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Significance Understanding the molecular response of plants to drought is critical to efforts to improve agricultural yields under increasingly frequent droughts. We grew 2 cultivars of the naturally drought-tolerant food crop sorghum in the field under drought stress. We sequenced the mRNA from weekly samples of these plants, resulting in a molecular profile of drought response over the growing season. We find molecular differences in the 2 cultivars that help explain their differing tolerances to drought and evidence of a disruption in the plant’s symbiosis with arbuscular mycorrhizal fungi. Our findings are of practical importance for agricultural breeding programs, while the resulting data are a resource for the plant and microbial communities for studying the dynamics of drought response., 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.

Sorghum bicolor (L.) Moench, the fifth most important cereal worldwide, is a multi-use crop for feed, food, forage and fuel. To enhance the sorghum and other important crop plants, establishing gene function is essential for their improvement. For sorghum, identifying genes associated with its notable abiotic stress tolerances requires a detailed molecular understanding of the genes associated with those traits. The limits of this knowledge became evident from our earlier in-depth sorghum transcriptome study showing that over 40% of its transcriptome had not been annotated. Here, we describe a full spectrum of tools to engineer, edit, annotate and characterize sorghum's genes. Efforts to develop those tools began with a morphogene-assisted transformation (MAT) method that led to accelerated transformation times, nearly half the time required with classical callus-based, non-MAT approaches. These efforts also led to expanded numbers of amenable genotypes, including several not previously transformed or historically recalcitrant. Another transformation advance, termed altruistic, involved introducing a gene of interest in a separate Agrobacterium strain from the one with morphogenes, leading to plants with the gene of interest but without morphogenes. The MAT approach was also successfully used to edit a target exemplary gene, phytoene desaturase. To identify single-copy transformed plants, we adapted a high-throughput technique and also developed a novel method to determine transgene independent integration. These efforts led to an efficient method to determine gene function, expediting research in numerous genotypes of this widely grown, multi-use crop.

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.

Determining gene function is an essential goal for the key bioenergy crop,Sorghum bicolor(L.) Moench - particularly for genes associated with its notable abiotic stress tolerances. However, detailed molecular understanding of the genes associated with those traits is limited. This was made clear in our in-depth transcriptome studies in sorghum, which indicated nearly 50% of its transcriptome is not annotated. In this report, we describe a full spectrum of tools needed to transform sorghum in order to validate and annotate genes. Efforts began with modifying a transformation method that uses the morphogenic genesBaby BoomandWuschel2(Ovule Development Protein2) to accelerate transformation speed and expand amenable genotypes. In our experience, transforming RTx430 without morphogenic genes requires ~18 to 21 weeks, compared with ~10 to 12 weeks to generate T0plants using methods with morphogenic genes. Utilizing morphogenic genes also allowed for the transformation of several sorghum genotypes not previously transformed or historically recalcitrant to transformation, i.e., rapid cycling SC187, stay-green BTx642, BTx623 and sweet sorghum Ramada. In order to validate candidate genes via engineering, while simultaneously introducing the morphogenic genes, a co-transformation strategy, termed altruistic transformation, was developed. To accomplish editing of the target gene, phytoene desaturase, novel constructs were created that also included morphogenic genes. To enable full characterization of transformed plants, we adapted techniques to determine copy number and independence of events at high-throughput levels. Through these efforts, we created a complete pathway from Agrobacterium infection to high-throughput molecular genotyping that can be used to ascertain gene function and expedite basic genetic research in this widely-grown bioenergy crop plant.