An isoflavone catabolism gene cluster underlying interkingdom interactions in the soybean rhizosphere.

Plant roots secrete various metabolites, including plant specialized metabolites, into the rhizosphere, and shape the rhizosphere microbiome, which is crucial for the plant health and growth. Isoflavones are major plant specialized metabolites found in legume plants, and are involved in interactions with soil microorganisms as initiation signals in rhizobial symbiosis and as modulators of the legume root microbiota. However, it remains largely unknown the molecular basis underlying the isoflavone-mediated interkingdom interactions in the legume rhizosphere. Here, we isolated Variovorax sp. strain V35, a member of the Comamonadaceae that harbors isoflavone-degrading activity, from soybean roots and discovered a gene cluster responsible for isoflavone degradation named ifc. The characterization of ifc mutants and heterologously expressed Ifc enzymes revealed that isoflavones undergo oxidative catabolism, which is different from the reductive metabolic pathways observed in gut microbiota. We further demonstrated that the ifc genes are frequently found in bacterial strains isolated from legume plants, including mutualistic rhizobia, and contribute to the detoxification of the antibacterial activity of isoflavones. Taken together, our findings reveal an isoflavone catabolism gene cluster in the soybean root microbiota, providing molecular insights into isoflavone-mediated legume-microbiota interactions.

α-Tomatine gradient across artificial roots recreates the recruitment of tomato root-associated Sphingobium.

α-Tomatine is a major saponin that accumulates in tomatoes (Solanum lycopersicum). We previously reported that α-tomatine secreted from tomato roots modulates root-associated bacterial communities, particularly by enriching the abundance of Sphingobium belonging to the family Sphingomonadaceae. To further characterize the α-tomatine-mediated interactions between tomato plants and soil bacterial microbiota, we first cultivated tomato plants in pots containing different microbial inoculants originating from three field soils. Four bacterial genera, namely, Sphingobium, Bradyrhizobium, Cupriavidus, and Rhizobacter, were found to be commonly enriched in tomato root-associated bacterial communities. We constructed a pseudo-rhizosphere system using a mullite ceramic tube as an artificial root to investigate the influence of α-tomatine in modifying bacterial communities. The addition of α-tomatine from the artificial root resulted in the formation of a concentration gradient of α-tomatine that mimicked the tomato rhizosphere, and distinctive bacterial communities were observed in the soil close to the artificial root. Sphingobium was enriched according to the α-tomatine concentration gradient, whereas Bradyrhizobium, Cupriavidus, and Rhizobacter were not enriched in α-tomatine-treated soil. The tomato root-associated bacterial communities were similar to the soil bacterial communities in the vicinity of artificial root-secreting exudates; however, hierarchical cluster analysis revealed a distinction between root-associated and pseudo-rhizosphere bacterial communities. These results suggest that the pseudo-rhizosphere device at least partially creates a rhizosphere environment in which α-tomatine enhances the abundance of Sphingobium in the vicinity of the root. Enrichment of Sphingobium in the tomato rhizosphere was also apparent in publicly available microbiota data, further supporting the tight association between tomato roots and Sphingobium mediated by α-tomatine.

Tomato root-associated Sphingobium harbors genes for catabolizing toxic steroidal glycoalkaloids.

IMPORTANCE: Saponins are a group of plant specialized metabolites with various bioactive properties, both for human health and soil microorganisms. Our previous works demonstrated that Sphingobium is enriched in both soils treated with a steroid-type saponin, such as tomatine, and in the tomato rhizosphere. Despite the importance of saponins in plant-microbe interactions in the rhizosphere, the genes involved in the catabolism of saponins and their aglycones (sapogenins) remain largely unknown. Here we identified several enzymes that catalyzed the degradation of steroid-type saponins in a Sphingobium isolate from tomato roots, RC1. A comparative genomic analysis of Sphingobium revealed the limited distribution of genes for saponin degradation in our saponin-degrading isolates and several other isolates, suggesting the possible involvement of the saponin degradation pathway in the root colonization of Sphingobium spp. The genes that participate in the catabolism of sapogenins could be applied to the development of new industrially valuable sapogenin molecules.

Apoplast-Localized ß-Glucosidase Elevates Isoflavone Accumulation in the Soybean Rhizosphere.

Plant specialized metabolites (PSMs) are often stored as glycosides within cells and released from the roots with some chemical modifications. While isoflavones are known to function as symbiotic signals with rhizobia and to modulate the soybean rhizosphere microbiome, the underlying mechanisms of root-to-soil delivery are poorly understood. In addition to transporter-mediated secretion, the hydrolysis of isoflavone glycosides in the apoplast by an isoflavone conjugate-hydrolyzing ß-glucosidase (ICHG) has been proposed but not yet verified. To clarify the role of ICHG in isoflavone supply to the rhizosphere, we have isolated two independent mutants defective in ICHG activity from a soybean high-density mutant library. In the root apoplastic fraction of ichg mutants, the isoflavone glycoside contents were significantly increased, while isoflavone aglycone contents were decreased, indicating that ICHG hydrolyzes isoflavone glycosides into aglycones in the root apoplast. When grown in a field, the lack of ICHG activity considerably reduced isoflavone aglycone contents in roots and the rhizosphere soil, although the transcriptomes showed no distinct differences between the ichg mutants and wild-types (WTs). Despite the change in isoflavone contents and composition of the root and rhizosphere of the mutants, root and rhizosphere bacterial communities were not distinctive from those of the WTs. Root bacterial communities and nodulation capacities of the ichg mutants did not differ from the WTs under nitrogen-deficient conditions either. Taken together, these results indicate that ICHG elevates the accumulation of isoflavones in the soybean rhizosphere but is not essential for isoflavone-mediated plant-microbe interactions.

Excretion of triacylglycerol as a matrix lipid facilitating apoplastic accumulation of a lipophilic metabolite shikonin.

Plants produce a large variety of lipophilic metabolites, many of which are secreted by cells and accumulated in apoplasts. These compounds often play a role to protect plants from environmental stresses. However, little is known about how these lipophilic compounds are secreted into apoplastic spaces. In this study, we used shikonin-producing cultured cells of Lithospermum erythrorhizon as an experimental model system to analyze the secretion of lipophilic metabolites, taking advantage of its high production rate and the clear inducibility in culture. Shikonin derivatives are lipophilic red naphthoquinone compounds that accumulate exclusively in apoplastic spaces of these cells and also in the root epidermis of intact plants. Microscopic analysis showed that shikonin is accumulated in the form of numerous particles on the cell wall. Lipidomic analysis showed that L. erythrorhizon cultured cells secrete an appreciable portion of triacylglycerol (24-38% of total triacylglycerol), composed predominantly of saturated fatty acids. Moreover, in vitro reconstitution assay showed that triacylglycerol encapsulates shikonin derivatives with phospholipids to form lipid droplet-like structures. These findings suggest a novel role for triacylglycerol as a matrix lipid, a molecular component involved in the secretion of specialized lipophilic metabolites.

Application of plant specialized metabolites to modulate soil microbiota.

Plant specialized metabolites (PSMs) are considerably diverse compounds with multifaceted roles in the adaptation of plants to various abiotic and biotic stresses. PSMs are frequently secreted into the rhizosphere, a small region around the roots, where they facilitate interactions between plants and soil microorganisms. PSMs shape the host-specific rhizosphere microbial communities that potentially influence plant growth and tolerance to adverse conditions. Plant mutants defective in PSM biosynthesis contribute to reveal the roles of each PSM in plant-microbiota interactions in the rhizosphere. Recently, various approaches have been used to directly supply PSMs to soil by in vitro methods or through addition in pots with plants. This review focuses on the feasibility of the direct PSM application methods to reveal rhizospheric plant-microbiota interactions and discusses the possibility of applying the knowledge gained to future engineering of rhizospheric traits.

Plant specialized metabolites in the rhizosphere of tomatoes: secretion and effects on microorganisms.

Plants interact with microorganisms in the phyllosphere and rhizosphere. Here the roots exude plant specialized metabolites (PSMs) that have diverse biological and ecological functions. Recent reports have shown that these PSMs influence the rhizosphere microbiome, which is essential for the plant's growth and health. This review summarizes several specialized metabolites secreted into the rhizosphere of the tomato plant (Solanum lycopersicum), which is an important model species for plant research and a commercial crop. In this review, we focused on the effects of such plant metabolites on plant-microbe interactions. We also reviewed recent studies on improving the growth of tomatoes by analyzing and reconstructing the rhizosphere microbiome and discussed the challenges to be addressed in establishing sustainable agriculture.

Inventory of ATP-binding cassette proteins in Lithospermum erythrorhizon as a model plant producing divergent secondary metabolites.

ATP-binding cassette (ABC) proteins are the largest membrane transporter family in plants. In addition to transporting organic substances, these proteins function as ion channels and molecular switches. The development of multiple genes encoding ABC proteins has been associated with their various biological roles. Plants utilize many secondary metabolites to adapt to environmental stresses and to communicate with other organisms, with many ABC proteins thought to be involved in metabolite transport. Lithospermum erythrorhizon is regarded as a model plant for studying secondary metabolism, as cells in culture yielded high concentrations of meroterpenes and phenylpropanoids. Analysis of the genome and transcriptomes of L. erythrorhizon showed expression of genes encoding 118 ABC proteins, similar to other plant species. The number of expressed proteins in the half-size ABCA and full-size ABCB subfamilies was ca. 50% lower in L. erythrorhizon than in Arabidopsis, whereas there was no significant difference in the numbers of other expressed ABC proteins. Because many ABCG proteins are involved in the export of organic substances, members of this subfamily may play important roles in the transport of secondary metabolites that are secreted into apoplasts.

Control of Fusarium and nematodes by entomopathogenic fungi for organic production of Zingiber officinale.

Ginger (genus Zingiber) is widely used as a spice and a medicinal herb worldwide and is the major ingredient of traditional local drinks such as jamu in Southeast Asia. Because ginger is frequently consumed, there is an increasing interest in organic ginger production without the use of synthetic agrochemicals. Recent studies have reported that certain kinds of entomopathogenic fungi (EPF) can establish endophytic- or mycorrhiza-like relationships with plants, thereby promoting plant growth and health, in addition to their typical role in crop protection as biological control agents. In this study, we explored the possibility of non-entomopathogenic effects of EPF Beauveria bassiana and Cordyceps fumosorosea on ginger plants (Zingiber officinale) via antagonism with Fusarium oxysporum or the parasitic nematode Meloidogyne incognita. The two EPF negatively affected the growth of F. oxysporum and survival of M. incognita in vitro. The application of EPF did not have any negative effect on the growth of ginger plants. Soil chemical properties were not different between the plots with or without EPF application, while the diversity of soil bacteria was observed to increase on application of EPF. At least C. fumosorosea appeared to persist in soil during the period of ginger cultivation. Thus, these EPF are potentially useful tools for producing chemical-free ginger.

Development of two-dimensional qualitative visualization method for isoflavones secreted from soybean roots using sheets with immobilized bovine serum albumin.

A visualization method for the qualitative evaluation of soybean isoflavones secreted from soybean roots by transferring them onto a sheet with immobilized bovine serum albumin (BSA) was developed. BSA was chemically bonded onto a glass microfiber filter. The fluorescence quenching resulting from the interaction of BSA with soybean isoflavones such as daidzein and daidzin was utilized. Fluorescence images before and after soybean roots were placed in contact with the sheets with immobilized BSA were taken with an electron-multiplying charge-coupled device camera. The fluorescence quenching in the images was visualized and analyzed. Soybean isoflavones were extracted from the sheets for quantitative analysis, and the correlation coefficient between the quenched fluorescence intensity per sheet and the total amount of soybean isoflavones was 0.78 (p < 0.01), indicating a high correlation. The quenched fluorescence intensity was lower in pumpkin roots, which do not secrete soybean isoflavone. It was found from analyzed images that soybean isoflavone is secreted in larger amounts from the basal region of the taproot and the tips of the lateral roots of soybean.

L-Canavanine, a Root Exudate From Hairy Vetch (Vicia villosa) Drastically Affecting the Soil Microbial Community and Metabolite Pathways.

L-Canavanine, a conditionally essential non-proteinogenic amino acid analog to L-arginine, plays important roles in cell division, wound healing, immune function, the release of hormones, and a precursor for the synthesis of nitric oxide (NO). In this report, we found that the L-canavanine is released into the soil from the roots of hairy vetch (Vicia villosa) and declines several weeks after growth, while it was absent in bulk proxy. Hairy vetch root was able to exudate L-canavanine in both pots and in vitro conditions in an agar-based medium. The content of the L-canavanine in pots and agar conditions was higher than the field condition. It was also observed that the addition of L-canavanine significantly altered the microbial community composition and diversity in soil. Firmicutes and Actinobacteria became more abundant in the soil after the application of L-canavanine. In contrast, Proteobacteria and Acidobacteria populations were decreased by higher L-canavanine concentration (500 nmol/g soil). Prediction of the soil metabolic pathways using PICRUSt2 estimated that the L-arginine degradation pathway was enriched 1.3-fold when L-canavanine was added to the soil. Results indicated that carbon metabolism-related pathways were altered and the degradation of nitrogen-rich compounds (i.e., amino acids) enriched. The findings of this research showed that secretion of the allelochemical L-canavanine from the root of hairy vetch may alter the soil microbial community and soil metabolite pathways to increase the survival chance of hairy vetch seedlings. This is the first report that L-canavanine acts as an allelochemical that affects the biodiversity of soil microbial community.

Triterpenoid and Steroidal Saponins Differentially Influence Soil Bacterial Genera.

Plant specialized metabolites (PSMs) are secreted into the rhizosphere, i.e., the soil zone surrounding the roots of plants. They are often involved in root-associated microbiome assembly, but the association between PSMs and microbiota is not well characterized. Saponins are a group of PSMs widely distributed in angiosperms. In this study, we compared the bacterial communities in field soils treated with the pure compounds of four different saponins. All saponin treatments decreased bacterial α-diversity and caused significant differences in ß-diversity when compared with the control. The bacterial taxa depleted by saponin treatments were higher than the ones enriched; two families, Burkholderiaceae and Methylophilaceae, were enriched, while eighteen families were depleted with all saponin treatments. Sphingomonadaceae, which is abundant in the rhizosphere of saponin-producing plants (tomato and soybean), was enriched in soil treated with α-solanine, dioscin, and soyasaponins. α-Solanine and dioscin had a steroid-type aglycone that was found to specifically enrich Geobacteraceae, Lachnospiraceae, and Moraxellaceae, while soyasaponins and glycyrrhizin with an oleanane-type aglycone did not specifically enrich any of the bacterial families. At the bacterial genus level, the steroidal-type and oleanane-type saponins differentially influenced the soil bacterial taxa. Together, these results indicate that there is a relationship between the identities of saponins and their effects on soil bacterial communities.

Evaluation of Growth, Yield, and Biochemical Attributes of Bitter Gourd (Momordica charantia L.) Cultivars under Karaj Conditions in Iran.

Vegetative and reproductive characteristics, fruit yield, and biochemical compounds of six bitter melon cultivars (Iranshahr, Mestisa, No. 486, Local Japanese, Isfahan, and Ilocano) were evaluated under Karaj conditions in Iran. The phytochemical properties of the cultivars were evaluated using both shade-dried and freeze-dried samples at three fruit developmental stages (unripe, semi-ripe, and ripe). There were significant differences in the vegetative and reproductive characteristics among cultivars, where cv. No. 486 was superior to most vegetative attributes. The fruit yield of cultivars varied from 2.98-5.22 kg/plant. The number of days to male and female flower appearance ranged from 19.00-25.33 and from 25-33 days, respectively. The leaf charantin content was in the range of 4.83-11.08 µg/g. Fruit charantin content varied with developmental stage, drying method, and cultivar. The highest charantin content (13.84 ± 3.55 µg/g) was observed at the semi-ripe fruit stage, and it was much higher in the freeze-dried samples than the shade-dried samples. Cultivar No. 486 had the highest (15.43 ± 2.4 µg/g) charantin content, whereas the lowest charantin content (8.51 ± 1.15 µg/g) was recorded in cultivar cv. Local Japanese. The highest total phenol content (25.17 ± 2.27 mg GAE/g) was recorded in freeze-dried samples of ripe fruits of cv. No. 486, whereas the lowest phenol content was detected in the shade-dried samples of semi-ripe fruits of Isfahan. cv. Flavonoid content was higher with the shade-drying method, irrespective of cultivar. In conclusion, considering the fruit yield and active biological compounds in the studied cultivars, cv. No. 486 should be grown commercially because of its higher yield and production of other secondary metabolites.

Production of raspberry ketone by redirecting the metabolic flux to the phenylpropanoid pathway in tobacco plants.

Raspberry ketone is one of the characteristic flavors of raspberry fruits, and it is an important and expensive ingredient in the flavor and fragrance industries. It is present at low levels in plant tissues, and its occurrence is limited to a few taxa. In this context, the stable production of nature-identical raspberry ketone using heterologous synthesis in plants hosts has recently garnered the attention of plant biochemists. In this study, we demonstrate the rational switching of the metabolic flow from anthocyanin pigments to volatile phenylbutanoid production via the phenylpropanoid pathway. This shift led to the efficient and stable production of raspberry ketone and its glycosides via heterologous expression of the biosynthetic enzymes benzalacetone synthase (BAS) and raspberry ketone/zingerone synthase 1 (RZS1) in the transgenic tobacco (Nicotiana tabacum 'Petit Havana SR-1'). Additionally, we achieved improved product titers by activating the phenylpropanoid pathway with the transcriptional factor, production of anthocyanin pigment 1 (PAP1), from Arabidopsis thaliana. We further demonstrated another metabolic-flow switching by RNA interference (RNAi)-mediated silencing of chalcone synthase (CHS) to increase pathway-intermediate p-coumaroyl-CoA in transgenic tobacco for raspberry-ketone production. The redirection of metabolic flux resulted in transgenic lines producing 0.45 µg/g of raspberry ketone and 4.5 µg/g, on the fresh weight basis, of its glycosides in the flowers. These results suggest that the intracellular enforcement of endogenous substrate supply is an important factor while engineering the phenylpropanoid pathway. This strategy might be useful for the production of other phenylpropanoids/polyketides that are produced via the pathway-intermediate p-coumaroyl-CoA, in tobacco plants.

Nitrogen Deficiency-induced Bacterial Community Shifts in Soybean Roots.

Nitrogen deficiency affects soybean growth and physiology, such as symbiosis with rhizobia; however, its effects on the bacterial composition of the soybean root microbiota remain unclear. A bacterial community analysis by 16S rRNA gene amplicon sequencing showed nitrogen deficiency-induced bacterial community shifts in soybean roots with the marked enrichment of Methylobacteriaceae. The abundance of Methylobacteriaceae was low in the roots of field-grown soybean without symptoms of nitrogen deficiency. Although Methylobacteriaceae isolated from soybean roots under nitrogen deficiency did not promote growth or nodulation when inoculated into soybean roots, these results indicate that the enrichment of Methylobacteriaceae in soybean roots is triggered by nitrogen-deficiency stress.

Flavonoids and saponins in plant rhizospheres: roles, dynamics, and the potential for agriculture.

Plants are in constant interaction with a myriad of soil microorganisms in the rhizosphere, an area of soil in close contact with plant roots. Recent research has highlighted the importance of plant-specialized metabolites (PSMs) in shaping and modulating the rhizosphere microbiota; however, the molecular mechanisms underlying the establishment and function of the microbiota mostly remain unaddressed. Flavonoids and saponins are a group of PSMs whose biosynthetic pathways have largely been revealed. Although these PSMs are abundantly secreted into the rhizosphere and exert various functions, the secretion mechanisms have not been clarified. This review summarizes the roles of flavonoids and saponins in the rhizosphere with a special focus on interactions between plants and the rhizosphere microbiota. Furthermore, this review introduces recent advancements in the dynamics of these metabolites in the rhizosphere and indicates potential applications of PSMs for crop production and discusses perspectives in this emerging research field.

Tobacco Root Endophytic Arthrobacter Harbors Genomic Features Enabling the Catabolism of Host-Specific Plant Specialized Metabolites.

Plant roots constitute the primary interface between plants and soilborne microorganisms and harbor microbial communities called the root microbiota. Recent studies have demonstrated a significant contribution of plant specialized metabolites (PSMs) to the assembly of root microbiota. However, the mechanistic and evolutionary details underlying the PSM-mediated microbiota assembly and its contribution to host specificity remain elusive. Here, we show that the bacterial genus Arthrobacter is predominant specifically in the tobacco endosphere and that its enrichment in the tobacco endosphere is partially mediated by a combination of two unrelated classes of tobacco-specific PSMs, santhopine and nicotine. We isolated and sequenced Arthrobacter strains from tobacco roots as well as soils treated with these PSMs and identified genomic features, including but not limited to genes for santhopine and nicotine catabolism, that are associated with the ability to colonize tobacco roots. Phylogenomic and comparative analyses suggest that these genes were gained in multiple independent acquisition events, each of which was possibly triggered by adaptation to particular soil environments. Taken together, our findings illustrate a cooperative role of a combination of PSMs in mediating plant species-specific root bacterial microbiota assembly and suggest that the observed interaction between tobacco and Arthrobacter may be a consequence of an ecological fitting process. IMPORTANCE Host secondary metabolites have a crucial effect on the taxonomic composition of its associated microbiota. It is estimated that a single plant species produces hundreds of secondary metabolites; however, whether different classes of metabolites have distinctive or common roles in the microbiota assembly remains unclear. Here, we show that two unrelated classes of secondary metabolites in tobacco play a cooperative role in the formation of tobacco-specific compositions of the root bacterial microbiota, which has been established as a consequence of independent evolutionary events in plants and bacteria triggered by different ecological effects. Our findings illustrate mechanistic and evolutionary aspects of the microbiota assembly that are mediated by an arsenal of plant secondary metabolites.

Parallel evolution of UbiA superfamily proteins into aromatic O-prenyltransferases in plants.

Plants produce ∼300 aromatic compounds enzymatically linked to prenyl side chains via C-O bonds. These O-prenylated aromatic compounds have been found in taxonomically distant plant taxa, with some of them being beneficial or detrimental to human health. Although their O-prenyl moieties often play crucial roles in the biological activities of these compounds, no plant gene encoding an aromatic O-prenyltransferase (O-PT) has been isolated to date. This study describes the isolation of an aromatic O-PT gene, CpPT1, belonging to the UbiA superfamily, from grapefruit (Citrus × paradisi, Rutaceae). This gene was shown responsible for the biosynthesis of O-prenylated coumarin derivatives that alter drug pharmacokinetics in the human body. Another coumarin O-PT gene encoding a protein of the same family was identified in Angelica keiskei, an apiaceous medicinal plant containing pharmaceutically active O-prenylated coumarins. Phylogenetic analysis of these O-PTs suggested that aromatic O-prenylation activity evolved independently from the same ancestral gene in these distant plant taxa. These findings shed light on understanding the evolution of plant secondary (specialized) metabolites via the UbiA superfamily.

Field multi-omics analysis reveals a close association between bacterial communities and mineral properties in the soybean rhizosphere.

The plant root-associated environments such as the rhizosphere, rhizoplane, and endosphere are different from the outer soil region (bulk soil). They establish characteristic conditions including microbiota, metabolites, and minerals, and they can directly affect plant growth and development. However, comprehensive insights into those characteristic environments, especially the rhizosphere, and molecular mechanisms of their formation are not well understood. In the present study, we investigated the spatiotemporal dynamics of the root-associated environment in actual field conditions by multi-omics analyses (mineral, microbiome, and transcriptome) of soybean plants. Mineral and microbiome analyses demonstrated a characteristic rhizosphere environment in which most of the minerals were highly accumulated and bacterial communities were distinct from those in the bulk soil. Mantel's test and co-abundance network analysis revealed that characteristic community structures and dominant bacterial taxa in the rhizosphere significantly interact with mineral contents in the rhizosphere, but not in the bulk soil. Our field multi-omics analysis suggests a rhizosphere-specific close association between the microbiota and mineral environment.

Enhancement of developmentally regulated daidzein secretion from soybean roots in field conditions as compared with hydroponic culture.

Analyses of metabolite secretions by field-grown plants remain scarce. We analyzed daidzein secretion by field-grown soybean. Daidzein secretion was higher during early vegetative stages than reproductive stages, a trend that was also seen for hydroponically grown soybean. Daidzein secretion was up to 10 000-fold higher under field conditions than hydroponic conditions, leading to a more accurate simulation of rhizosphere daidzein content.