Are Symbiotic Methanotrophs Key Microbes for N Acquisition in Paddy Rice Root

Flooded fields such as rice (Oryza sativa) paddies are a major source of atmospheric CH4, a powerful greenhouse gas, via biogeochemical processes that are mediated by soil and plant microbial communities (31, 45, 61). Microbial processes relevant to the CH4 cycle are not fully understood even by omic–driven and culturing approaches (43). The ecosystem of rice paddies has been regarded as an ideal model system for studies on the fundamental aspects of microbial ecology (29, 36, 41). The rhizosphere is regarded as a hot spot for the transformation of a number of inorganic and organic substances, including C1 compounds such as methane (CH4), by means of redox reactions (29, 36, 41). CH4 produced from anoxic soils by methanogenic archaea is transported from the roots to the leaf sheaths via the aerenchyma of the rice plant (44). On the other hand, rice roots in paddies and rhizosphere soil grow under partially oxic conditions, allowing the growth of aerobic methanotrophic bacteria that utilize CH4 and methanol as their carbon and energy sources (17). Up to 90% of CH4 is consumed by aerobic methanotrophs in the rice root (21, 38, 61). Nitrogen (N) is one of the most important nutrients for plant growth (30). Although modern agriculture depends heavily on an adequate supply of N to sustain high crop yields, this is accompanied by well-documented high energy costs and environmental damage (30). Thus, reduced fertilizer usage is one of the objectives of field management to promote sustainable agriculture. Bodelier et al. (6) found that ammonium-based fertilizers stimulated CH4 oxidation in the soil around rice roots and reduced the emission of CH4. Other researchers also reported that N fertilization levels affect CH4 emission from rice fields; however, the details of this topic are being debated (3, 53, 62). A mechanistic understanding of CH4–N cycle interactions is a key unresolved issue in biogeochemical research on rice paddies and natural wetlands (7–9, 15). Recent multi-omic approaches have provided insights into the functional dynamics of CH4–N cycles in freshwater lakes (12) and permafrost ecosystems (22, 32). However, few studies have examined CH4–N cycle interactions in rice paddies and wetland soils (e.g., 9). Plant-associated bacteria often occupy endophytic niches in the plant roots and shoots (25). Until recently, few such analyses, including those based on metagenomics and metaproteomics, had been applied to endophytes due to the technical difficulties associated with preparing metagenomic microbial DNA and proteins without serious contamination by plant materials. A technique to enrich bacterial cells from plant tissues has been developed (24) and was shown to be useful for analyses of the microbiomes associated with rice roots, including those of bacterial endophytes and epiphytes (25, 27–29, 45). A metagenomic study (28) indicated that low-N fertilization management strongly affected the biogeochemical processes in rice roots in a paddy field ecosystem, in which three key players (including methanotrophic Methylosinus sp.) were identified in the bacteria associated with rice roots under low levels of N fertilizer application (28). Subsequent research (4, 5) suggested interplay between a plant symbiosis gene, CH4 oxidation, and N2 fixation in rice roots in paddy fields. Since this interplay occurred exclusively under low-N fertilization management, mediated through the plant symbiosis gene, these processes are likely to be similar to symbiotic N2 fixation between rhizobia and legumes. Based on these studies, we propose a hypothesis for unanswered questions on the interplay between rice plants, root microbiomes, and their biogeochemical functions.