Nitrite Reductase

For the 793 bins passing completeness and contamination criteria, carbohydrate active enzymes (CAZy) were annotated using hmmsearch against the dbCAN v6 HMM database with default parameters^{69 (link)}. The results were filtered to remove hits with an e-value ≥ 1 × 10⁻¹⁴ and HMM coverage of ≤0.35. For CAZy domains overlapping the same region of sequence, the domain with the lower e-value was selected. Carbon and nitrogen metabolic functions were annotated by using HMMER3 against an in house HMM database built from KEGG orthology groups (KOs). Briefly, all KEGG database proteins with KOs were compared with all-v-all global similarity search using USEARCH. MCL was then used to sub-cluster KOs (inflation_value = 1.1). Each sub-cluster was aligned using MAFFT, and HMMs were constructed from sub-cluster alignments. HMMs were then scored against all KEGG sequences with KOs and a score threshold was set for each HMM at the score of the highest scoring hit outside of that HMMs sub-cluster. Access to the proprietary KEGG database was secured via contract, so only our procedure to profile them can be made public.
For methanol dehydrogenase (xoxF), CO dehydrogenase (coxL) and nitrite reductase (nirK) we constructed individual phylogenetic trees to discriminate homologous, but functionally distinct, proteins that can be identified by HMM search alone. XoxF sequences were initially identified in genomes using a custom HMM for PQQ-binding alcohol dehydrogenases^{21 (link)}. Angelo sequences were combined with reference sequences from refs. ^{33 (link),78 (link)} and aligned using MAFFT. A phylogenetic tree was constructed using FastTree (Supplementary Fig. 7 and Supplementary Data 12) and xoxF sequences were manually discriminated from mxaF and general ADH sequences by their position relative to reference sequences in the tree.
Putative coxL sequences were identified by KEGG HMM hits to K03520. Angelo hits were combined with reference sequences from ref. ^{9 (link)}, and aligned using MAFFT. A phylogenetic tree was constructed using FastTree (Supplementary Fig. 8 and Supplementary Data 13) and coxL-typeI sequences were manually identified by a known sequence motif ‘AYRCSFR’^{22 (link)} and their position relative to reference sequences in the tree.
Putative nirK sequences were identified by KEGG HMM hits to K00368. Angelo hits were combined with reference sequences from ref. ^{79 (link)} and aligned using MAFFT. A phylogenetic tree was constructed using FastTree (Supplementary Fig. 9 and Supplementary Data 14), and true NirK sequences were manually identified by the presence of properly aligned catalytic residues and their position relative to reference sequences in the tree.
C₁ carbon and inorganic nitrogen metabolism were assessed by looking at a specific set of 28 targeted functions. For further information on annotation criteria and functional assignments to genomes see Supplementary Tables 9–13.

Activities of enzymes related to C metabolism, including PEPC (Osuna et al., 1996 (link)),ERS (Ratinaud et al., 1983 (link)), TPS (Goddijn et al., 1997 (link)), and SPS (Feng et al., 2019 (link)) in rice tissues were assayed (detailed information is shown in Supplementary material M1).
Activities of enzymes activated in N metabolism, namely, NR (Ahanger et al., 2021 (link)), nitrite reductase (NiR) (Lin et al., 2022a (link)), and GS (Hou et al., 2019 (link)) in rice tissues were determined (detailed information is shown in Supplementary material M1).
Activities of enzymes involved in 2-OG biosynthesis, i.e., NADP-ICDH (Gálvez et al., 1994 (link)), isocitrate dehydrogenases (NAD-IDH) (Gálvez et al., 1994 (link)), and glutamate dehydrogenases (GDH) (Turano et al., 1996 (link)), were also measured (detailed information is shown in Supplementary material M1).

We used WT and transgenic lines to understand microbial population dynamics under field conditions in root zones. We collected soil from the rhizosphere after transplanting seedlings from nursery (day1), at the vegetative stage (day 14 and 28) and at the reproductive stage (day 43). A quantitative analysis of soil microorganism was conducted. We quantified the relative abundance of particulate methane monooxygenase (pmoA), methyl coenzyme M reductase (mcrA) used as functional marker genes to determine methanotrophs in soil. Nitrite reductase genes (nirK, nirS) were used as functional marker genes to determine denitrifying bacteria in soil samples, and for active and total N₂O consuming bacteria in soil, nitrous oxide reductase genes (nosZ) were used as a biomarker. Soil samples were also taken before and after plantation for chemical analysis to determine the nutrient content. Nylon bags were used to collect rhizosphere soil in the field. Fresh soil with a weight of about 0.5g attached to the roots was scratched, and the total DNA was extracted from the soil using FastDNA Spin Kit for soil (MP Biomedicals LLC USA). A QuantStudio 6Flex instrument was used to conduct a soil DNA quantitative experiment, which was repeated twice for each sample. The 20ul qRT-PCR system is as follows: 10.0ul SYBR PremixExTaq (TaKaRa Norrie Biotech Auckland New Zealand), 0.4ul each of the forward and reverse primers, 2.0ul template DNA, 0.4ul DyII, 6.8ul ddH₂O (Iqbal et al., 2021 (link)).