To control agronomic N losses and reduce environmental pollution, biological nitrification inhibition (BNI) is a promising strategy. BNI is an ecological phenomenon by which certain plants release bioactive compounds that can suppress nitrifying soil microbes. Herein, we report on two hydrophobic BNI compounds released from maize root exudation (1 and 2), together with two BNI compounds inside maize roots (3 and 4). On the basis of a bioassay-guided fractionation method using a recombinant nitrifying bacterium Nitrosomonas europaea, 2,7-dimethoxy-1,4-naphthoquinone (1, ED₅₀ = 2 μM) was identified for the first time from dichloromethane (DCM) wash concentrate of maize root surface and named “zeanone.” The benzoxazinoid 2-hydroxy-4,7-dimethoxy-2H-1,4-benzoxazin-3(4H)-one (HDMBOA, 2, ED₅₀ = 13 μM) was isolated from DCM extract of maize roots, and two analogs of compound 2, 2-hydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (HMBOA, 3, ED₅₀ = 91 μM) and HDMBOA-β-glucoside (4, ED₅₀ = 94 μM), were isolated from methanol extract of maize roots. Their chemical structures (1–4) were determined by extensive spectroscopic methods. The contributions of these four isolated BNI compounds (1–4) to the hydrophobic BNI activity in maize roots were 19%, 20%, 2%, and 4%, respectively. A possible biosynthetic pathway for zeanone (1) is proposed. These results provide insights into the strength of hydrophobic BNI activity released from maize root systems, the chemical identities of the isolated BNIs, and their relative contribution to the BNI activity from maize root systems.

Cucurbitacins are highly oxygenated triterpenoids characteristic of plants in the family Cucurbitaceae and responsible for the bitter taste of these plants. Fruits of bitter melon (Momordica charantia) contain various cucurbitacins possessing an unusual ether bridge between C5 and C19, not observed in other Cucurbitaceae members. Using a combination of next-generation sequencing and RNA-Seq analysis and gene-to-gene co-expression analysis with the ConfeitoGUIplus software, we identified three P450 genes, CYP81AQ19, CYP88L7, and CYP88L8, expected to be involved in cucurbitacin biosynthesis. CYP81AQ19 co-expression with cucurbitadienol synthase in yeast resulted in the production of cucurbita-5,24-diene-3β,23α-diol. A mild acid treatment of this compound resulted in an isomerization of the C23-OH group to C25-OH with the concomitant migration of a double bond, suggesting that a nonenzymatic transformation may account for the observed C25-OH in the majority of cucurbitacins found in plants. The functional expression of CYP88L7 resulted in the production of hydroxylated C19 as well as C5-C19 ether-bridged products. A plausible mechanism for the formation of the C5-C19 ether bridge involves C7 and C19 hydroxylations, indicating a multifunctional nature of this P450. On the other hand, functional CYP88L8 expression gave a single product, a triterpene diol, indicating a monofunctional P450 catalyzing the C7 hydroxylation. Our findings of the roles of several plant P450s in cucurbitacin biosynthesis reveal that an allylic hydroxylation is a key enzymatic transformation that triggers subsequent processes to produce structurally diverse products.

Background and aims: Biological nitrification inhibition (BNI) is a chemical ecological phenomenon whereby plants specifically suppress nitrification by releasing inhibiting compounds from roots, an effective strategy for improving nitrogen uptake by limiting nitrogen losses from agricultural fields. During this study, we have aimed at characterizing hydrophilic BNI activity released from maize roots to understand the chemical basis for BNI function in maize.Methods: Maize plants were grown hydroponically and root exudates were collected using water-based solutions and hydrophilic BNI activity was extracted. We isolated the target BNI compounds by a combination of chromatographic techniques and bioassays using a recombinant luminescent ammonia-oxidizing bacteriumNitrosomonas europaea(pHLUX20).Results: We identified 6-methoxy-2(3H)-benzoxazolone (MBOA) as the responsible BNI compound with a median effective dose (ED₅₀) = 0.76 μM. MBOA inhibited the conversion of NH₃to NH₂OH as well as NH₂OH to NO₂⁻inN. europaea, suggesting that MBOA blocks both ammonia monooxygenase and hydroxylamine oxidoreductase enzymatic pathways. Treatment with MBOA significantly suppressed NO₂⁻and NO₃⁻production during soil incubation, but this activity was reduced subsequently due to biodegradation of MBOA by soil microbes. A quantification experiment revealed that MBOA accounted for nearly 50% of the total BNI activity in hydrophilic and hydrophobic exudates from maize roots. A soil incubation test showed that two previously identified benzoxazinoids, HDMBOA and HDMBOA-β-glucoside, can be eventually transformed into MBOA.Conclusion: We elucidated MBOA as the key component of BNI in maize. Collectively, the present findings will serve as the groundwork for construction of an advanced environment-friendly agricultural system.

α-1,3-Glucan, a component of the fungal cell wall, is a refractory polysaccharide for most plants. Previously, we showed that various fungal plant pathogens masked their cell wall surfaces with α-1,3-glucan to evade plant immunity. This surface accumulation of α-1,3-glucan was infection specific, suggesting that plant factors might induce its production in fungi. Through immunofluorescence observations of fungal cell walls, we found that carrot (Daucus carota) extract induced the accumulation of α-1,3-glucan on germlings in Colletotrichum fioriniae, a polyphagous fungal pathogen that causes anthracnose disease in various dicot plants. Bioassay-guided fractionation of carrot leaf extract successfully identified two active substances that caused α-1,3-glucan accumulation in this fungus: lutein, a carotenoid widely distributed in plants, and stigmasterol, a plant-specific membrane component. Lutein, which had a greater effect on C. fioriniae, also induced α-1,3-glucan accumulation in other Colletotrichum species and in the phylogenetically distant rice pathogen Cochliobolus miyabeanus, but not in the rice pathogen Magnaporthe oryzae belonging to the same phylogenetic subclass as Colletotrichum. Our results suggested that fungal plant pathogens reorganize their cell wall components in response to specific plant-derived compounds, which these pathogens may encounter during infection.