Plant microbiomes harbor potential to promote nutrient turnover in impoverished substrates of a Brazilian biodiversity hotspot

Abstract

The substrates of the Brazilian campos rupestres, a grassland ecosystem, have extremely low concentrations of phosphorus and nitrogen, imposing restrictions to plant growth. Despite that, this ecosystem harbors almost 15% of the Brazilian plant diversity, raising the question of how plants acquire nutrients in such a harsh environment. Here, we set out to uncover the taxonomic profile, the compositional and functional differences and similarities, and the nutrient turnover potential of microbial communities associated with two plant species of the campos rupestres-dominant family Velloziaceae that grow over distinct substrates (soil and rock). Using amplicon sequencing data, we show that, despite the pronounced composition differentiation, the plant-associated soil and rock communities share a core of highly efficient colonizers that tend to be highly abundant and is enriched in 21 bacterial families. Functional investigation of metagenomes and 522 metagenome-assembled genomes revealed that the microorganisms found associated to plant roots are enriched in genes involved in organic compound intake, and phosphorus and nitrogen turnover. We show that potential for phosphorus transport, mineralization, and solubilization are mostly found within bacterial families of the shared microbiome, such as Xanthobacteraceae and Bryobacteraceae. We also detected the full repertoire of nitrogen cycle-related genes and discovered a lineage of Isosphaeraceae that acquired nitrogen-fixing potential via horizontal gene transfer and might be also involved in nitrification via a metabolic handoff association with Binataceae. We highlight that plant-associated microbial populations in the campos rupestres harbor a genetic repertoire with potential to increase nutrient availability and that the microbiomes of biodiversity hotspots can reveal novel mechanisms of nutrient turnover.

Fig. 1. Composition and novelty of the V. epidendroides and B. macrantha microbiomes.

A Sampling was conducted in the campos rupestres grasslands ecoregion (left). Vellozia epidendroides (center) specimens were collected in patches of shallow soil. Barbacenia macrantha (right) was found in a rocky area, where it grows over exposed rocks. B Community composition inferred from 16S rRNA gene ASVs at the phylum level. Samples were grouped according to their environment. Bar heights are proportional to the relative abundance of the phylum. Low abundance phyla (relative abundance < 2%) were grouped under the “Other” category. C Maximum-likelihood phylogenetic tree of the bacterial MAGs presented in this study, rooted at the Patescibacteria clade. The innermost ring indicates the phylum associated with each node. The center ring shows the genomic GC content. The outermost ring represents the scaled means of log-transformed relative genomic coverages across the four environments. D Weighted average community identity (WACI) computed from 16S rRNA gene ASV data. The blue and green dashed lines represent the median intra-rank 16S rRNA gene identity at the genus and family levels, respectively. E Phylogenetic gain (PG) contributed by the MAGs to different taxa at the phylum, class, and order levels. Only taxa with PG higher than the following cut-offs are shown: 5% at the phylum level, 30% at the class level, and 40% at the order level. RX root (external), RN root (internal), SX stem (external), SN stem (internal), LX leaf (external), LN leaf (internal).

Fig. 2. The differentiation between V. epidendroides and B. macrantha microbiomes is taxonomically structured.

A Multidimensional scaling of the Bray–Curtis dissimilarities computed from 16S rRNA gene ASV abundance data. Samples are colored according to their associated plant species, and shape indicates whether they were from below ground (substrate and root) or above ground (stem and leaves) environments. The p values of the groupings were obtained from PERMANOVA tests. B Bar plots representing the fraction of V. epidendroides-exclusive, shared, and B. macrantha-exclusive 16S rRNA gene ASVs across all sample types. The absolute numbers of ASVs within each group are shown. C Enrichment of bacterial families (grouped by phyla) in one or the other plant across all environments (circle colors). The enrichment score in the x-axis was computed using the Kolmogorov–Smirnov test and represent the deviation from a null model where ASVs from a given family are uniformly distributed in a list ranked by the ratio between the abundances in each plant. No family was found to be enriched in the internal leaf communities of either plant. RX root (external), RN root (internal), SX stem (external), SN stem (internal), LX leaf (external), LN leaf (internal).

Fig. 3. V. epidendroides and B. macrantha share a core microbiome that encompasses multiple families of efficient colonizers.

A Proportion of the total number of 16S rRNA gene ASVs (light yellow) and of the ASV abundance (dark yellow) shared between the communities associated with both plants. B Bacterial families (grouped by phyla) enriched within the shared ASV sets. The x-axis shows the false discovery rate (FDR) obtained from hypergeometric tests. The extent of the enrichment for each family, represented by the circle areas, was quantified as the ratio between the number of ASVs in the shared fraction and the number of ASVs observed in both the shared and exclusive fractions. No family was found to be enriched in the internal leaf communities of either plant. RX root (external), RN root (internal), SX stem (external), SN stem (internal), LX leaf (external), LN leaf (internal).

Fig. 4. Mean total abundance (sum of RPKGs) of the investigated transporter genes in the roots (x-axis) and substrates (y-axis) of both plants.

Circles are colored according to their assigned substrate class: amino acids (23 substrates) and organic acids (16 substrates). Horizontal and vertical lines represent the standard error of the mean in roots and substrates, respectively.

Fig. 5. Root-associated microbiomes exhibit increased P-turnover potential in comparison to substrate communities.

A Root exudates both solubilize phosphorus (P) in the plant substrate and recruit microorganisms that consume this nutrient. As the recruited microbes can mobilize phosphorus that would otherwise be unavailable for the plants (in pink), the total bioaccessible phosphorus concentration increases over time. B Mean total abundances (sum of RPKGs) of proteins and pathways involved in processes linked to phosphate turnover (transport, mineralization, and solubilization) in the substrate and root-associated communities. Abundances of multiprotein complexes (pstABCS, phnCDE, ugpABCE, and phnGHIJKLM) were computed by averaging the abundances of their subunits. Vertical lines represent the standard error of the mean. C Phylogenetic regressions of the number of phosphate turnover-related genes. The MAGs retrieved in this study were compared to GTDB genomes to identify differences in the numbers of gene copies associated with phosphorus turnover. The color scale indicates the magnitude of the enrichment (blue) or depletion (red) of each process in the MAGs, and the area of the circles represent the statistical significance of the regression coefficient. Regression coefficients with p value > 0.05 are omitted. Phylogenetic regressions were performed on the whole set of bacterial genomes and on the phyla containing at least 5 MAGs. RX root (external), CR campos rupestres.

Fig. 6. Structural diversity of siderophore biosynthetic gene clusters (BGCs) identified in the campos rupestres metagenomes.

BGC regions containing siderophore clusters were hierarchically clustered using UPGMA with BiG-SCAPE distances. Groups of highly similar regions were identified based on their inconsistency coefficient and only the medoids are shown. Blue labels indicate the BGC regions that belong to the Pseudonocardiaceae-associated gene cluster clan. Taxonomies are presented at the family and phylum (in parenthesis) levels, except for two BGC regions whose contigs were assigned to the Bacteria domain. Heatmaps represent scaled means of log-transformed relative contig coverages in the four environments. Gene clusters are shown as arrays of genes (arrows) and their protein domains (colored blocks) centered at the siderophore biosynthesis protein. BGC regions containing other types of biosynthetic clusters (rightmost column) were trimmed to display only the loci assigned to the siderophore clusters.

Fig. 7. The N-turnover landscape of the campos rupestres microbiomes.

A Phyla predicted to be involved in nitrogen-cycling reactions: fixation (black arrow), nitrification (blue arrows), denitrification (red arrows), and assimilatory nitrate reduction (gray arrow). Compounds that can be taken up by plants roots are depicted in green. Phyla that contributed less than 5% of the genes involved in each reaction were grouped under the “Other” category. B Total abundances (sum of RPKGs) of reactions involved in nitrogen turnover in the substrate and external root-associated communities. The abundances of multiprotein complexes (nifHDK, amoABC, nxrAB, narGHI, napAB, nasAB, norBC, nirBD, and nrfAH) were computed by averaging the abundances of their subunits. Vertical lines represent the standard error of the mean. RX = root (external). C Cladogram of phylogenies inferred from metagenomic nifH orthologs. Branches are colored according to the major group they belong to. The dominant clades are indicated by the outer rings. Orthologs encoded by MAGs encoding nif are indicated by greek letters: (α): Verrucomicrobiota, (β): Enterobacteriaceae, (γ and δ): Isosphaeraceae. D In the V. epidendroides-associated communities, nitrogen is mostly fixed by endophytic and free-living Bradyrhizobium, and by Isosphaeraceae, which likely received their nif complex via horizontal gene transfer (HGT). Ammonium is then oxidized into hydroxylamine by methylotrophic Binataceae. This molecule is then released from the cell and is oxidized into nitrite by Isosphaeraceae. In the B. macrantha-associated communities, nitrogen is likely converted into ammonium by endophytic Bradyrhizobium. Ammonia originated from organic matter decomposition is oxidized into hydroxylamine and, subsequently, into nitrite by Nitrososphaeraceae. In both plants, the oxidation of nitrite into nitrate is likely performed by a several taxa.