Forest floor community metatranscriptomes identify fungal and bacterial responses to N deposition in two maple forests

Abstract

Anthropogenic N deposition alters patterns of C and N cycling in temperate forests, where forest floor litter decomposition is a key process mediated by a diverse community of bacteria and fungi. To track forest floor decomposer activity we generated metatranscriptomes that simultaneously surveyed the actively expressed bacterial and eukaryote genes in the forest floor, to compare the impact of N deposition on the decomposers in two natural maple forests in Michigan, USA, where replicate field plots had been amended with N for 16 years. Site and N amendment responses were compared using about 74,000 carbohydrate active enzyme transcript sequences (CAZymes) in each metatranscriptome. Parallel ribosomal RNA (rRNA) surveys of bacterial and fungal biomass and taxonomic composition showed no significant differences in either biomass or OTU richness between the two sites or in response to N. Site and N amendment were not significant variables defining bacterial taxonomic composition, but they were significant for fungal community composition, explaining 17 and 14% of the variability, respectively. The relative abundance of expressed bacterial and fungal CAZymes changed significantly with N amendment in one of the forests, and N-response trends were also identified in the second forest. Although the two ambient forests were similar in community biomass, taxonomic structure and active CAZyme profile, the shifts in active CAZyme profiles in response to N-amendment differed between the sites. One site responded with an over-expression of bacterial CAZymes, and the other site responded with an over-expression of both fungal and different bacterial CAZymes. Both sites showed reduced representation of fungal lignocellulose degrading enzymes in N-amendment plots. The metatranscriptome approach provided a holistic assessment of eukaryote and bacterial gene expression and is applicable to other systems where eukaryotes and bacteria interact.

Assembly of Active Bacterial and Fungal Communities Along a Natural Environmental Gradient

“…To compare patterns from the ribosomal survey and a functional gene, we utilized existing metatranscriptome data generated from the same samples used in the current study [18]. We compared the proportion active OTUs using rRNA to rDNA ratios to the relative abundance of a gene associated with transitions from dormant to active states, the resuscitation promoting factor C (rpfC), which has been found in numerous metagenomes and is prevalent in soils [3].…”

“…We compared the proportion active OTUs using rRNA to rDNA ratios to the relative abundance of a gene associated with transitions from dormant to active states, the resuscitation promoting factor C (rpfC), which has been found in numerous metagenomes and is prevalent in soils [3]. Methods for preparation of samples for sequencing on the Illumina HiSeq 2000 can be found in Hesse et al (2015) [18]. Of the 12 samples from the ambient plots, three failed to produce libraries, reducing the number of matched ribosomal-transcriptome samples to 7.…”

“…Detailed explanation of molecular and sequence processing methods can be found in Hesse et al (2015) [18]. Briefly, total DNA and RNA were co-extracted from three 2-g litter subsamples using a beat beating and phenol chloroform extraction method.…”

Tree species select diverse soil fungal communities expressing different sets of lignocellulolytic enzyme-encoding genes

“…Among soil microorganisms, saprotrophic fungi are considered to be the most efficient decomposers of these biopolymers due to their important lignocellulolytic potential, filamentous growth form and wide diversity in soils Talbot et al, 2015). Indeed, despite the presence of diversified bacterial communities in forest litters and soils (Urbanov a et al, 2015), fungal lignocellulolytic transcripts are more abundant in soils (Hesse et al, 2015) and a study using a metaproteomic approach reported that all hydrolytic enzymes identified from beech litter extracts were likely produced by saprotrophic fungi (Schneider et al, 2012).…”

“…Therefore, the assemblage of the active fungal communities was assessed by the sequencing of a fragment of the Elongation Factor 1-alpha (EF1a) gene, a housekeeping and constitutively expressed eukaryotic gene (Rehner and Buckley, 2005). Indeed, neither the intron-like rDNA internal transcribed spacer (ITS) region nor the nuclear large and small-subunit rDNA encoding genes (28S and 18S), widely used as barcode markers for fungal identification in environmental studies (Schmidt et al, 2013;Hesse et al, 2015), can be amplified from environmental cDNAs synthesized from eukaryotic poly-A mRNA using oligo(dT) primers. Although the EF1a gene sequence has a poor species-level resolution in fungi compared to the ITS (Schoch et al, 2012), EF1a sequence assignation to the main fungal phyla or sub-phyla can be confidently performed (Roger et al, 1999).…”