Isolation and characterization of the hydrophilic BNI compound, 6-methoxy-2(3H)-benzoxazolone (MBOA), from maize roots

Plant Soil https://doi.org/10.1007/s11104-023-06021-7 RESEARCH ARTICLE Isolation and characterization of the hydrophilic BNI compound, 6‑methoxy‑2(3H)‑benzoxazolone (MBOA), from maize roots Junnosuke Otaka · Guntur Venkata Subbarao · Jiang MingLi · Hiroshi Ono · Tadashi Yoshihashi Received: 16 January 2023 / Accepted: 5 April 2023 © The Author(s) 2023 Abstract 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 Responsible Editor: Devrim Coskun. Supplementary Information The online version contains supplementary material available at https://doi. org/10.1007/s11104-023-06021-7. J. Otaka (*) · T. Yoshihashi (*) Biological Resources and Post‑harvest Division, Japan International Research Center for Agricultural Sciences (JIRCAS), 1‑1 Ohwashi, City of Tsukuba, Ibaraki, Japan e-mail: T. Yoshihashi e-mail: G. V. Subbarao · J. MingLi Crop, Livestock and Environment Division, Japan International Research Center for Agricultural Sciences (JIRCAS), 1‑1 Ohwashi, City of Tsukuba, Ibaraki, Japan H. Ono National Agriculture and Food Research Organization (NARO), 2‑1‑12 Kannondai, City of Tsukuba, Ibaraki, Japan 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 bacterium Nitrosomonas 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 N H3 to NH2OH as − well as N H2OH to N O2 in N. europaea, suggesting that MBOA blocks both ammonia monooxygenase and hydroxylamine oxidoreductase enzymatic pathways. Treatment with MBOA significantly suppressed O3− production during soil incubation, NO2− and N 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-βglucoside, 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. Vol.: (0123456789) 13 Plant Soil Keywords Biological nitrification inhibition · BNI · Maize · MBOA · Nitrification inhibitors · Nitrogen pollution Introduction In agriculture, large amounts of nitrogen fertilizers are used for crop production to feed the growing world population. Nearly 50% of the nitrogen fertilizer applied to Poaceae crops (e.g., maize, wheat, and rice) is lost largely because of two soil microbial transformations, nitrification and denitrification, resulting in loss of soil-nitrogen and low nitrogen use efficiency (NUE) (Coskun et al. 2017; Subbarao et al. 2013b; Thakur and Medhi 2019). Nitrification, a stepwise oxidation process from NH3 to nitrate ( NO3−) by soil microbes, plays an important role in the nitrogen cycle. However, excessive production of N O3− and its high mobility leads to groundwater contamination and generation of harmful greenhouse gas such as N 2O and NO (Kuypers et al. 2018; Rivett et al. 2008; Scheer et al. 2020; Stayner et al. 2017; Tian et al. 2020). Thus, overapplication of nitrogen fertilizer (as N H3) together with generation of excess soil-NO3− provoke serious economic and environmental damage (Subbarao and Searchinger 2021). Therefore, a strategy is needed for suppression of nitrification to increase NUE to correct the imbalances in nitrogen cycle. Biological nitrification inhibition (BNI) provides an innovative way to reduce nitrogen loss from agricultural system (Ghatak et al. 2022; Subbarao and Searchinger 2021). BNI is a chemical ecological phenomenon by which specific natural products (secondary metabolites) secreted from the plant root system, including terpenoids, alkaloids, fatty acids, and phenylpropanoids, that inhibit nitrification and growth of nitrifiers (Subbarao et al. 2009; Subbarao et al. 2013b; Wendeborn 2020). The BNI-possessing crops can retain more NH4 in the rhizosphere through circumvention of N O3− production. Therefore, utilization of BNI has several major advantages. (a) It is eco-friendly: the environmental pollution risk is reduced through using the activity of phytochemicals released from crop root systems. (b) The effect is sustainable: plant crops can continuously biosynthesize and secrete BNI compounds from roots into rhizosphere to keep nitrifier activity under check. (c) Costs are reduced: application of additional agrochemicals is not needed. To date, BNI has been observed in certain plants, including main Vol:. (1234567890) 13 staple food crops, namely maize, wheat, rice, and sorghum (Otaka et al. 2022; Subbarao et al. 2021; Subbarao et al. 2013a; Sun et al. 2016; Zakir et al. 2008). Understanding BNI function in maize is important due to: (a) Among staple crops, maize is the most productive crop providing food and feed, (b) It consumes a major portion of nitrogen fertilizer produced globally, and (c) Maize production systems contribute to nitrogen pollution in a major way globally. To develop a BNI strengthened crop with a higher yield of BNI compounds, understanding the chemical identity of BNI compounds released from roots is essential. For isolation of BNI compounds, a crucial property is whether the root exudates and compounds are water-insoluble (hydrophobic) or water-soluble (hydrophilic) (Subbarao et al. 2013a). Water-insoluble hydrophobic compounds with lower mobility are predominant in the rhizosphere, whereas hydrophilic compounds in water can move more farther from the roots and have wide-reaching influence. Chemical identities of both hydrophobic and hydrophilic BNI compounds in plant crops will lead to a deeper understanding of BNI function in the soil. We have recently reported two major hydrophobic BNI-contributing compounds from the root surface in maize, namely 2,7-dimethoxy-1,4-naphthoquinone (zeanone; ED50 = 2 μM) and 2-hydroxy-4,7-dimethoxy-2H-1,4benzoxazin-3(4H)-one (HDMBOA; ED50 = 13 μM), together with two analogs of HDMBOA from inside the roots, namely 7-methoxy-2H-1,4-benzoxazin3(4H)-one (HMBOA; ED50 = 91 μM) and HDMBOAβ-glucoside (ED50 = 94 μM) (Fig. S1) (Otaka et al. 2022). This study is (...truncated)