Rhizosphere and detritusphere habitats modulate expression of soil N-cycling genes during plant development

Abstract

Plant roots modulate microbial nitrogen (N) cycling by regulating the supply of root-derived carbon and nitrogen uptake. These differences in resource availability cause distinct micro-habitats to develop: soil near living roots, decaying roots, near both, or outside the direct influence of roots. While many environmental factors and genes control the microbial processes involved in the nitrogen cycle, most research has focused on single genes and pathways, neglecting the interactive effects these pathways have on each other. The processes controlled by these pathways determine consumption and production of N by soil microorganisms. We followed the expression of N-cycling genes in four soil microhabitats over a period of active root growth for an annual grass. We found that the presence of root litter and living roots significantly altered gene expression involved in multiple nitrogen pathways, as well as tradeoffs between pathways, which ultimately regulate N availability to plants.

Keywords: detritusphere; gene expression; metatranscriptomics; nitrification; plant litter; rhizosphere; soil microbiome; soil nitrogen.

Fig 1

Principal coordinates analysis of nitrogen cycling gene expression. Influence of living roots and root detritus on soil microbial gene expression during 3 weeks of Avena fatua root growth (harvests at 3, 6, 12, and 22 days) in a California annual grassland soil. Input data are Bray-Curtis dissimilarity of expression of N-cycling genes variance normalized to the sequencing depth and gene length, determined by mapping reads to ORFs assembled from metatranscriptomes, in (A) all timepoints and (B) excluding the last timepoint (22 days). n = 3 for each habitat and timepoint. Ellipses represent a 90% confidence interval of the ordination coordinates (calculated by stat_ellipse).

Fig 2

Expression level and upregulation by soil habitat of nitrogen cycling genes from assembled transcripts. Bold font indicates high expression levels (mean counts in each timepoint and habitat >5 × 10⁴ after normalization to the gene length and sequencing depth). Significant upregulation compared to unamended bulk soil at the same timepoint was determined by ANOVA and Tukey post hoc test (adjusted P < 0.05; Table S3). Numbers denote pathways: 1, ammonium oxidation; 2, nitrite oxidation; 3, assimilatory nitrate reduction; 4, denitrification; 5, anammox; 6, nitrogen fixation; 7, DNRA; 8, ammonium assimilation (GDH and GS/GOGAT); 9, macromolecular N mineralization; amt, ammonium transporter. The functional role of each enzyme is explained in Table S2. The nirK homologue is an archaeal enzyme which participates in archaeal nitrification, but unlike its bacterial homologue, it does not generate nitrate (43).

Fig 3

Phylogenetic determination of nitrifiers by amoA, nirK, and 16S rRNA. Phylogeny of assembled ORFs (black) (A) amoA and (B) nirK placed into phylogenetic trees containing curated reference sequences from RefSeq (gray). Colors represent phylogenetic clades. Bars around the trees represent cumulative coverage from all samples to specific assembled transcripts. (C) Relative abundance of AOA, AOB, and nitrite oxidizing bacteria (NOB) of the entire microbial community by 16S-rRNA gene abundance. (D) Positive correlation between expression of amoA and nirK transcripts by location and amendment (R ² = 0.7). (E) Positive correlation between expression of amoA and Thaumarchaeota extracellular protease transcripts by location and amendment (R ² = 0.61). (F) Normalized expression of amoA in the rhizosphere (with or without litter amendment) over time. The legend in panel D applies to panels C–F.

Fig 4

Expression of ammonium assimilation pathways and their regulatory genes. (A) Simplified conceptual representation of glnA activation via the regulatory cascade. (B) Variance-normalized expression of glnA in amended/unamended rhizosphere and bulk soil. Asterisks denote a significant increase compared to the same litter amendment in bulk soil (e.g., unamended rhizosphere vs unamended bulk). (C) Ratio of GS/GOGAT to GDH (glnA:gudB) expression in the metatranscriptomes. (D) Mean log ratio transformed differential expression compared to unamended bulk soil of glnA, and the regulatory genes that activate it under N limitation.