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

Involvement of the leaf-specific multidrug and toxic compound extrusion (MATE) transporter Nt-JAT2 in vacuolar sequestration of nicotine in Nicotiana tabacum

Alkaloids play a key role in higher plant defense against pathogens and herbivores. Following its biosynthesis in root tissues, nicotine, the major alkaloid of Nicotiana species, is translocated via xylem transport toward the accumulation sites, leaf vacuoles. Our transcriptome analysis of methyl jasmonate-treated tobacco BY-2 cells identified several multidrug and toxic compound extrusion (MATE) transporter genes. In this study, we characterized a MATE gene, Nicotiana tabacum jasmonate-inducible alkaloid transporter 2 (Nt-JAT2), which encodes a protein that has 32% amino acid identity with Nt-JAT1. Nt-JAT2 mRNA is expressed at a very low steady state level in whole plants, but is rapidly upregulated by methyl jasmonate treatment in a leaf-specific manner. To characterize the function of Nt-JAT2, yeast cells were used as the host organism in a cellular transport assay. Nt-JAT2 was localized at the plasma membrane in yeast cells. When incubated in nicotine-containing medium, the nicotine content in Nt-JAT2-expressing cells was significantly lower than in control yeast. Nt-JAT2-expressing cells also showed lower content of other alkaloids like anabasine and anatabine, but not of flavonoids, suggesting that Nt-JAT2 transports various alkaloids including nicotine. Fluorescence assays in BY-2 cells showed that Nt-JAT2-GFP was localized to the tonoplast. These findings indicate that Nt-JAT2 is involved in nicotine sequestration in leaf vacuoles following the translocation of nicotine from root tissues

Diurnal metabolic regulation of isoflavones and soyasaponins in soybean roots

Isoflavones and soyasaponins are major specialized metabolites accumulated in soybean roots and secreted into the rhizosphere. Unlike the biosynthetic pathway, the transporters involved in metabolite secretion remain unknown. The developmental regulation of isoflavone and soyasaponin secretions has been recently reported, but the diurnal regulation of their biosynthesis and secretion still needs to be further studied. To address these challenges, we conducted transcriptome and metabolite analysis using hydroponically grown soybean plants at 6-hr intervals for 48 hr in a 12-hr-light/12-hr-dark condition. Isoflavone and soyasaponin biosynthetic genes showed opposite patterns in the root tissues; that is, the former genes are highly expressed in the daytime, while the latter ones are strongly induced at nighttime. GmMYB176 encoding a transcription factor of isoflavone biosynthesis was upregulated from ZT0 (6:00 a.m.) to ZT6 (12:00 a.m.), followed by the induction of isoflavone biosynthetic genes at ZT6. The isoflavone aglycone content in the roots accordingly increased from ZT6 to ZT18 (0:00 a.m.). The isoflavone aglycone content in root exudates was kept consistent throughout the day, whereas that of glucosides increased at ZT6, which reflected the decreased expression of the gene encoding beta-glucosidase involved in the hydrolysis of apoplast-localized isoflavone conjugates. Co-expression analysis revealed that those isoflavone and soyasaponin biosynthetic genes formed separate clusters, which exhibited a correlation to ABC and MATE transporter genes. In summary, the results in this study indicated the diurnal regulation of isoflavone biosynthesis in soybean roots and the putative transporter genes responsible for isoflavone and soyasaponin transport

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

ダイズ根圏細菌のイソフラボン代謝遺伝子クラスターを発見--根圏形成メカニズムの理解や有用物質生産に貢献--.京都大学プレスリリース. 2024-04-24.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

トマト根に定着する細菌からトマトの毒を分解する酵素を発見 --土壌微生物が植物の分泌する有害成分を解毒するメカニズムの理解に貢献--. 京都大学プレスリリース. 2023-10-02.Roots of Bloody Mary. Tomato root-associated Sphingobium harbors genes for neutralizing toxic compound. 京都大学プレスリリース. 2023-10-06.Plant roots exude various organic compounds, including plant specialized metabolites (PSMs), into the rhizosphere. The secreted PSMs enrich specific microbial taxa to shape the rhizosphere microbiome, which is crucial for the healthy growth of the host plants. PSMs often exhibit biological activities; in turn, some microorganisms possess the capability to either resist or detoxify them. Saponins are structurally diverse triterpene-type PSMs that are mainly produced by angiosperms. They are generally considered as plant defense compounds. We have revealed that α-tomatine, a steroid-type saponin secreted from tomato (Solanum lycopersicum) roots, increases the abundance of Sphingobium bacteria. To elucidate the mechanisms underlying the α-tomatine-mediated enrichment of Sphingobium, we isolated Sphingobium spp. from tomato roots and characterized their saponin-catabolizing abilities. We obtained the whole-genome sequence of Sphingobium sp. RC1, which degrades steroid-type saponins but not oleanane-type ones, and performed a gene cluster analysis together with a transcriptome analysis of α-tomatine degradation. The in vitro characterization of candidate genes identified six enzymes that hydrolyzed the different sugar moieties of steroid-type saponins at different positions. In addition, the enzymes involved in the early steps of the degradation of sapogenins (i.e., aglycones of saponins) were identified, suggesting that orthologs of the known bacterial steroid catabolic enzymes can metabolize sapogenins. Furthermore, a comparative genomic analysis revealed that the saponin-degrading enzymes were present exclusively in certain strains of Sphingobium spp., most of which were isolated from tomato roots or α-tomatine-treated soil. Taken together, these results suggest a catabolic pathway for highly bioactive steroid-type saponins in the rhizosphere

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