ABSTRACTReductive soil disinfestation (RSD) is an effective method to inhibit soilborne pathogens. However, it remains unclear how RSD combined with different types of organic materials affects the soil ecosystems of perennial plants. Pot experiments were conducted to investigate the effects of RSD incorporated with perilla (PF), alfalfa (MS), ethanol, and acetic acid on soil properties, enzyme activities, microbial communities and functions, and seedling growth. Results showed that RSD-related treatments improved soil properties and enzyme activities, changed microbial community composition and structure, enhanced microbial interactions and functions, and facilitated seedling growth. Compared with CK, RSD-related treatments increased soil pH, available nitrogen, and available potassium contents, sucrase and catalase activities, and decreased soil electric conductivity values. Meanwhile, RSD-related treatment also significantly reduced the relative abundance of Fusarium while increasing the relative abundance of Arthrobacter, Terrabacter, and Gemmatimonas. The reduction was more evident in PF and MS treatment, suggesting the potential for RSD combined with solid agricultural wastes to suppress pathogens. Furthermore, the microbial network of RSD-related treatment was more complex and interconnected, and the functions related to carbon, nitrogen, sulfur, and hydrogen cycling were significantly increased, while the functions of bacterial and fungal plant pathogens were decreased. Importantly, RSD-related treatments also significantly promoted seed germination and seedling growth. In summary, RSD combined with solid agricultural wastes is better than liquid easily degradable compounds by regulating the composition and function of microbial communities to improve soil quality and promote plant growth.IMPORTANCEReductive soil disinfestation (RSD) is an effective agricultural practice. We found that RSD combined with solid agricultural wastes is better than that of liquid easily degradable compounds, may improve soil quality and microbial community structure, inhibit the proliferation of pathogenic bacteria, and contribute to the growth of replanted crops. Thus, RSD combined with solid agricultural wastes is more effective than liquid easily degradable compounds.

Ginsenoside Rb1 is an allelopathic self-toxic substance that can affect the growth and development of ginseng. This study investigated whether the application of exogenous Rb1 enhances the pathogenicity of ginseng by regulating the antioxidant system and endogenous hormones. Rb1 can inhibit the growth and development of Panax ginseng by inducing Fusarium oxysporum under three concentrations. At the same time, the activities of four antioxidant enzymes and the contents of three endogenous hormones in the roots of Panax ginseng decreased significantly. Compared with the control group, the incidence of ginseng in different treatment groups was significantly increased and the underground growth was significantly inhibited. In conclusion, exogenous ginsenoside Rb1 with different concentrations can enhance the pathogenicity of ginseng.

To meet societal needs, a large number of medicinal plants are cultivated artificially. However, issues such as diseases and continuous cropping obstacles (CCO) have severely impacted their quality and yield. Exploring and innovating the cultivation technology for medicinal plants is essential to meet their high demand and ensure sustainable development. The role of titanium dioxide nanoparticles (nano-TiO2) in medicinal plant cultivation remains unclear. To advance the application of nanotechnology in this field, a comprehensive exploration of its potential benefits is necessary. In this study, nano-TiO2 was applied to ginseng (Panax ginseng C.A. Meyer) to acquire a holistic comprehension of its impact on ginseng growth, rhizosphere, and ginseng-used soil. Our findings reveal that nano-TiO2 significantly enhances ginseng root activity and has notable effects on antioxidant enzyme systems. The two concentrations of nano-TiO2 markedly influenced the structure and composition of microbial communities in the rhizosphere and ginseng-used soil, including key microorganisms such as Chloroflexi and Acidobacteriota, which are closely involved in soil function. Furthermore, nano-TiO2 altered the competitive and cooperative relationships within microbial networks. Nano-TiO2 application significantly increased soil organic matter (SOM) content in rhizosphere and ginseng-used soils and affected the activities of several important soil enzymes. Environmental factors, such as EC, pH, and soil nutrients, were found to be the main factors influencing the microbial community. In conclusion, our findings illuminate the complex effects of nano-TiO2 on the “plant-microbial-soil” system in the context of ginseng cultivation. This work offers novel strategies for optimizing medicinal plant growth and development, as well as improving cultivated soil by using nanomaterials.

To meet societal needs, a large number of medicinal plants are cultivated artificially. However, issues such as diseases and continuous cropping obstacles (CCO) have severely impacted their quality and yield. Exploring and innovating the cultivation technology for medicinal plants is essential to meet their high demand and ensure sustainable development. The role of titanium dioxide nanoparticles (nano-TiO2) 2 ) in medicinal plant cultivation remains unclear. To advance the application of nanotechnology in this field, a comprehensive exploration of its potential benefits is necessary. In this study, nano-TiO2 2 was applied to ginseng ( Panax ginseng C.A. Meyer) to acquire a holistic comprehension of its impact on ginseng growth, rhizosphere, and ginseng-used soil. Our findings reveal that nano-TiO2 2 significantly enhances ginseng root activity and has notable effects on antioxidant enzyme systems. The two concentrations of nano-TiO2 2 markedly influenced the structure and composition of microbial communities in the rhizosphere and ginseng-used soil, including key microorganisms such as Chloroflexi and Acidobacteriota, which are closely involved in soil function. Furthermore, nano-TiO2 2 altered the competitive and cooperative relationships within microbial networks. Nano-TiO2 2 application significantly increased soil organic matter (SOM) content in rhizosphere and ginseng-used soils and affected the activities of several important soil enzymes. Environmental factors, such as EC, pH, and soil nutrients, were found to be the main factors influencing the microbial community. In conclusion, our findings illuminate the complex effects of nano-TiO2 2 on the "plant-microbial-soil" system in the context of ginseng cultivation. This work offers novel strategies for optimizing medicinal plant growth and development, as well as improving cultivated soil by using nanomaterials.

Ginseng is a plant with a long history of use in traditional Chinese medicine. However, large-scale cultivation of ginseng is limited by widespread issues with continuous cropping. To explore the physiological and molecular mechanisms behind ginseng crop disorders, transcriptomics and metabolomics were combined in this study to analyze the effects of continuous cropping on changes in ginseng metabolites, differential gene expression, and regulation of root secretions. The results showed that 106 metabolites and 1175 genes were significantly altered at the metabolic and transcriptional levels in ginseng from continuous cropping compared with the control group. More organic acids, alkaloids, flavonoids, and phenolics accumulated in the ginseng from continuous cropping than the control ginseng. Furthermore, significant differences were found in the expression of genes involved in phytohormone signaling, phenylpropanoid biosynthesis, and plant–pathogen interaction pathways. The results provide information on the regulation of expression of genes associated with continuous cropping disorders, and suggest that continuous cropping disorders may lead to severe damage to ginseng roots by pathogens and result in slow growth. Our research provides new insight into the response of ginseng to crop disorders, and reveals potential molecular mechanisms of the ginseng response to crop disorders.

The allelopathic autotoxicity of ginsenosides is an important cause of continuous cropping obstacles in ginseng planting. There is no report on the potential molecular mechanism of the correlation between polarity of ginsenoside components and their allelopathic autotoxicity. This study applied a combination of metabolomics and transcriptomics analysis techniques, combined with apparent morphology, physiological indexes, and cell vitality detection of the ginseng hairy roots, through which the molecular mechanism of correlation between polarity and allelopathic autotoxicity of ginsenosides were comprehensively studied. The hairy roots of ginseng presented more severe cell apoptosis under the stress of low-polarity ginsenoside components (ZG70). ZG70 exerted allelopathic autotoxicity by regulating the key enzyme genes of cis-zeatin (cZ) synthesis pathway, indole-3-acetic acid (IAA) synthesis pathway, and jasmonates (JAs) signaling transduction pathway. The common pathway for high-polarity ginsenoside components (ZG50) and ZG70 to induce the development of allelopathic autotoxicity was through the expression of key enzymes in the gibberellin (GA) signal transduction pathway, thereby inhibiting the growth of ginseng hairy roots. cZ, indole-3-acetamid (IAM), gibberellin A1 (GA1), and jasmonoyl-L-isoleucine (JA-ILE) were the key response factors in this process. It could be concluded that the polarity of ginsenoside components were negatively correlated with their allelopathic autotoxicity.

Continuous cropping of ginseng leads to serious declines in yield and quality because of self-toxicity of alle-lochemicals and other factors in soil. However, because of the long growth cycle and low survival rate of ginseng, rapid screening of autotoxic activity is difficult. Therefore, it is important to analyze the allelochemicals and identify a model plant with autotoxic responses similar to those of ginseng. In this study, UPLC-Orbitrap-HRMS targeted metabolomics and verification of autotoxic activity were used to analyze a problem soil from contin-uously cropped ginseng. Allelochemical markers were screened by OPLS-DA. Seeds and seedlings of maize, Chinese cabbage, cucumber, green beans, wheat, sunflower, and oats were selected to identify potential model plants. Model plants with autotoxic responses similar to those of ginseng were evaluated by comparing morphological, physiological, and biochemical characteristics. The n-butanol extract of the continuously cropped problem soil had the most significant autotoxic activity. Twenty-three ginsenosides and the contributions to autotoxic effects were screened and evaluated. Of potential model plants, seeds and seedlings of cucumber showed similar growth inhibition to that of ginseng under the action of allelochemicals. Thus, metabolomics can be used to screen allelochemicals in soil and predict the autotoxic effects, and the cucumber plant model can be used to rapidly screen allelopathic activity of ginseng. The study will provide reference for methodology in allelopathy research on ginseng.

Root exudates contain plant metabolites secreted by the roots into the soil, such as ginsenosides secreted by the ginseng root. However, little is known about ginseng root exudate and its impact on the chemical and microbial properties of soil. In this study, the effect of increasing concentrations of ginsenosides on the chemical and microbial properties of soil was tested. Chemical analysis and high-throughput sequencing techniques were used to evaluate the soil chemical properties and microbial characteristics following exogenous application of 0.1 mg·L −1 , 1 mg·L −1 , and 10 mg·L −1 ginsenosides. Ginsenosides application significantly altered soil enzyme activities; SOM-dominated physicochemical properties were significantly reduced which altered the composition and structure of the soil microbial community. In particular, treatment with 10 mg∙L −1 ginsenosides significantly increased the relative abundance of pathogenic fungi such as Fusarium, Gibberella and Neocosmospora . These findings indicate that ginsenosides in root exudates are important factors that may lead to increased deterioration of soil during ginseng cultivation and provided new research direction for the subsequent study on the mechanism of interaction between ginsenosides and soil microbial communities.

There are serious soil sickness in ginseng cultivation. Crop rotation is an effective agricultural management to improve soil sustainability and reduce soil sickness. To explore an appropriate ginseng rotation system, Medicago sativa (alfalfa) and Perilla frutescens (perilla) were planted on ginseng cultivation soil for 1 year to evaluate the improvement effect of both. Through chemical analysis and high-throughput sequencing technology, we found that after alfalfa and perilla cultivation for one-year, various nutrients and enzyme activities in ginseng cultivation soil were significantly improved. In addition, perilla significantly increased the diversity and richness of soil fungal communities. Cultivation of alfalfa and perilla significantly changed the composition of soil bacterial and fungal communities and significantly reduced the abundance of the potentially pathogenic fungi Ilyonectria. Further pot experiments also showed that the improved soil could significantly increase root activity of ginseng plant after two plants were planted. It should be noted that, unlike alfalfa, perilla decreased soil electrical conductivity, increased soil organic matter, soil urease, and may significantly improve the diversity and richness of soil fungal community. Moreover, in the pot experiment, the root fresh weight of ginseng cultured in perilla treated soil increased significantly. This study highlights that perilla may have better soil improvement effect than alfalfa and it has the potential to be used in the soil improvement of ginseng cultivation.

Background Long-term cultivation of ginseng can cause severe crop disorders and soil sickness. Crop rotation is an effective agricultural management measure to improve soil sustainability and decrease pathogens. However, the suitable ginseng rotation system and the changes in soil microbial community and soil characteristics under the rotation system need to be further explored. Methods To explore suitable ginseng crop rotation systems and improve soil utilization, Allium fistulosum and Brassica napus were planted on ginseng cultivation soil for one year. The effects of the two crops on the chemical properties and enzyme activities of the ginseng cultivation soil were evaluated by chemical analysis. In addition, amplicon sequencing targeting 16 s rDNA genes of bacteria and ITS of fungi has been used to characterize the functional and compositional diversity of microbial communities. Results The results elucidated that the levels of available phosphorus (AP) and available potassium (AK) in the soil increased significantly after one year of cultivation for both crops and Allium fistulosum cultivation may also have reduced soil salinity. In addition, the effects of the two crops on the activities of key soil enzymes were different. Catalase (CAT), urease (URE), and acid phosphatase (A-PHO) activities were significantly reduced and sucrase (SUC), and laccase (LAC) activities were significantly increased after Allium fistulosum planting. While A-PHO activity was significantly increased and LAC activity was significantly decreased after Brassica napus planting. Allium fistulosum significantly reduced the abundance of soil fungal communities. The cultivation of Allium fistulosum and Brassica napus significantly altered the composition of soil bacterial and fungal communities, where changes in the abundance of dominant microorganisms, such as Ascomycota , and Mortierellomycota , etc., were closely related to soil chemistry and enzyme activity. Moreover, both significantly reduced the abundance of the pathogenic fungus Ilyonectria . Conclusions Our study clarified the effects of Allium fistulosum and Brassica napus on the microbial community and physicochemical properties of ginseng cultivated soil and provides a basis for the sustainable application of ginseng cultivation soil and the development of ginseng crop rotation systems.

为探究不同改良土壤方式对连作人参生长发育的影响,于吉林省吉林市昌邑区左家镇北方药用植物资源圃进行大田试验,设老参地土壤为对照(CK),研究强还原土壤灭菌加熏蒸(RSD+SF)、强还原土壤灭菌加复合菌(RSD+F)、熏蒸加复合菌(SF+F)3种处理对人参生长发育、生理抗性、根系活力、发病及地下生长情况的影响.结果表明,3种改土方式对人参的生长发育均有一定的促进作用.其中,RSD+SF组的人参根长、根粗、须根数、干质量和湿质量均明显优于其他处理组,但是,人参的折干率表现为RSD+F组显著高于其他处理组;此外,3种处理的人参MDA含量差异不显著(P>0.05),但是SOD和CAT活性差异显著,表现为RSD+F组人参的SOD及CAT活性最强,SF+F组人参的SOD活性最低,为186.70 U/g,RSD+SF组人参的CAT活性最低,为122.89 U/g;同时,RSD+F组人参根系活力最强,为99.53 mg/(g·h),其次为SF+F组,根系活力为61.90 mg/(g·h),RSD+SF组根系活力最弱,为54.42 mg/(g·h);RSD+SF组人参发病率最低,仅为23.3％,其次是SF+F组,RSD+F组人参发病率最高(35％);3种处理下的人参病情严重情况无显著性差异(P>0.05),SF+F组人参地下根部长势最好且无病斑,RSD+SF组人参地下部分长势良好,个别人参出现病斑,而RSD+F组人参地下生长受抑制,须根数明显减少,且6株人参均出现病斑.由此可知,RSD+SF及SF+F改良连作人参土壤的效果要优于RSD+F组的改良效果,并能更好地促进人参的生长发育.

薤白是药食两用植物,具有丰富的营养价值和药用功效,因此如何培育出优质高产的薤白,受到人们的广泛关注.本文对薤白的营养价值、药用价值、植物学特征和生物学特性进行简述,从选地、整地、繁殖方法、田间管理和采收等方面对其栽培技术进行总结,并对未来研究方向进行展望,以期为薤白的生产、开发和利用提供参考.

P. ginseng C.A. Meyer is a valuable Chinese herbal medicine that belongs to the Araliaceae family. Major obstacles to the continuous cropping of ginseng have severely restricted the sustainable development of the ginseng industry. The allelopathic effects of triterpenoid saponins play an important role in disorders related to continuous cropping; however, the mechanisms underlying the allelopathic autotoxicity of triterpenoid ginsenosides remain unknown. In this study, we performed mRNA and miRNA sequencing analyses to identify candidate genes and miRNAs that respond differentially to ginsenoside Ro stress in ginseng and their targets. The growth of the ginseng hairy roots was significantly inhibited under Ro stress (0.5 mg/L, Ro-0.5). The inhibition of root growth and injury to root-tip cells promoted the accumulation of the endogenous hormones indole-3-acetic acid and salicylic acid and inhibited the accumulation of abscisic acid and jasmonate acid. The accumulation of ginsenosides, except Rg3, was significantly inhibited under Ro-0.5 stress. An mRNA analysis of the Ro-0.5 and control groups showed that differentially expressed genes were mostly concentrated in the hormone signal transduction pathway. ARF7 and EFM were upregulated, whereas XTH23 and ZOX1 were downregulated. These genes represent important potential candidates for hormone-responsive continuous cropping diseases. In total, 74 differentially expressed miRNAs were identified based on the miRNA sequencing analysis, of which 22 were upregulated and 52 were downregulated. The target genes of ptc-miR156k_L + 1, mtr-miR156b-5p, gma-miR156a_R + 1, and mtr-miR156e all belonged to TRINITY_DN14567_c0_g4, which is a gene in the plant hormone signal transduction pathway. These four miRNAs were all negatively correlated with mRNA, indicating their likely involvement in the response of ginseng to continuous cropping disorders and the regulation of ginsenoside synthesis. Our findings provide useful insights for removing the barriers to continuous ginseng cropping and have important implications in the genetic engineering of plant stress responses.

强还原土壤灭菌(reductive soil disinfestation,RSD)和土壤熏蒸(soil fumigation,SF)是缓解人参连作障碍的常用方法.为研究2种方法对土壤细菌群落和土壤酶活性的影响,采用高通量测序技术和化学分析方法对强还原土壤灭菌加氯化苦熏蒸(RSD+SF)、强还原土壤灭菌加复合菌(RSD+F)、氯化苦熏蒸加复合菌(SF+F)3种方式改良的土壤细菌群落和土壤酶活性进行分析.结果表明,RSD+F组细菌群落多样性与丰富度均最高,SF+F组均最低,3组拥有相同细菌菌属431个.RSD+SF组中,丰富度最高的细菌为Gemmatimonas,其丰富度为9.17％;RSD+F组中丰富度最高的细菌为norank_f_noranko_Gaiellales,其丰富度为8.72％;RSD+F组中丰富度最高的细菌为Bacillus,其丰富度为9.16％;Baci llus为3种方式改良土壤前10种优势菌群中共有的优势菌群.土壤酶活性与土壤细菌群落结构存在显著性关系,随着生长时间的增加,不同方式改良后的连作人参土壤酶活性均具有显著性差异(P＜0.05).由此可知,3种土壤改良方式均能在不同程度地增加有益细菌属的丰富度并提高土壤酶活性,其中RSD+SF组和RSD+F组的有益细菌属数量及土壤酶活性均高于SF+F组.

Saposhnikovia divaricata is derived from the dried roots of Saposhnikovia divaricata (Turcz.) Schischk and used as a Chinese herbal medicine for treating respiratory, immune, and nervous system diseases. The continuously increasing market demand for traditional Chinese medicine requires the commercial cultivation of Saposhnikovia divaricata using standardized methods and high yielding genotypes, such as double-headed root plants, for achieving consistent quality and a reliable supply. In this study, we aimed to identify the quantitative differences in chromone, a precursor of flavonoid biosynthesis, between plants with single- and double-headed roots using high-performance liquid chromatography and further explore the two phenotypes at the transcriptomic and metabolomic levels. Our results showed that the chromone content was significantly higher in plants with double-headed roots than in those with single-headed roots. Transcriptomic analysis revealed six significantly differentially expressed genes between the two phenotypes, including five key genes in the flavonoid biosynthesis pathway (4-coumarate-CoA ligase, chalcone synthase 1, vinorine synthase, chalcone-flavonone isomerase 1, and flavanone 3 beta-hydroxylase) and one key gene in the abscisic acid biosynthetic pathway (zeaxanthin epoxidase). Moreover, metabolomic analysis showed that the 126 differentially expressed metabolites were mainly enriched in the biosynthesis of secondary metabolites and phytohormones. Overall, our results suggest that plants with double-headed roots have higher medicinal value than those with single-headed roots, probably due to differences in various biosynthetic pathways. These data might help select the genotypes with superior yield and therapeutic properties.

Reductive soil disinfestation (RSD) and soil fumigant chloropicrin (SFC) are two common agricultural strategies for the elimination of soil-borne pathogens. However, the differences in soil environmental factors, soil bacterial microbiome, and root performance between SFC and RSD are poorly understood. In this study, three soil treatments, untreated control (CK), SFC with 0.5 t⋅ha–1 chloropicrin, and RSD with 15 t⋅ha–1 animal feces, were compared. We evaluated their effects on soil environmental factors, bacterial community structure, and root activity using chemical analysis and high-throughput sequencing. RSD treatment improved soil composition structure, bacterial diversity, and root performance to a greater extent. Carbon source utilization preference and bacterial community structure were strikingly altered by SFC and RSD practices. Bacterial richness, diversity, and evenness were notably lowered in the SFC- and RSD-treated soil compared with the CK-treated soil. However, RSD-treated soil harbored distinct unique and core microbiomes that were composed of more abundant and diverse potentially disease-suppressive and organic-decomposable agents. Also, soil bacterial diversity and composition were closely related to soil physicochemical properties and enzyme activity, of which pH, available Na (ANa), available Mg (AMg), available Mn (AMn), total Na (TNa), total Ca (TCa), total Cu (TCu), total Sr (TSr), urease (S-UE), acid phosphatase (S-ACP), and sucrase (S-SC) were the main drivers. Moreover, RSD treatment also significantly increased ginseng root activity. Collectively, these results suggest that RSD practices could considerably restore soil nutrient structure and bacterial diversity and improve root performance, which can be applied as a potential agricultural practice for the development of disease-suppressive soil.

Panax ginseng is a valuable traditional herbal medicine material with numerous applications. Ginsenosides are the key bioactive compounds in ginseng. Cold stress can activate stress tolerance mechanisms that regulate biomass and biosynthesis in ginseng tissue. In this study, the effects of short- and long-term cold stress (5°C) on the physiological characteristics, tissue-specific ginsenoside distributions, and ginsenoside synthesis gene expressions of 3-year-old P. ginseng during the flowering period were investigated. Short-term cold stress significantly reduced ginseng biomass (root fresh weight and dry weight), and increased malondialdehyde, proline, soluble sugar, and soluble protein concentrations. Superoxide dismutase, peroxidase, and catalase activities also increased significantly under cold stress. With prolongation of the cold stress period, all antioxidant enzyme activity decreased. The protopanaxatriol-type ginsenoside concentrations in the taproots (phloem and xylem) and fibrous roots, as well as the protopanaxadiol-type ginsenoside concentrations in the leaves, increased significantly under short-term cold stress. The key genes (SE, DS-II, CYP716A52v2, and CYP716A53v2) involved in the ginsenoside biosynthesis pathway were significantly positively correlated with the ginsenoside accumulation trends. Thus, short-term cold stress can stimulate membrane lipid peroxidation, in turn stimulating the antioxidant enzyme system to alleviate oxidative damage and increasing the expression of key enzyme genes involved in ginsenoside biosynthesis. During agricultural production, protopanaxadiol/protopanaxatriol ratios could be manipulated by low-temperature storage or treatments.

Jasmonates (JAs) are plant hormones that regulate the biosynthesis of many secondary metabolites, such as hydroxycinnamic acid amides (HCAAs), through jasmonic acid (JA)-responsive transcription factors (TFs). HCAAs are renowned for their role in plant defense against pathogens. The multidrug and toxic compound extrusion transporter DETOXIFICATION18 (DTX18) has been shown to mediate the extracellular accumulation of HCAAs p-coumaroylagmatine (CouAgm) at the plant surface for defense response. However, little is known about the regulatory mechanism of DTX18 gene expression by TFs. Yeast one-hybrid screening using the DTX18 promoter as bait isolated the key positive regulator redox-responsive TF 1 (RRTF1), which is a member of the AP2/ethylene-response factor family of proteins. RRTF1 is a JA-responsive factor that is required for the transcription of the DTX18 gene, and it thus promotes CouAgm secretion at the plant surface. As a result, overexpression of RRTF1 caused increased resistance against the fungus Botrytis cinerea, whereas rrtf1 mutant plants were more susceptible. Using yeast two-hybrid screening, we identified the BTB/POZ-MATH (BPM) protein BPM1 as an interacting partner of RRTF1. The BPM family of proteins acts as substrate adaptors of CUL3-based E3 ubiquitin ligases, and we found that only BPM1 and BPM3 were able to interact with RRTF1. In addition, we demonstrated that RRTF1 was subjected to degradation through the 26S proteasome pathway and that JA stabilized RRTF1. Knockout of BPM1 and BPM3 in bpm1/3 double mutants enhanced RRTF1 accumulation and DTX18 gene expression, thus increasing resistance to the fungus B. cinerea. Our results provide a better understanding of the fine-tuned regulation of JA-induced TFs in HCAA accumulation.

Fagopyrum tataricum is an ancient dicotyledonous crop and is well known to enrich in flavonoids, such as rutin. The Subgroup 4 of R2R3-MYB transcription factors, FtMYB11, FtMYB13, FtMYB14, FtMYB15 and FtMYB16 are key repressors of rutin biosynthesis in buckwheat. However, the regulatory mechanism of FtMYB16 on rutin biosynthesis is still unclear. Transcriptomics analysis showed that FtMYB16 is highly expressed in root and tightly co-expressed with the multidrug and toxic compound extrusion (MATE) transporter gene FtMATE1, which could transport isoquercitrin and promote rutin accumulation. Electrophoretic mobility shift assay (MESA), chromatin immunoprecipitation (ChIP) assay, transient activation assay and transgenic hairy roots showed that FtMYB16 directly repressed the FtMATE1 gene expression and rutin biosynthesis pathway. Yeast two hybrid screening and bimolecular fluorescence complementation assay showed that the FtMYB16 repressor interacted with an importin-α protein Ftimportin-α1 in the nucleus and cytoplasm, depending on the nuclear localization signal (NLS) of FtMYB16. Translocation assay and transient activation assay demonstrated that Ftimportin-α1 directly mediated the nuclear localization of FtMYB16 to execute the repression activity. Our findings figure out the role of MATE transporter in rutin metabolism and establish a model of MYB TFs/importin-α complex in the nucleo-cytoplasmic trafficking pathway in plants, which may be useful for the design of artificially modified metabolic pathway. Keywords: Importin, MATE transporter, MYB transcription factor, Nucleo-cytoplasmic trafficking, Rutin, Buckwheat. This article is protected by copyright. All rights reserved.

Jasmonates (JAs) are plant hormones which regulate biosynthesis of many secondary metabolites, such as glucosinolates (GLSs), through JAs-responsive transcription factors (TFs). The JAs-responsive gene, has been shown to catalyze the conversion of indole-3-acetaldoxime (IAOx) to indolic glucosinolates (IGLSs). However, little is known about the regulatory mechanism of gene expression by JAs. In yeast one-hybrid screens using the promoter as bait we isolated two JAs-responsive TFs ERF109 and MYB51 that are involved in JAs-regulated IGLS biosynthesis. Furthermore, using a yeast two-hybrid assay, we identified ERF109 as an interacting partner of MYB51, and Jasmonate ZIM-domain (JAZ) proteins as interactors of MYB51, and BTB/POZ-MATH (BPM) proteins as interactors of ERF109. Both JAZ and BPM proteins are necessary for the full repression of the ERF109-MYB51-MYC3 ternary complex activity on gene expression and JA-regulated IGLS biosynthesis. Biochemical analysis showed that the 26S proteasome-mediated degradation of ERF109 protein is mediated by a CRL3 E3 ligase independently of JA signaling. Genetic and physiological evidence shows that MYB51 acts as an adaptor and activator to bridge the interaction with the co-activators MYC3 and ERF109, for synergistically activating the gene expression, and all three factors are essential and exert a coordinated control in JAs-induced IGLS biosynthesis. Overall, this study provides insights into the molecular mechanisms of JAs-responsive ERF109-MYB51-MYC3 ternary complexes in controlling JAs-regulated GLSs biosynthesis, which provides a better understanding of plant secondary metabolism.