Metabolomic, photoprotective, and photosynthetic acclimatory responses to post-flowering drought in sorghum.

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 California's 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.

Successional adaptive strategies revealed by correlating arbuscular mycorrhizal fungal abundance with host plant gene expression.

The shifts in adaptive strategies revealed by ecological succession and the mechanisms that facilitate these shifts are fundamental to ecology. These adaptive strategies could be particularly important in communities of arbuscular mycorrhizal fungi (AMF) mutualistic with sorghum, where strong AMF succession replaces initially ruderal species with competitive ones and where the strongest plant response to drought is to manage these AMF. Although most studies of agriculturally important fungi focus on parasites, the mutualistic symbionts, AMF, constitute a research system of human-associated fungi whose relative simplicity and synchrony are conducive to experimental ecology. First, we hypothesize that, when irrigation is stopped to mimic drought, competitive AMF species should be replaced by AMF species tolerant to drought stress. We then, for the first time, correlate AMF abundance and host plant transcription to test two novel hypotheses about the mechanisms behind the shift from ruderal to competitive AMF. Surprisingly, despite imposing drought stress, we found no stress-tolerant AMF, probably due to our agricultural system having been irrigated for nearly six decades. Remarkably, we found strong and differential correlation between the successional shift from ruderal to competitive AMF and sorghum genes whose products (i) produce and release strigolactone signals, (ii) perceive mycorrhizal-lipochitinoligosaccharide (Myc-LCO) signals, (iii) provide plant lipid and sugar to AMF, and (iv) import minerals and water provided by AMF. These novel insights frame new hypotheses about AMF adaptive evolution and suggest a rationale for selecting AMF to reduce inputs and maximize yields in commercial agriculture.

Interplay of starch debranching enzyme and its inhibitor is mediated by Redox-Activated SPL transcription factor.

The germination process is of central importance across the cultivated species involving several key enzymes for mobilization of stored food reserves. Pullulanase (PUL), a starch-debranching enzyme, plays an important role in mobilizing stored endosperm food reserves during germination. Pullulanase inhibitor (PULI) hinders PUL's activity through an unknown mechanism. Barley has one PUL and two PULI genes. During the time-dependent processes of seed germination, only PULI-1 expression shows an antagonistic relationship with that of PUL. Our data have indicated that the expression of PULI-1 is modulated by SPL (Squamosa-promoter-binding Protein Like) transcription factors, known to be targeted by miR156. We show that the binding of recombinant HvSPL3 protein to the PULI-1 promoter occurs under reducing, but not under oxidizing conditions. Replacement of Cys residues with threonine in HvSPL3 abolishes the binding, indicating an essential role of the redox state in the expression of PULI. Our findings may have important implications for the industrial use of starch.

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.

Stress-associated developmental reprogramming in moss protonemata by synthetic activation of the common symbiosis pathway.

Symbioses between angiosperms and rhizobia or arbuscular mycorrhizal fungi are controlled through a conserved signaling pathway. Microbe-derived, chitin-based elicitors activate plant cell surface receptors and trigger nuclear calcium oscillations, which are decoded by a calcium/calmodulin-dependent protein kinase (CCaMK) and its target transcription factor interacting protein of DMI3 (IPD3). Genes encoding CCaMK and IPD3 have been lost in multiple non-mycorrhizal plant lineages yet retained among non-mycorrhizal mosses. Here, we demonstrated that the moss Physcomitrium is equipped with a bona fide CCaMK that can functionally complement a Medicago loss-of-function mutant. Conservation of regulatory phosphosites allowed us to generate predicted hyperactive forms of Physcomitrium CCaMK and IPD3. Overexpression of synthetically activated CCaMK or IPD3 in Physcomitrium led to abscisic acid (ABA) accumulation and ectopic development of brood cells, which are asexual propagules that facilitate escape from local abiotic stresses. We therefore propose a functional role for Physcomitrium CCaMK-IPD3 in stress-associated developmental reprogramming.

The recent evolutionary rescue of a staple crop depended on over half a century of global germplasm exchange.

Rapid environmental change can lead to population extinction or evolutionary rescue. The global staple crop sorghum (Sorghum bicolor) has recently been threatened by a global outbreak of an aggressive new biotype of sugarcane aphid (SCA; Melanaphis sacchari). We characterized genomic signatures of adaptation in a Haitian breeding population that had rapidly adapted to SCA infestation, conducting evolutionary population genomics analyses on 296 Haitian lines versus 767 global accessions. Genome scans and geographic analyses suggest that SCA adaptation has been conferred by a globally rare East African allele of RMES1, which spread to breeding programs in Africa, Asia, and the Americas. De novo genome sequencing revealed potential causative variants at RMES1. Markers developed from the RMES1 sweep predicted resistance in eight independent commercial and public breeding programs. These findings demonstrate the value of evolutionary genomics to develop adaptive trait technology and highlight the benefits of global germplasm exchange to facilitate evolutionary rescue.

Morphogene-assisted transformation of Sorghum bicolor allows more efficient genome editing.

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.

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.

Broadening the impact of plant science through innovative, integrative, and inclusive outreach.

Population growth and climate change will impact food security and potentially exacerbate the environmental toll that agriculture has taken on our planet. These existential concerns demand that a passionate, interdisciplinary, and diverse community of plant science professionals is trained during the 21st century. Furthermore, societal trends that question the importance of science and expert knowledge highlight the need to better communicate the value of rigorous fundamental scientific exploration. Engaging students and the general public in the wonder of plants, and science in general, requires renewed efforts that take advantage of advances in technology and new models of funding and knowledge dissemination. In November 2018, funded by the National Science Foundation through the Arabidopsis Research and Training for the 21st century (ART 21) research coordination network, a symposium and workshop were held that included a diverse panel of students, scientists, educators, and administrators from across the US. The purpose of the workshop was to re-envision how outreach programs are funded, evaluated, acknowledged, and shared within the plant science community. One key objective was to generate a roadmap for future efforts. We hope that this document will serve as such, by providing a comprehensive resource for students and young faculty interested in developing effective outreach programs. We also anticipate that this document will guide the formation of community partnerships to scale up currently successful outreach programs, and lead to the design of future programs that effectively engage with a more diverse student body and citizenry.

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.

Holo-omics for deciphering plant-microbiome interactions.

Host-microbiome interactions are recognized for their importance to host health. An improved understanding of the molecular underpinnings of host-microbiome relationships will advance our capacity to accurately predict host fitness and manipulate interaction outcomes. Within the plant microbiome research field, unlocking the functional relationships between plants and their microbial partners is the next step to effectively using the microbiome to improve plant fitness. We propose that strategies that pair host and microbial datasets-referred to here as holo-omics-provide a powerful approach for hypothesis development and advancement in this area. We discuss several experimental design considerations and present a case study to highlight the potential for holo-omics to generate a more holistic perspective of molecular networks within the plant microbiome system. In addition, we discuss the biggest challenges for conducting holo-omics studies; specifically, the lack of vetted analytical frameworks, publicly available tools, and required technical expertise to process and integrate heterogeneous data. Finally, we conclude with a perspective on appropriate use-cases for holo-omics studies, the need for downstream validation, and new experimental techniques that hold promise for the plant microbiome research field. We argue that utilizing a holo-omics approach to characterize host-microbiome interactions can provide important opportunities for broadening system-level understandings and significantly inform microbial approaches to improving host health and fitness. Video abstract.

Agricultural Soil Management Practices Differentially Shape the Bacterial and Fungal Microbiome of Sorghum bicolor.

Soils play important roles in biological productivity. While past work suggests that microbes affect soil health and respond to agricultural practices, it is not well known how soil management shapes crop host microbiomes. To elucidate the impact of management on microbial composition and function in the sorghum microbiome, we performed 16S rRNA gene and ITS2 amplicon sequencing and metatranscriptomics on soil and root samples collected from a site in California's San Joaquin Valley that is under long-term cultivation with 1) standard (ST) or no tilling (NT) and 2) cover-cropping (CC) or leaving the field fallow (NO). Our results revealed that microbial diversity, composition, and function change across tillage and cover type, with a heightened response in fungal communities, versus bacterial. Surprisingly, ST harbored greater microbial alpha diversity than NT, indicating that tillage may open niche spaces for broad colonization. Across management regimes, we observed class-level taxonomic level shifts. Additionally, we found significant functional restructuring across treatments, including enrichment for microbial lipid and carbohydrate transport and metabolism and cell motility with NT. Differences in carbon cycling were also observed, with increased prevalence of glycosyltransferase and glycoside hydrolase carbohydrate active enzyme families with CC. Lastly, treatment significantly influenced arbuscular mycorrhizal fungi, which had the greatest prevalence and activity under ST, suggesting that soil practices mediate known beneficial plant-microbe relationships. Collectively, our results demonstrate how agronomic practices impact critical interactions within the plant microbiome and inform future efforts to configure trait-associated microbiomes in crops.Importance While numerous studies show that farming practices can influence the soil microbiome, there are often conflicting results on how microbial diversity and activity respond to treatment. In addition, there is very little work published on how the corresponding crop plant microbiome is impacted. With bacteria and fungi known to critically affect soil health and plant growth, we concurrently compared how the practices of no and standard tillage, in combination with either cover-cropping or fallow fields, shape soil and plant-associated microbiomes between the two classifications. In determining not only the response to treatment in microbial diversity and composition, but for activity as well, this work demonstrates the significance of agronomic practice in modulating plant-microbe interactions, as well as encourages future work on the mechanisms involved in community assemblages supporting similar crop outcomes.

Author Correction: A calcium signalling network activates vacuolar K+ remobilization to enable plant adaptation to low-K environments.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A calcium signalling network activates vacuolar K+ remobilization to enable plant adaptation to low-K environments.

Potassium (K) is an essential nutrient, but levels of the free K ions (K+) in soil are often limiting, imposing a constant stress on plants. We have discovered a calcium (Ca2+)-dependent signalling network, consisting of two calcineurin B-like (CBL) Ca2+ sensors and a quartet of CBL-interacting protein kinases (CIPKs), which plays a key role in plant response to K+ starvation. The mutant plants lacking two CBLs (CBL2 and CBL3) were severely stunted under low-K conditions. Interestingly, the cbl2 cbl3 mutant was normal in K+ uptake but impaired in K+ remobilization from vacuoles. Four CIPKs-CIPK3, 9, 23 and 26-were identified as partners of CBL2 and CBL3 that together regulate K+ homeostasis through activating vacuolar K+ efflux to the cytoplasm. The vacuolar two-pore K+ (TPK) channels were directly activated by the vacuolar CBL-CIPK modules in a Ca2+-dependent manner, presenting a mechanism for the activation of vacuolar K+ remobilization that plays an important role in plant adaptation to K+ deficiency.

Genetic Engineering and Editing of Plants: An Analysis of New and Persisting Questions.

Genetic engineering is a molecular biology technique that enables a gene or genes to be inserted into a plant's genome. The first genetically engineered plants were grown commercially in 1996, and the most common genetically engineered traits are herbicide and insect resistance. Questions and concerns have been raised about the effects of these traits on the environment and human health, many of which are addressed in a pair of 2008 and 2009 Annual Review of Plant Biology articles. As new science is published and new techniques like genome editing emerge, reanalysis of some of these issues, and a look at emerging issues, is warranted. Herein, an analysis of relevant scientific literature is used to present a scientific perspective on selected topics related to genetic engineering and genome editing.

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.