A comparative transcriptomic analysis was conducted for the nitrogen-efficient (BNI-Munal) and derivative parent Munal wheat genotypes to unravel the gene expression patterns across four nitrogen levels (0%, 50%, 75%, and 100%). Analyzing the genes of BNI-enabled wheat helps us understand how they are expressed differently, which heavily influences BNI activity. Grain yield and 1000-grain weight were higher in BNI Munal than in Munal. All the other traits were similar in performance. Varying nitrogen dosages led to significant differences in gene expression patterns between the two genotypes. Genes related to binding and catalytic activity were prevalent among molecular functions, while genes corresponding to cellular anatomical entities dominated the cellular component category. Differential expression was observed in 371 genes at 0%N, 261 genes at 50%N, 303 genes at 75%N, and 736 genes at 100%N. Five unigenes (three upregulated and two downregulated) were consistently expressed across all nitrogen levels. Further analysis of upregulated unigenes identified links to the NrpA gene (involved in nitrogen regulation), tetratricopeptide repeat-containing protein (PPR), and cytokinin dehydrogenase 2. Analysis of downregulated genes pointed to associations with the Triticum aestivum 3BS-specific BAC library, which encodes the NPF (Nitrate and Peptide Transporter Family) and the TaVRN gene family (closely related to the TaNUE1 gene). The five unigenes and one unigene highlighted in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were validated in Munal and BNI Munal. The results obtained will enhance our understanding about gene expression patterns across different nitrogen levels in BNI wheat and help us breed wheat varieties with the BNI trait for improved NUE.

Biological nitrification inhibitors (BNIs) assist in enhancing nitrogen availability in the wheat (Triticum aestivum L.) rhizosphere by suppressing nitrification in the rhizosphere through root-derived metabolites that may alter microbial populations associated with the nitrogen cycle and nitrogen-use efficiency. Plant roots interact with a variety of microbial communities in the rhizosphere, which is a significant hotspot for microbial diversity and activity. These microbial communities play an important role in the transformation and cycle of carbon and essential nutrients, including nitrogen and phosphorus, within the rhizosphere. Rhizospheric soil samples from three BNI-enriched genotypes, viz., BNI Munal, BNI Roelfs, and BNI Vorobey, along with their respective parental genotypes Munal, Roelfs, and Vorobey and three commercial cultivars, viz., DBW 187, DBW 303, and DBW 371, were evaluated in a pot experiment using 16S rRNA gene amplicon sequencing at four nitrogen levels (0%, 50%, 75%, and 100%) in order to study the population dynamics of the bacterial community. In the present study, the bacterial range related to the rhizosphere of the wheat plant was analyzed via PCR amplification, production of a library, and sequencing of 16S rDNA genes. The comparative analysis of bacterial abundance levels of different samples was carried out at phylum, class, order, family, genus, and species levels. Both host genotype and nitrogen availability had an impact on the organization of the bacterial community in the wheat rhizosphere, as evidenced by the differences in the relative abundance of key bacterial taxa with these factors. Diversity indices indicated differences in the relative abundance of bacterial taxa in the rhizosphere of the BNI-enriched genotypes, their parents, and the three commercial cultivars. The heatmap revealed differences in the relative abundance of bacterial taxa among rhizosphere soil samples, with Proteobacteria being one of the predominant phyla. The second ring of the Krona chart indicated that the bacterial population of the Nitrosomonadaceae family of the Proteobacteria phylum was found to be the most affected among BNI-enriched genotypes when nitrogen level was altered. This suggests that, when using 16S r RNA gene amplicon sequencing, the wheat genotype and nitrogen availability were associated with changes in bacterial taxa related to nitrogen cycling, but their functional effect remains to be confirmed. These findings have laid the groundwork for analyzing the impact of associated microbiota on future wheat production systems, particularly concerning nitrification.