Metatranscriptomes of California grassland soil microbial communities in response to rewetting

In Mediterranean grasslands, the first rainfall after the dry season is a time of high activity (1) and stress (2) for soil microbial communities, when a large portion of previously fixed organic carbon is mineralized and lost as CO₂ (3). Despite its importance, we have minimal understanding of the genetic controls that dictate soil microbial response to rewetting. Here, we describe a total of 47 metatranscriptomes that illustrate the short-term rewetting response of soil microbial communities.

Samples were collected in an annual grassland at the Hopland Extension and Research Center, Hopland, California, USA (39.00092,–123.07962) from an established field experiment with reduced and controlled precipitation treatments. The site contained 16 3.25 m² plots covered by rainout shelters; half received 50% of annual precipitation, and the rest received 100% of annual precipitation. Soils were collected in September 2018, at the end of the Mediterranean climate dry season. The top 0–15 cm of a 0.5 m³ area was collected from four plots per treatment and transported to Lawrence Livermore National Lab, Livermore, CA, USA.

Soils were homogenized via a 2-mm sieve and weighed into 44 incubation chambers per treatment, each containing 5 g. Samples were rewet to 22% gravimetric water content with either H₂¹⁸O (98 atom% ¹⁸O) or natural abundance water and then 4 samples per treatment were destructively harvested at 3, 24, 48, 72, or 168 h post-rewetting, with four samples harvested per treatment just prior to rewetting. Harvested soils were immediately frozen in liquid N₂ and stored at −80°C.

Samples treated with H₂¹⁸O were used in a quantitative stable isotope probing study (4); however, this treatment was not predicted to influence transcription and RNA was therefore extracted from four randomly selected samples according to the time and precipitation treatments only. RNA was extracted using an RNEasy PowerSoil Total RNA Kit (Qiagen) using the standard protocol. RNA quality was assessed using a Nanodrop One Spectrophotometer (Thermo Fisher Scientific Inc) and gel electrophoresis, and quantity was determined using a Qubit Fluorometer (Invitrogen).

High-quality RNA samples were shipped to the Department of Energy Joint Genome Institute (JGI; Berkeley, CA, USA) for sequencing. From 100 ng of RNA, rRNA was depleted using QIAseq FastSelect kits (Qiagen): 5S/16S/23S, rRNA Plant, and rRNA Yeast. The TruSeq Stranded mRNA kit (Illumina) was used with heat-fragmented 300–400 bp RNA to create the first strand of cDNA using random hexamers and SuperScript Reverse Transcriptase (Thermo Fisher Scientific) followed by the second strand. cDNA was A-tailed and ligated with JGI’s dual index adapters. Prior to sequencing, one RNA sample was discarded because of low quality, leaving 47 samples. Prepared libraries were quantified with a KAPA Biosystem NGS library qPCR kit (Roche) and run on a LightCycler 480 real-time PCR instrument (Roche). Samples were sequenced on an Illumina NovaSeq sequencer using a NovaSeq XP V1.5 reagent kits on S4 flow cells, following the 2 × 151 read protocol. Raw reads were QC filtered using BBTools (v 38.86)—by trimming and removing contaminants (human, mouse, cat, dog), duplicate, ribosomal, and low-quality reads. QC-filtered reads were assembled into contiguous sequences using MEGAHIT v 1.2.9 (5) (--k-list 23, 43, 63, 83, 103, 123). The input reads were then mapped to assemblies using bbmap (v. 38.86; with option --ambiguous=random) (6). Assembled contiguous sequences were annotated using the IMG Annotation Pipeline v.5.1.5 (7).

We acknowledge that Hopland Research and Extension Center (HREC) sits on the traditional, unceded land of the Pomo Indians. Research conducted at the University of California, Berkeley took place on the territory of xučyun (Huichin), the ancestral and unceded land of the Chochenyo-speaking Ohlone people, the successors of the sovereign Verona Band of Alameda County. This land was and continues to be of great importance to the Muwekma Ohlone Tribe and other familial descendants of the Verona Band.

We thank members of the Firestone and Pett-Ridge groups who helped with field sampling and soil processing, Donald Herman, Mengting Yuan, John Bailey, and staff of HREC who helped to design and manage the precipitation experiment, and Xiao Bin Max Li and Marissa Lafler who helped with the large-scale wet-up microcosm experiment.

This research was supported by the U.S. Department of Energy (DOE), Office of Biological and Environmental Research (BER), Genomic Science Program Lawrence Livermore National Laboratory (LLNL) "Microbes Persist" Soil Microbiome Scientific Focus Area SCW1632, and a subaward to UC Berkeley. Field plots and precipitation management were initially generated via DOE BER awards DE-SC0020163 and DE-SC0016247 (to M.K.F.) and awards SCW1589, and SCW1421 (to J.P.-R.). Sequencing was conducted at the Joint Genome Institute (JGI) via a DOE Biological and Environmental Support Science award (proposal #508594; to J.P.-R.). Research Work at Lawrence Livermore National Laboratory was conducted under the auspices of the U.S. DOE under contract DE-AC52-07NA27344. The work (proposal 10.46936/10.25585/60008139) conducted by the U.S. DOE Joint Genome Institute (https://ror.org/04xm1d337), a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. DOE operated under Contract No. DE-AC02-05CH11231.