations Conflicts of Interest: The authors declare no conflicts of interest. Acknowledgments: We thank National Key R&D Program of China (2023YFD1901002) for financial support. 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Mycorrhiza 29:251–261 Wang Y, Zhang W, Liu W, Ahammed GJ, Wen W, Guo S, Shu S, Sun J (2021) Auxin is involved in arbuscular mycorrhizal fungi-promoted tomato growth and NADP-malic enzymes expression in continuous cropping substrates. Bmc Plant Biol 21:48 Xu Y, Liu F, Wu F, Zhao M, Zou R, Wu J, Li X (2022) A novel SCARECROW-LIKE3 transcription factor LjGRAS36 in Lotus japonicus regulates the development of arbuscular mycorrhizal symbiosis. Physiol Mol Biol Plants 28:573–583 Xu Y, Liu F, Wu F, Zou R, Zhao M, Wu J, Cheng B, Li X (2024) Zinc finger protein LjRSDL regulates arbuscule degeneration of arbuscular mycorrhizal fungi in Lotus japonicus. Plant Physiology:kiae487 Xue L, Cui H, Buer B, Vijayakumar V, Delaux PM, Junkermann S, Bucher M (2015) Network of GRAS transcription factors involved in the control of arbuscule development in Lotus japonicus. Plant Physiol 167:854–871 Yang K, Fu R, Feng H, Jiang G, Finkel O, Sun T, Liu M, Huang B, Li S, Wang X, Yang T, Wang Y, Wang S, Xu Y, Shen Q, Friman VP, Jousset A, Wei Z (2023) RIN enhances plant disease resistance via root exudate-mediated assembly of disease-suppressive rhizosphere microbiota. Mol Plant 16:1379–1395 You J, Pan X, Yang C, Du Y, Osire T, Yang T, Zhang X, Xu M, Xu G, Rao Z (2021) Microbial production of riboflavin: Biotechnological advances and perspectives. Metab Eng 68:46–58 Zhang G, Raza W, Wang X, Ran W, Shen Q (2012) Systemic modification of cotton root exudates induced by arbuscular mycorrhizal fungi and Bacillus vallismortis HJ-5 and their effects on Verticillium wilt disease. Appl Soil Ecol 61:85–91 Zhao M, Zhao J, Yuan J, Hale L, Wen T, Huang Q, Vivanco JM, Zhou J, Kowalchuk GA, Shen Q (2021) Root exudates drive soil-microbe-nutrient feedbacks in response to plant growth. Plant Cell Environ 44:613–628 Supplementary Files Supplementaryinformation.docx Cite Share Download PDF Status: Published Journal Publication published 19 Apr, 2026 Read the published version in Journal of Plant Research → Version 1 posted Editorial decision: Major revision 07 Dec, 2025 Reviewers agreed at journal 27 Sep, 2025 Reviewers invited by journal 11 Sep, 2025 Editor assigned by journal 02 Sep, 2025 First submitted to journal 01 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7506078","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":513463046,"identity":"b9ecf527-e29e-422c-8bac-8b72dbc7abdb","order_by":0,"name":"Xinhao Luo","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYBACPgYehgMfKmzkwDzGBjBlgFcLGwMP48EZZ9KMgSzitTAf5m07lNhAvBaJ3ANALQfS58/vMWD4ucMusYG9eZsEQ80d3Fp4ziUcnHPuTu6GYzwGjL1nkhMbeI6VSTAce4ZbC3uPwYE3Zc9yN7DxGDDwtjEnNkjkmEkwNhzGrYWZx+AAD9vhdPk2oC1/2+oTG+TfENACtOUgT9vhBAagw5h52w4DbeEhoIXnjAEokA03HEsrOCzbdty4jSet2CLhGG4t/BI5xh+AUSkv33x448O3bdWy/eyHN974UINbCwo4ALYXRCQQp2EUjIJRMApGAQ4AAOmtVjKCSqtQAAAAAElFTkSuQmCC","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Xinhao","middleName":"","lastName":"Luo","suffix":""},{"id":513463047,"identity":"632c75e6-6536-49b6-8631-1db9e67b1d86","order_by":1,"name":"Xiaowan Geng","email":"","orcid":"","institution":"Chuzhou University","correspondingAuthor":false,"prefix":"","firstName":"Xiaowan","middleName":"","lastName":"Geng","suffix":""},{"id":513463048,"identity":"dc8ca56f-dfd6-4bad-b190-f60233fc1fd8","order_by":2,"name":"Jing Zhou","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhou","suffix":""},{"id":513463049,"identity":"bc147ca4-2c92-40a8-a946-7519eea80ff7","order_by":3,"name":"Jin Chen","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jin","middleName":"","lastName":"Chen","suffix":""},{"id":513463050,"identity":"96a5a90a-fcdb-4f4b-9dbe-eac41fb4468c","order_by":4,"name":"Beijiu Cheng","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Beijiu","middleName":"","lastName":"Cheng","suffix":""},{"id":513463051,"identity":"7ea5eafd-75f8-45d9-82ff-91107810fc44","order_by":5,"name":"Xiaoyu Li","email":"","orcid":"","institution":"Anhui Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyu","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-09-01 08:33:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7506078/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7506078/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10265-026-01705-4","type":"published","date":"2026-04-19T15:57:29+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":91621024,"identity":"69f383b5-9d79-44c2-9baf-4964cdae2451","added_by":"auto","created_at":"2025-09-18 11:28:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2373113,"visible":true,"origin":"","legend":"\u003cp\u003eCircular semi-hydroponic plant root exudates sterile culture device. (a) Schematic diagram of each part. (b) Vertical view. (c) End view.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7506078/v1/c0d74863750f75069335469c.png"},{"id":91621021,"identity":"074e0e51-bc15-42c2-9f4f-45a62e83c56a","added_by":"auto","created_at":"2025-09-18 11:28:23","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1543093,"visible":true,"origin":"","legend":"\u003cp\u003eApplication example (A) and the use process (B) of semi-hydroponic secretion device.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7506078/v1/24cf1387659f3ea148a96d4e.jpeg"},{"id":91621044,"identity":"4e9e85c8-39cd-4691-9dfd-381392d4da22","added_by":"auto","created_at":"2025-09-18 11:28:24","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":16119392,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic analysis of maize inoculated and uninoculated with AMF for 60 days. (a) The growth of maize; (b) Plant height; (c) Root length; (d) Aboveground biomass; (e) Underground biomass; (f) Blade length; (g) Total chlorophyll level. *P\u0026lt;0.05, **P\u0026lt;0.01, ***P\u0026lt;0.001, ****P\u0026lt;0.0001 (n=3, data are the means±SEM). Analysis of colonization structure of maize inoculated and uninoculated with AMF (h) Not infected; (i) Arbuscular structure; (j) Vesicle structure.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7506078/v1/9c7a2856409d2a2076e90a9c.png"},{"id":91621382,"identity":"21dba8ea-af7b-4ce0-abb9-0e2b9b0ed6e5","added_by":"auto","created_at":"2025-09-18 11:36:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":929065,"visible":true,"origin":"","legend":"\u003cp\u003eMultivariate statistical analysis between B73 and RiB73. PCA score plot of (a) positive (R2X = 0.879, Q2 = -0.325) and (d) negative (R2X = 0.836, Q2 = 0.153) ionization mode, respectively; PLS-DA score plot of (b) positive (R2X = 0.695, R2Y = 0.999, Q2 = 0.953) and (e) negative (R2X = 0.911, R2Y = 0.999, Q2 = 0.992) ionization mode, respectively; cross-validation plot of PLS-DA model of (c) positive (intercepts of R2 and Q2 were 0.998 and 0.509, respectively) and (f) negative (intercepts of R2 and Q2 were 0.998 and 0.738, respectively) ionization mode, respectively. Data are presented as the mean ± SEM (n=3).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7506078/v1/e00f122aa6c016d30bd6636e.png"},{"id":91621385,"identity":"658111c9-5c9b-4151-beed-a95a100d537a","added_by":"auto","created_at":"2025-09-18 11:36:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":995306,"visible":true,"origin":"","legend":"\u003cp\u003eHeat-map of the metabolite contents that showed coordinated changes between B73 and RiB73 in (a) positive mode and (b) negative mode.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7506078/v1/fae30530d1a37f9ef66459d8.png"},{"id":91621023,"identity":"22459ad8-3ace-480e-a7f7-785c6f496511","added_by":"auto","created_at":"2025-09-18 11:28:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1254182,"visible":true,"origin":"","legend":"\u003cp\u003ePathway analyses between B73 and RiB73. (a) KEGG enrichment bubble diagram analysis of differential metabolites between inoculated and uninoculated AMF. The y-axis (−log (P-value)) and x-axis represent the significance of the pathway and pathway impact between B73 and RiB73, respectively. (b-g) Relative content of certain signature metabolites. *P\u0026lt;0.05 (n=3, data are the means±SEM).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7506078/v1/2ba1660d5de59135d0c5fcf7.png"},{"id":107352613,"identity":"33a672e3-becd-4909-a424-f7d9d2083f3d","added_by":"auto","created_at":"2026-04-20 16:14:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":22146579,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7506078/v1/50920b8b-9359-4103-8129-4766ddc39f54.pdf"},{"id":91621028,"identity":"bb89041e-877f-4810-8073-d8d4c32a7f32","added_by":"auto","created_at":"2025-09-18 11:28:23","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":1717112,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7506078/v1/8555092b8c858ff45ce65e73.docx"}],"financialInterests":"","formattedTitle":"A semi-hydroponic cultivation system designed for collecting root exudates from maize in symbiosis with arbuscular mycorrhiza fungi","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePlants continuously release various compounds into their surrounding environment through a process known as exudation. This procedure plays a pivotal role in mediating diverse interactions within the rhizosphere, contributing to the cycling of carbon and nitrogen. (Chai and Schachtman \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Panchal et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) These compounds, known as root exudates, encompass a diverse array of high- and low-molecular-weight substances spanning various chemical classes, including amino acids, organic acids, alcohols, polypeptides, sugars, phenolics, enzymes, proteins, and hormones. (Baetz and Martinoia \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) And there is still uncertainty regarding whether these compounds secreted by the roots or absorbed from the soil. Under specific conditions, root exudates are harnessed to boost crop yields and enhance tolerance to environmental stresses. Therefore, obtaining pure, uncontaminated root exudates for research is of paramount importance.\u003c/p\u003e\u003cp\u003e According to existing research, the methods for collecting root exudates primarily include hydroponics and soil substrate cultivation. Hydroponics typically utilizes pure water or nutrient solutions for cultivation, facilitating the detection of root exudates, as it avoids mechanical damage caused by removing roots from solid substrates. (Strehmel et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) However, this approach cannot emulate the natural mechanical constraints of soil on root development, and the substantial volume of nutrient solution complicates the collection of root exudates. Soil substrate cultivation offers greater versatility and serves effectively as a preliminary screening tool, as it is applied to plants grown in field conditions. (Canarini et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) Whereas, the microbial communities in soil not only affect root exudates, but also secrete their own compounds, which can easily mix with the root exudates, leading to impure samples and interference in subsequent analysis. (Eichmann et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) Additionally, during the washing process, some degree of root damage is inevitable. (Oburger et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) Therefore, to simulate the natural mechanical constraints of soil, avoid root damage, and eliminate the confounding influence of soil microbes, the use of hydroponic systems or supported hydroponics with substrates such as gels, glass beads, or vermiculite presents a superior approach for studying root exudates. (Vranova et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2013\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eThe symbiotic relationship between plants and arbuscular mycorrhizal fungi (AMF) is a widespread and mutually beneficial interaction, present in over two-thirds of terrestrial plant species. At the ecosystem level, AMF play a crucial role in nutrient cycling and regulate the ecosystem's response to environmental fluctuations, highlighting their irreplaceable ecological significance. (Luo et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) Symbiosis with AMF offers multifaceted benefits to maize, primarily including enhanced nutrient uptake (Cheng et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), improved stress resistance (Colombo et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), better root development (Han et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), strengthened soil health and increased yield and quality (Wang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMetabolomics approaches offer a comprehensive view of chemical profiles and have been widely applied in medicine, plant sciences, and food research. (Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e) Nowadays, many studies employ metabolomics to analyze various plant root exudates, aiming to identify all metabolites present in the rhizosphere. Kaur et al has reported that AMF symbiosis can significantly influence the levels of key metabolites such as sugars, organic acids, and amino acids, thereby enhancing plant resistance. (Kaur and Suseela \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) Zhang et al found that AMF can influence the levels of phenolic acids in cotton root exudates, thereby reducing the incidence of Fusarium wilt in cotton. (Zhang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) Meanwhile, Shukla et al discovered that after AMF colonization, the release of root exudates can be altered to restrict nematode movement, thereby reducing nematode invasion in tomatoes. (Shukla et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)\u003c/p\u003e\u003cp\u003eBased on previous research, we designed a semi-hydroponic system for collecting root exudates from maize or other plants, utilizing perlite as a solid support to simulate soil separation. SHCS is integrated with drip irrigation to support symbiosis and facilitate the collection of maize root exudates. At the same time, non-targeted metabolomics analysis of the extracted root exudates was conducted to preliminarily investigate the differences in the presence or absence of AMF symbiosis, thereby validating the applicability of SHCS we designed\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Experimental materials and planting treatments\u003c/h2\u003e\u003cp\u003eThe study material consisted of maize wild-type B73, which were provided by the National Engineering Laboratory of Crop Stress Resistance at Anhui Agricultural University. And the AMF species used was \u003cem\u003eRhizophagus irregularis\u003c/em\u003e (Ri) DAOM 197198, provided by Sun Yat-Sen University, Guangzhou, China. Mycorrhizal colonization and arbuscule size analyses were performed as previously described. (Xue et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Semi-hydroponic\u003c/h2\u003e\u003cp\u003eThe experiment was conducted in a greenhouse to prevent airborne contamination. All assembly equipment was sterilized in an autoclave prior to use. The components of the system all consist of common items, including a 1 L culture bottle, several silicone tubes, an angle iron assembly for the device shelf, a flatjaw pinchcock to control circulation, a 500 mL solution bottle, and a multi-channel peristaltic pump. The growth substrate is perlite, which is sieved through a 60-mesh screen and then sterilized at 121\u0026deg;C for 60 minutes. After cooling for 2 days, it is ready for use. (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) Select B73 maize seedlings with similar growth, and secure them using perlite. First, place the filter membrane into the culture vessel, followed by a 1\u0026ndash;2 cm layer of sterile perlite. Position the seedlings in an appropriate location and add more perlite to secure them. To maintain sterile conditions, the culture vessel should be thoroughly cleaned and then wiped with 75% ethanol before use. Rinse the vessel filled with perlite twice with sterile full-strength Hoagland nutrient solution, then add 350 mL of sterile nutrient solution for cultivation. The vessel should be wrapped with aluminum foil and black opaque tape to prevent algal growth. Finally, cover the bottom of the culture vessel with aluminum foil to reserve space for the plants to protrude. The high-phosphorus Hoagland nutrient solution consists of 5 mmol/L KNO₃, 5 mmol/L Ca(NO₃)₂\u0026middot;4H₂O, 2 mmol/L MgSO₄\u0026middot;7H₂O, 20 \u0026micro;mol/L FeEDTA, 1 mmol/L KH₂PO₄, 1 \u0026micro;mol/L H₃BO₃, and trace elements including 2 \u0026micro;mol/L MnCl₂\u0026middot;4H₂O, 2 \u0026micro;mol/L ZnSO₄, 0.2 \u0026micro;mol/L CuSO₄\u0026middot;5H₂O, and 0.2 \u0026micro;mol/L (NH₄)₆Mo₇O₂₄. A constant nutrient solution flow rate of 350 mL/h was maintained. For each AMF maize plant, a mixture of 40 g of spore-sand (200 spores) was added. Wrap the solution bottle with aluminum foil and black light-blocking tape to simulate the darkness of the soil environment as closely as possible. After planting, keep the apparatus in a greenhouse with a 16-hour light/26\u0026deg;C and 8-hour dark/18\u0026deg;C cycle, and a relative humidity of approximately 60%. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Trypan Blue Staining\u003c/h2\u003e\u003cp\u003e Trypan blue staining of mycorrhizal roots from maize was performed according to established protocols. (Sportes et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Xu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) Root segments of maize were initially fixed in FAA (formalin acetic acid alcohol) for 4 hours. Subsequently, the segments were incubated at high temperature with 10% KOH, acidified, and clarified. Finally, the segments were stained with a 0.05% trypan blue solution. The AM fungal structures within the root segments were subsequently visualized using a DM5000B microscope (Leica, Wetzlar, Germany). The mycorrhizal infection rate and infection intensity of maize inoculated with Ri for 30 and 60 days were assessed following published methods. (Wang et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Sample preparation\u003c/h2\u003e\u003cp\u003eOn the 60 days after planting, fill the culture bottles with 300 mL of ultrapure water to collect maize root exudates. After 12 hours, transfer the liquid to labeled sealed bags and store at -80\u0026deg;C for 24 hours. Next, freeze-dry the samples for 72 hours to obtain the lyophilized root exudate powder. Store the powder in centrifuge tubes for subsequent metabolomic sequencing.\u003c/p\u003e\u003cp\u003e50 mg solid sample was added to a 2 mL centrifuge tube along with a 6 mm diameter grinding bead. Metabolite extraction was performed using 400 \u0026micro;L of extraction solution (methanol: water\u0026thinsp;=\u0026thinsp;4:1 (v:v)) containing 0.02 mg/mL of internal standard (L-2-chlorophenylalanine). The samples were ground using the Wonbio-96c frozen tissue grinder (Shanghai Wanbo Biotechnology Co., LTD) for 6 minutes (-10\u0026deg;C, 50 Hz), followed by low-temperature ultrasonic extraction for 30 minutes (5\u0026deg;C, 40 kHz). The samples were then placed at -20\u0026deg;C for 30 minutes, centrifuged for 15 minutes (4\u0026deg;C, 13000 g), and the supernatant was transferred to the injection vial for LC-MS/MS analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Metabolomics analysis of UPLC-Orbitrap-MS/MS\u003c/h2\u003e\u003cp\u003eThe LC-MS/MS analysis of the sample was conducted on a Thermo UHPLC-Q Exactive system equipped with an ACQUITY HSS T3 column (100 mm \u0026times; 2.1 mm i.d., 1.8 \u0026micro;m; Waters, USA). The mobile phases consisted of 0.1% formic acid in water (95:5, v/v) (solvent A) and 0.1% formic acid in acetonitrile: isopropanol (47.5:47.5:5, v/v) (solvent B). Positive ion mode separation gradient: 0\u0026ndash;3 min (20% B), 3-4.5 min (35% B), 4.5-5 min (100% B), 5-6.3 min (maintained at 100% B), 6.3\u0026ndash;6.4 min (0% B), 6.4-8 min (maintained at 0% B); Negative ion mode separation gradient: 0-1.5 min (5% B), 1.5-2 min (10% B), 2-4.5 min (30% B), 4.5-5 min (100% B), 5-6.3 min (maintained at 100% B), 6.3\u0026ndash;6.4 min (0% B), 6.4-8 min (maintained at 0% B). The flow rate was 0.40 mL/min and the column temperature was 40℃.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Statistical analysis\u003c/h2\u003e\u003cp\u003eRaw data were obtained from UPLC-Orbitrap-MS/MS. The preprocessed datasets, encompassing both positive and negative modes, were analyzed using unsupervised principal component analysis (PCA), supervised orthogonal partial least squares discriminant analysis (OPLS-DA) with SIMCA-P software (version 14.1) (Umetrics, Ume\u0026aring;, Sweden). Potential differential metabolites among B73 and RiB73 were identified based on VIP\u0026thinsp;\u0026gt;\u0026thinsp;1.0, p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and FC\u0026thinsp;\u0026gt;\u0026thinsp;1.5. VIP is a metric used in multivariate statistical methods like PLS-DA. It quantifies the contribution of each variable (e.g., metabolites, genes, or features) to the model, and fold change measures the ratio of change between two conditions, typically expressed as a magnitude of up- or down-regulation. It is commonly used in biological and omics studies. (Li et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e) A heat map was generated using OECloud tools at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://cloud.oebiotech.com\u003c/span\u003e\u003cspan address=\"https://cloud.oebiotech.com\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e\u003cp\u003eMetabolite peaks were annotated using accurate mass measurements through online metabolite databases such as HMDB (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://hmdb.ca/\u003c/span\u003e\u003cspan address=\"https://hmdb.ca/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and PubChem (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubchem.ncbi.nlm.nih.gov/\u003c/span\u003e\u003cspan address=\"https://pubchem.ncbi.nlm.nih.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAll results were analyzed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. T-tests were employed to determine significant differences among different groups or between two specific groups, respectively, using GraphPad Prism 9 (GraphPad Software, USA). Statistical significance was established at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Maize grows normally in a SHCS\u003c/h2\u003e\u003cp\u003eTo assess the growth of wild-type maize B73 under semi-hydroponic conditions, we measured several basic physiological parameters, including plant height, root length, aboveground biomass, underground biomass, blade length, and total chlorophyll level. Under semi-hydroponic conditions, maize showed a significant increase in plant height, root length, shoot biomass, root biomass, and leaf length at 30 days compared to 15 days. Additionally, total chlorophyll level remained stable, with no significant difference observed between 15 and 30 days. (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) These results demonstrate that maize can grow normally in the semi-hydroponic system we established.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.2 AMF promoted maize growth\u003c/h2\u003e\u003cp\u003eThe colonization of AM fungi can significantly enhance plant growth. At 60 days, RiB73 (wild-type maize B73 colonize \u003cem\u003eRhizophagus irregularis\u003c/em\u003e) showed a significant increase in plant height, root length, shoot biomass, root biomass, and leaf length at 60 days compared to B73. Meanwhile, total chlorophyll level show no significant difference between B73 and RiB73. (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) The inoculation effectiveness was assessed through trypan blue staining. No fungal structures of AMF were observed under a standard microscope after trypan blue staining in the untreated B73 group. Following AMF inoculation, structures such as arbuscules, vesicles, and hyphaws were observed in the maize. (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eh-j) Simultaneously, we assessed the mycorrhizal infection rate and infection intensity in maize inoculated with Ri to ensure successful colonization. (Fig.S4)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Significant different chemical profiles among B73 and RiB73 by UPLC-Orbitrap-MS/MS\u003c/h2\u003e\u003cp\u003eChemical profiles of B73 and RiB73 were detected by UPLC-Orbitrap-MS/MS system. A total of 3313 ion features in positive ion mode and 3687 ion features negative ion mode were detected, respectively. (Fig.\u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) Firstly, the PCA was applied to evaluate intra- and inter-group differences and to reduce background noises. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b) Secondly, the OPLS-DA statistical analysis methods were further employed to evaluate the deference of chemical profiles between B73 and RiB73. Furthermore, R2X, R2Y and Q2 were calculated as 0.695, 0.999 and 0.953 in positive and 0.911, 0.999, 0.992 in negative, respectively which indicated that the models were established successfully without over-fitting B73 and RiB73 exhibited significant separation, indicating distinct chemical profiles. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec, d) And R2Y and Q2 of permutation were calculated as 0.998 and 0.509 in positive and 0.998 and 0.738 in negative. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee, f)\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Characteristic metabolites of B73 and RiB73\u003c/h2\u003e\u003cp\u003eVariables with VIP\u0026thinsp;\u0026gt;\u0026thinsp;1, p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.5 were identified as potential differential metabolites. These metabolites were characterized by comparing their retention time (RT) and m/z values with relevant data published in the literature or available in databases. A total of 54 metabolites were tentatively identified, comprising 34 in positive mode and 20 in negative mode.\u003c/p\u003e\u003cp\u003eThe heat map based on the 54 differentially abundant metabolites offered a comprehensive overview of the differences in metabolite content between B73 and RiB73. (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b) In the plot, red and blue colors represent values higher and lower than the mean, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Metabolic pathway analysis between B73 and RiB73\u003c/h2\u003e\u003cp\u003eThe analysis of metabolic pathways based on differential metabolites helps us understand how metabolic pathways were enriched between B73 and RiB73. It presented an enrichment analysis of metabolic pathways in B73 and RiB73. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea) The data were visualized with the logarithm of the significance level (p-value) on the y-axis and the pathway impact on the x-axis. Through enrichment analysis and topology, we identified 15 metabolic pathways, including phenylalanine, tyrosine, and tryptophan biosynthesis, biosynthesis of various plant secondary metabolites, phenylpropanoid biosynthesis, linoleic acid metabolism, arachidonic acid metabolism, riboflavin metabolism, histidine metabolism, biotin metabolism, pentose phosphate pathway, carbon fixation in photosynthetic organisms, sphingolipid metabolism, α-linolenic acid metabolism, diterpenoid biosynthesis, glycerophospholipid metabolism, and cysteine and methionine metabolism. In these enriched pathways, we focused on metabolites of interest. For instance, riboflavin and lumichrome in the riboflavin metabolism pathway, L-histidinol phosphate in the histidine metabolism pathway, gibberellin A116 in the diterpenoid biosynthesis pathway, as well as key plant hormones like indolelactic acid (iaa) and abscisic acid (aba). (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb-g) except for L-histidinol phosphate, the levels of all other metabolites significantly increased after AMF inoculation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe collection of root exudates poses a significant challenge in studying AMF metabolism. Hydroponic systems lack the natural mechanical barriers provided by soil, while soil cultivation cannot avoid microbial influences and mechanical damage during sampling. (Barros et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) To address these challenges, a SHCS was designed to collect rhizosphere exudates from maize in symbiosis with AMF. This system utilizes perlite as a solid support to simulate soil barriers, combined with drip irrigation to facilitate symbiosis and the collection of maize root exudates. Then it was used to collect root exudates from B73 and AMF-inoculated B73, followed by metabolomics analysis using LC-MS/MS. Through comparative analysis, we identified significant differences in metabolite levels between B73 and RiB73.\u003c/p\u003e\u003cp\u003eOur recirculating semi-hydroponic system effectively supports the healthy growth of maize plants and is well-suited for the collection of root exudates. The advantages of this system are comprehensive. Firstly, the nutrient solution supplied to the plants throughout the experiment remains sterile, as the system uses a peristaltic pump to distribute the sterilized nutrient solution directly to the plants through silicone tubing. Additionally, a 240-mesh filter membrane is placed over the top of the plant culture bottle, creating a barrier to prevent the loss of the substrate. The system is placed in a greenhouse to ensure optimal plant growth conditions, including controlled humidity, as fluctuations in humidity may affect both plant growth and the distribution of root exudates. Furthermore, this controlled environment allows for standardized collection procedures, including specific plant secretion collection times, to eliminate the influence of confounding factors on root exudates. A similar system used for tomato plants has been successful in maintaining sterility in the rhizosphere. (Kuijken et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) Secondly, this system allows for continuous collection of root exudates without disturbing the roots. Like the non-invasive root box method, it benefits the study of plant root exudates by minimizing the risk of root damage and artificial interference with exudate secretion, as seen in sampling systems under soil or sand cultivation conditions. (Tiziani et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) Lastly, the use of a recirculating semi-hydroponic system better simulates soil conditions compared to hydroponic or aeroponic systems.\u003c/p\u003e\u003cp\u003eCurrently, the collection devices for plant root exudates are still not fully optimized. In some studies, pure hydroponic systems with nutrient solutions have been employed. the root exudates in the nutrient solution were collected. (Yang et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Zhao et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) But the primary shortcomings of this method are its inability to replicate the natural mechanical barriers experienced by roots in soil, and the large volume of nutrient solution, which makes the collection of root exudates challenging. Additionally, some studies have attempted to collect exudates in situ from field soil or greenhouse potted plants. (Barros et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Phillips et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) In our study, root exudates are metabolized by the soil microbial community, which, along with the compounds secreted by microorganisms into the rhizosphere, complicates the analysis of root exudates in soil. Therefore, there is an urgent need for a new root exudate collection device to address the issues present in existing technologies.\u003c/p\u003e\u003cp\u003eIn metabolomic studies, a total of 54 differentiated metabolites were putatively identified between B73 and RiB73. Among them, a few of the differential metabolites have been reported in previous studies. Riboflavin, also known as vitamin B2, is an essential nutrient for humans and animals and can be synthesized by plants and microorganisms. (You et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) In a study on tomatoes, vitamins such as riboflavin were found to be related to defense mechanisms in AMF. (Sanchez-Bel et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) Although our study did not impose stress treatments, we similarly observed higher levels of riboflavin in RiB73, this could be an interesting finding. The role of lumichrome is often associated with riboflavin, as the latter can be easily converted enzymatically or photochemically into lumichrome. (Dakora et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2015\u003c/span\u003e) They are considered a novel molecule that stimulates plant development, particularly in the relationship between leguminous plants and rhizobia. (Matiru and Dakora \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Sanchez-Bel et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) In our study, lumichrome has been reported for the first time in AMF, and it has the potential to serve as a differential metabolite distinguishing between AMF-treated and untreated samples. Two common plant hormones, indoleacetic acid and abscisic acid, along with other substances related to hormone signaling, such as gibberellin A116, was found to be present at higher levels in RiB73. In tomato roots, AMF induced an increase in endogenous IAA levels. (Wang et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) In Funneliformis mosseae, mycorrhiza stimulates root hair growth as well as IAA synthesis and transport in trifoliate orange. (Liu et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) In mycorrhizal roots, the ABA content was consistently higher compared to that in the roots of nonmycorrhizal control plants. This has been confirmed in plants such as tomatoes, maize, and soybeans. (Charpentier et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Danneberg et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; Herrera-Medina et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) Dihydrophaseic acid were identified as metabolites of ABA. (Baek et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) Its content follows the same trend as ABA. Gibberellin A116 is also known as GA12. In Arabidopsis, the root-to-shoot translocation of GA12 allows for a flexible growth response to changes in ambient temperature, (Camut et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) may have implications for controlling developmental phase transitions and adapting to adverse environments. (Regnault et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2015\u003c/span\u003e)\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eOverall, a new plant growth system was established, combining perlite-supported semi-hydroponics and a semi-automated drip irrigation system for the repetitive collection of plant root exudates. This growth system offers several advantages over other systems, as it simulates certain aspects of mechanical barrier rooting in soil. It also allows for the collection of root exudates without disturbing or damaging the roots. Maize can grow normally in the semi-hydroponic system established. Meanwhile, the growth-promoting effect of AMF inoculation on maize was most pronounced at 60 days. This study analyzed and compared the chemical profiles between B73 and RiB73, identifying a total of 54 differentially abundant metabolites. Furthermore, a total of 54 metabolites exhibited AMF-related characteristics, enriched across 15 metabolic pathways. Key metabolites include lumichrome, riboflavin, indolelactic acid, abscisic acid, gibberellin A116, and L-histidinol phosphate. These metabolites have the potential to serve as biomarkers for studying maize root exudates following AMF inoculation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eConflicts of Interest:\u003c/h2\u003e\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAcknowledgments:\u003c/h2\u003e\u003cp\u003eWe thank National Key R\u0026amp;D Program of China (2023YFD1901002) for financial support.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBaek SC, Lee BS, Yi SA, Yu JS, Lee J, Ko YJ, Pang C, Kim KH (2020) Discovery of Dihydrophaseic Acid Glucosides from the Florets of Carthamus tinctorius. Plants (Basel) 9\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBaetz U, Martinoia E (2014) Root exudates: the hidden part of plant defense. 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Plant Cell Environ 44:613\u0026ndash;628\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpre","sideBox":"Learn more about [Journal of Plant Research](http://link.springer.com/journal/10265)","snPcode":"10265","submissionUrl":"https://www.editorialmanager.com/jpre/default2.aspx","title":"Journal of Plant Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Semi-hydroponic, root exudates, maize, arbuscular mycorrhizal fungi, Metabolomics","lastPublishedDoi":"10.21203/rs.3.rs-7506078/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7506078/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe collection of root exudates, particularly those from plants symbiotically associated with arbuscular mycorrhizal fungi (AMF), were notably challenging. A semi-hydroponic cultivation system (SHCS) was designed to collect rhizosphere exudates from maize in symbiosis with AMF. This system utilizes perlite as a solid support to simulate soil barriers, combined with drip irrigation to facilitate symbiosis and the collection of maize root exudates. SHCS consists of a culture bottle, a solution bottle providing nutrients, a peristaltic pump for powering the system, silicone tubes connecting all components, a flat-jaw pinchcock for operation, and a device shelf for placing all items. Then it was used to collect root exudates from maize-wild type B73 and AMF-inoculated B73, followed by metabolomics analysis using LC-MS/MS. Through comparative analysis, we identified significant differences in metabolite levels between B73 and RiB73. Briefly, a total of 54 metabolites exhibited AMF-related characteristics, and these metabolites were enriched in 15 metabolic pathways. Key metabolites include lumichrome, riboflavin, indolelactic acid, abscisic acid, gibberellin a116, and l-histidinol phosphate. Among them, l-histidinol phosphate significantly decreased after AMF inoculation, while the other metabolites showed a notable increase in content.\u003c/p\u003e","manuscriptTitle":"A semi-hydroponic cultivation system designed for collecting root exudates from maize in symbiosis with arbuscular mycorrhiza fungi","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-18 11:28:19","doi":"10.21203/rs.3.rs-7506078/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2025-12-07T21:20:35+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-09-28T03:09:59+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-11T06:51:51+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-02T07:12:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Plant Research","date":"2025-09-01T04:32:30+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-plant-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jpre","sideBox":"Learn more about [Journal of Plant Research](http://link.springer.com/journal/10265)","snPcode":"10265","submissionUrl":"https://www.editorialmanager.com/jpre/default2.aspx","title":"Journal of Plant Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"96132dde-dc95-40f4-b19b-e8eb52095c7a","owner":[],"postedDate":"September 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T16:12:21+00:00","versionOfRecord":{"articleIdentity":"rs-7506078","link":"https://doi.org/10.1007/s10265-026-01705-4","journal":{"identity":"journal-of-plant-research","isVorOnly":false,"title":"Journal of Plant Research"},"publishedOn":"2026-04-19 15:57:29","publishedOnDateReadable":"April 19th, 2026"},"versionCreatedAt":"2025-09-18 11:28:19","video":"","vorDoi":"10.1007/s10265-026-01705-4","vorDoiUrl":"https://doi.org/10.1007/s10265-026-01705-4","workflowStages":[]},"version":"v1","identity":"rs-7506078","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7506078","identity":"rs-7506078","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}