Co-occurrence networks reveal more complexity than community composition in resistance and resilience of microbial communities.

Plant response to drought stress involves fungi and bacteria that live on and in plants and in the rhizosphere, yet the stability of these myco- and micro-biomes remains poorly understood. We investigate the resistance and resilience of fungi and bacteria to drought in an agricultural system using both community composition and microbial associations. Here we show that tests of the fundamental hypotheses that fungi, as compared to bacteria, are (i) more resistant to drought stress but (ii) less resilient when rewetting relieves the stress, found robust support at the level of community composition. Results were more complex using all-correlations and co-occurrence networks. In general, drought disrupts microbial networks based on significant positive correlations among bacteria, among fungi, and between bacteria and fungi. Surprisingly, co-occurrence networks among functional guilds of rhizosphere fungi and leaf bacteria were strengthened by drought, and the same was seen for networks involving arbuscular mycorrhizal fungi in the rhizosphere. We also found support for the stress gradient hypothesis because drought increased the relative frequency of positive correlations.

Cell Wall Compositions of Sorghum bicolor Leaves and Roots Remain Relatively Constant Under Drought Conditions.

Renewable fuels are needed to replace fossil fuels in the immediate future. Lignocellulosic bioenergy crops provide a renewable alternative that sequesters atmospheric carbon. To prevent displacement of food crops, it would be advantageous to grow biofuel crops on marginal lands. These lands will likely face more frequent and extreme drought conditions than conventional agricultural land, so it is crucial to see how proposed bioenergy crops fare under these conditions and how that may affect lignocellulosic biomass composition and saccharification properties. We found that while drought impacts the plant cell wall of Sorghum bicolor differently according to tissue and timing of drought induction, drought-induced cell wall compositional modifications are relatively minor and produce no negative effect on biomass conversion. This contrasts with the cell wall-related transcriptome, which had a varied range of highly variable genes (HVGs) within four cell wall-related GO categories, depending on the tissues surveyed and time of drought induction. Further, many HVGs had expression changes in which putative impacts were not seen in the physical cell wall or which were in opposition to their putative impacts. Interestingly, most pre-flowering drought-induced cell wall changes occurred in the leaf, with matrix and lignin compositional changes that did not persist after recovery from drought. Most measurable physical post-flowering cell wall changes occurred in the root, affecting mainly polysaccharide composition and cross-linking. This study couples transcriptomics to cell wall chemical analyses of a C4 grass experiencing progressive and differing drought stresses in the field. As such, we can analyze the cell wall-specific response to agriculturally relevant drought stresses on the transcriptomic level and see whether those changes translate to compositional or biomass conversion differences. Our results bolster the conclusion that drought stress does not substantially affect the cell wall composition of specific aerial and subterranean biomass nor impede enzymatic hydrolysis of leaf biomass, a positive result for biorefinery processes. Coupled with previously reported results on the root microbiome and rhizosphere and whole transcriptome analyses of this study, we can formulate and test hypotheses on individual gene candidates' function in mediating drought stress in the grass cell wall, as demonstrated in sorghum.

Genome-resolved metagenomics reveals role of iron metabolism in drought-induced rhizosphere microbiome dynamics.

Recent studies have demonstrated that drought leads to dramatic, highly conserved shifts in the root microbiome. At present, the molecular mechanisms underlying these responses remain largely uncharacterized. Here we employ genome-resolved metagenomics and comparative genomics to demonstrate that carbohydrate and secondary metabolite transport functionalities are overrepresented within drought-enriched taxa. These data also reveal that bacterial iron transport and metabolism functionality is highly correlated with drought enrichment. Using time-series root RNA-Seq data, we demonstrate that iron homeostasis within the root is impacted by drought stress, and that loss of a plant phytosiderophore iron transporter impacts microbial community composition, leading to significant increases in the drought-enriched lineage, Actinobacteria. Finally, we show that exogenous application of iron disrupts the drought-induced enrichment of Actinobacteria, as well as their improvement in host phenotype during drought stress. Collectively, our findings implicate iron metabolism in the root microbiome's response to drought and may inform efforts to improve plant drought tolerance to increase food security.

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers.

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

Fungal community assembly in drought-stressed sorghum shows stochasticity, selection, and universal ecological dynamics.

Community assembly of crop-associated fungi is thought to be strongly influenced by deterministic selection exerted by the plant host, rather than stochastic processes. Here we use a simple, sorghum system with abundant sampling to show that stochastic forces (drift or stochastic dispersal) act on fungal community assembly in leaves and roots early in host development and when sorghum is drought stressed, conditions when mycobiomes are small. Unexpectedly, we find no signal for stochasticity when drought stress is relieved, likely due to renewed selection by the host. In our experimental system, the host compartment exerts the strongest effects on mycobiome assembly, followed by the timing of plant development and lastly by plant genotype. Using a dissimilarity-overlap approach, we find a universality in the forces of community assembly of the mycobiomes of the different sorghum compartments and in functional guilds of fungi.

Top-down mass spectrometry of histone modifications in sorghum reveals potential epigenetic markers for drought acclimation.

Sorghum [Sorghum bicolor (L.) Moench] is an important cereal crop noted for its ability to survive water-limiting conditions. Herein, we present an analytical workflow to explore the changes in histone modifications through plant developmental stages and two drought stresses in two sorghum genotypes that differ in their response to drought. Top-down mass spectrometry (MS) is an ideal method to profile histone modifications and distinguish closely related histone proteoforms. We analyzed leaves of 48 plants and identified 26 unique histone proteins and 677 unique histone proteoforms (124 full-length and 553 truncated proteoforms). We detected trimethylation on nearly all H2B N-termini where acetylation is commonly expected. In addition, an unexpected modification on H2A histones was assigned to N-pyruvic acid 2-iminylation based on its unique neutral loss of CO2. Interestingly, some of the truncated histones, in particular H4 and H3.2, showed significant changes that correlated with the growth and water conditions. The histone proteoforms could serve as targets in search of chromatin modifiers and ultimately have important ramifications in future attempts of studying plant epigenetic reprogramming under stress.

Transcriptomic analysis of field-droughted sorghum from seedling to maturity reveals biotic and metabolic responses.

Drought is the most important environmental stress limiting crop yields. The C4 cereal sorghum [Sorghum bicolor (L.) Moench] is a critical food, forage, and emerging bioenergy crop that is notably drought-tolerant. We conducted a large-scale field experiment, imposing preflowering and postflowering drought stress on 2 genotypes of sorghum across a tightly resolved time series, from plant emergence to postanthesis, resulting in a dataset of nearly 400 transcriptomes. We observed a fast and global transcriptomic response in leaf and root tissues with clear temporal patterns, including modulation of well-known drought pathways. We also identified genotypic differences in core photosynthesis and reactive oxygen species scavenging pathways, highlighting possible mechanisms of drought tolerance and of the delayed senescence, characteristic of the stay-green phenotype. Finally, we discovered a large-scale depletion in the expression of genes critical to arbuscular mycorrhizal (AM) symbiosis, with a corresponding drop in AM fungal mass in the plants' roots.

Strong succession in arbuscular mycorrhizal fungal communities.

The ecology of fungi lags behind that of plants and animals because most fungi are microscopic and hidden in their substrates. Here, we address the basic ecological process of fungal succession in nature using the microscopic, arbuscular mycorrhizal fungi (AMF) that form essential mutualisms with 70-90% of plants. We find a signal for temporal change in AMF community similarity that is 40-fold stronger than seen in the most recent studies, likely due to weekly samplings of roots, rhizosphere and soil throughout the 17 weeks from seedling to fruit maturity and the use of the fungal DNA barcode to recognize species in a simple, agricultural environment. We demonstrate the patterns of nestedness and turnover and the microbial equivalents of the processes of immigration and extinction, that is, appearance and disappearance. We also provide the first evidence that AMF species co-exist rather than simply co-occur by demonstrating negative, density-dependent population growth for multiple species. Our study shows the advantages of using fungi to test basic ecological hypotheses (e.g., nestedness v. turnover, immigration v. extinction, and coexistence theory) over periods as short as one season.

Drought delays development of the sorghum root microbiome and enriches for monoderm bacteria.

Drought stress is a major obstacle to crop productivity, and the severity and frequency of drought are expected to increase in the coming century. Certain root-associated bacteria have been shown to mitigate the negative effects of drought stress on plant growth, and manipulation of the crop microbiome is an emerging strategy for overcoming drought stress in agricultural systems, yet the effect of drought on the development of the root microbiome is poorly understood. Through 16S rRNA amplicon and metatranscriptome sequencing, as well as root metabolomics, we demonstrate that drought delays the development of the early sorghum root microbiome and causes increased abundance and activity of monoderm bacteria, which lack an outer cell membrane and contain thick cell walls. Our data suggest that altered plant metabolism and increased activity of bacterial ATP-binding cassette (ABC) transporter genes are correlated with these shifts in community composition. Finally, inoculation experiments with monoderm isolates indicate that increased colonization of the root during drought can positively impact plant growth. Collectively, these results demonstrate the role that drought plays in restructuring the root microbiome and highlight the importance of temporal sampling when studying plant-associated microbiomes.