Plant-associated microbial communities shift in composition as a result of environmental perturbations, such as drought. It has been shown that Actinobacteria are enriched in plant roots and rhizospheres during drought stress. However, the correlations between microbiome dynamics and plant response to drought are poorly understood. Here we apply a combination of bacterial community composition analysis and plant metabolite profiling in Sorghum bicolor roots, rhizospheres, and soil during drought and drought recovery to investigate potential contributions of host metabolism to shifts in bacterial composition. Our results provide a detailed view of metabolic shifts across the plant root during drought and show that the response to rewatering differs between root and soil; additionally, we identify drought-responsive metabolites that are highly correlated with the observed changes in Actinobacteria abundance. Furthermore, we find that pipecolic acid is a drought-enriched metabolite in sorghum roots, and that exogenous application of pipecolic acid inhibits root growth. Finally, we show that this activity functions independent of the systemic acquired resistance pathway and has the potential to impact Actinobacterial taxa within the root microbiome.

Background

Sorghum bicolor is the fifth most commonly grown cereal worldwide and is remarkable for its drought and abiotic stress tolerance. For these reasons and the large size of biomass varieties, it has been proposed as a bioenergy crop. However, little is known about the genes underlying sorghum's abiotic stress tolerance and biomass yield.

Results

To uncover the genetic basis of drought tolerance in sorghum at a genome-wide level, we undertook a high-density phenomics genome wide association study (GWAS) in which 648 diverse sorghum lines were phenotyped at two locations in California once per week by drone over the course of a growing season. Biomass, height, and leaf area were measured by drone for individual field plots, subjected to two drought treatments and a well-watered control. The resulting dataset of ~ 171,000 phenotypic data-points was analyzed along with 183,989 genotype by sequence markers to reveal 213 high-quality, replicated, and conserved GWAS associations.

Conclusions

The genomic intervals defined by the associations include many strong candidate genes, including those encoding heat shock proteins, antifreeze proteins, and other domains recognized as important to plant stress responses. The markers identified by our study can be used for marker assisted selection for drought tolerance and biomass. In addition, our results are a significant step toward identifying specific sorghum genes controlling drought tolerance and biomass yield.

Climate change is globally affecting rainfall patterns, necessitating the improvement of drought tolerance in crops. Sorghum bicolor is a relatively drought-tolerant cereal. Functional stay-green sorghum genotypes can maintain green leaf area and efficient grain filling during terminal post-flowering water deprivation, a period of ~10 weeks. To obtain molecular insights into these characteristics, two drought-tolerant genotypes, BTx642 and RTx430, were grown in replicated control and terminal post-flowering drought field plots in Californias Central Valley. Photosynthetic, photoprotective, and water dynamics traits were quantified and correlated with metabolomic data collected from leaves, stems, and roots at multiple timepoints during control and drought conditions. Physiological and metabolomic data were then compared to longitudinal RNA sequencing data collected from these two genotypes. The unique metabolic and transcriptomic response to post-flowering drought in sorghum supports a role for the metabolite galactinol in controlling photosynthetic activity through regulating stomatal closure in post-flowering drought. Additionally, in the functional stay-green genotype BTx642, photoprotective responses were specifically induced in post-flowering drought, supporting a role for photoprotection in the molecular response associated with the functional stay-green trait. From these insights, new pathways are identified that can be targeted to maximize yields under growth conditions with limited water.

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.

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.

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.