Seedling root system adaptation to water availability during maize domestication and global expansion.

The maize root system has been reshaped by indirect selection during global adaptation to new agricultural environments. In this study, we characterized the root systems of more than 9,000 global maize accessions and its wild relatives, defining the geographical signature and genomic basis of variation in seminal root number. We demonstrate that seminal root number has increased during maize domestication followed by a decrease in response to limited water availability in locally adapted varieties. By combining environmental and phenotypic association analyses with linkage mapping, we identified genes linking environmental variation and seminal root number. Functional characterization of the transcription factor ZmHb77 and in silico root modeling provides evidence that reshaping root system architecture by reducing the number of seminal roots and promoting lateral root density is beneficial for the resilience of maize seedlings to drought.

Heritable microbiome variation is correlated with source environment in locally adapted maize varieties.

Beneficial interactions with microorganisms are pivotal for crop performance and resilience. However, it remains unclear how heritable the microbiome is with respect to the host plant genotype and to what extent host genetic mechanisms can modulate plant-microbiota interactions in the face of environmental stresses. Here we surveyed 3,168 root and rhizosphere microbiome samples from 129 accessions of locally adapted Zea, sourced from diverse habitats and grown under control and different stress conditions. We quantified stress treatment and host genotype effects on the microbiome. Plant genotype and source environment were predictive of microbiome abundance. Genome-wide association analysis identified host genetic variants linked to both rhizosphere microbiome abundance and source environment. We identified transposon insertions in a candidate gene linked to both the abundance of a keystone bacterium Massilia in our controlled experiments and total soil nitrogen in the source environment. Isolation and controlled inoculation of Massilia alone can contribute to root development, whole-plant biomass production and adaptation to low nitrogen availability. We conclude that locally adapted maize varieties exert patterns of genetic control on their root and rhizosphere microbiomes that follow variation in their home environments, consistent with a role in tolerance to prevailing stress.

Transcriptomic changes in barley leaves induced by alcohol ethoxylates indicate potential pathways of surfactant detoxification.

Hardly anything is known regarding the detoxification of surfactants in crop plants, although they are frequently treated with agrochemical formulations. Therefore, we studied transcriptomic changes in barley leaves induced in response to spraying leaf surfaces with two alcohol ethoxylates (AEs). As model surfactants, we selected the monodisperse tetraethylene glycol monododecyl (C12E4) ether and the polydisperse BrijL4. Barley plants were harvested 8 h after spraying with a 0.1% surfactant solution and changes in gene expression were analysed by RNA-sequencing (RNA-Seq). Gene expression was significantly altered in response to both surfactants. With BrijL4 more genes (9724) were differentially expressed compared to C12E4 (6197). Gene families showing pronounced up-regulation were cytochrome P450 enzymes, monooxygenases, ABC-transporters, acetyl- and methyl- transferases, glutathione-S-transferases and glycosyltransferases. These specific changes in gene expression and the postulated function of the corresponding enzymes allowed hypothesizing three potential metabolic pathways of AE detoxification in barley leaves. (i) Up-regulation of P450 cytochrome oxidoreductases suggested a degradation of the lipophilic alkyl residue (dodecyl chain) of the AEs by ω- and ß- oxidation. (ii) Alternatively, the polar PEG-chain of AEs could be degraded. (iii) Instead of surfactant degradation, a further pathway of detoxification could be the sequestration of AEs into the vacuole or the apoplast (cell wall). Thus, our results show that AEs lead to pronounced changes in the expression of genes coding for proteins potentially being involved in the detoxification of surfactants.

Genetic regulation of the root angle in cereals.

The root angle plays a critical role in efficiently capturing nutrients and water from different soil layers. Steeper root angles enable access to mobile water and nitrogen from deeper soil layers, whereas shallow root angles facilitate the capture of immobile phosphorus from the topsoil. Thus, understanding the genetic regulation of the root angle is crucial for breeding crop varieties that can efficiently capture resources and enhance yield. Moreover, this understanding can contribute to developing varieties that effectively sequester carbon in deeper soil layers, supporting global carbon mitigation efforts. Here we review and consolidate significant recent discoveries regarding the molecular components controlling root angle in cereal crop species and outline the remaining research gaps in this field.

Contrasting cytosolic glutathione redox dynamics under abiotic and biotic stress in barley as revealed by the biosensor Grx1-roGFP2.

Barley is a staple crop of major global importance and relatively resilient to a wide range of stress factors in the field. Transgenic reporter lines to investigate physiological parameters during stress treatments remain scarce. We generated and characterized transgenic homozygous barley lines (cv. Golden Promise Fast) expressing the genetically encoded biosensor Grx1-roGFP2, which indicates the redox potential of the major antioxidant glutathione in the cytosol. Our results demonstrated functionality of the sensor in living barley plants. We determined the glutathione redox potential (EGSH) of the cytosol to be in the range of -308 mV to -320 mV. EGSH was robust against a combined NaCl (150 mM) and water deficit treatment (-0.8 MPa) but responded with oxidation to infiltration with the phytotoxic secretome of the necrotrophic fungus Botrytis cinerea. The generated reporter lines are a novel resource to study biotic and abiotic stress resilience in barley, pinpointing that even severe abiotic stress leading to a growth delay does not automatically induce cytosolic EGSH oxidation, while necrotrophic pathogens can undermine this robustness.

Spatiotemporal transcriptomic plasticity in barley roots: unravelling water deficit responses in distinct root zones.

BACKGROUND: Drought poses a major threat to agricultural production and thus food security. Understanding the processes shaping plant responses to water deficit is essential for global food safety. Though many studies examined the effect of water deficit on the whole-root level, the distinct functions of each root zone and their specific stress responses remain masked by this approach. RESULTS: In this study, we investigated the effect of water deficit on root development of the spring barley (Hordeum vulgare L.) cultivar Morex and examined transcriptomic responses at the level of longitudinal root zones. Water deficit significantly reduced root growth rates after two days of treatment. RNA-sequencing revealed root zone and temporal gene expression changes depending on the duration of water deficit treatment. The majority of water deficit-regulated genes were unique for their respective root zone-by-treatment combination, though they were associated with commonly enriched gene ontology terms. Among these, we found terms associated with transport, detoxification, or cell wall formation affected by water deficit. Integration of weighted gene co-expression analyses identified differential hub genes, that highlighted the importance of modulating energy and protein metabolism and stress response. CONCLUSION: Our findings provide new insights into the highly dynamic and spatiotemporal response cascade triggered by water deficit and the underlying genetic regulations on the level of root zones in the barley cultivar Morex, providing potential targets to enhance plant resilience against environmental constraints. This study further emphasizes the importance of considering spatial and temporal resolution when examining stress responses.

Molecular dissection of heterosis in cereal roots and their rhizosphere.

KEY MESSAGE: Heterosis is already manifested early in root development. Consistent with the dominance model of heterosis, gene expression complementation is a general mechanism that contributes to phenotypic heterosis in maize hybrids. Highly heterozygous F1-hybrids outperform their parental inbred lines, a phenomenon known as heterosis. Utilization of heterosis is of paramount agricultural importance and has been widely applied to increase yield in many crop cultivars. Plant roots display heterosis for many traits and are an important target for further crop improvement. To explain the molecular basis of heterosis, several genetic hypotheses have been proposed. In recent years, high-throughput gene expression profiling techniques have been applied to investigate hybrid vigor. Consistent with the classical genetic dominance model, gene expression complementation has been demonstrated to be a general mechanism to contribute to phenotypic heterosis in diverse maize hybrids. Functional classification of these genes supported the notion that gene expression complementation can dynamically promote hybrid vigor under fluctuating environmental conditions. Hybrids tend to respond differently to available nutrients in the soil. It was hypothesized that hybrid vigor is promoted through a higher nutrient use efficiency which is linked to an improved root system performance of hybrids in comparison to their inbred parents. Recently, the interaction between soil microbes and their plant host was added as further dimension to disentangle heterosis in the belowground part of plants. Soil microbes influenced the performance of maize hybrids as illustrated in comparisons of sterile soil and soil inhabited by beneficial microorganisms.

Bacterium-enabled transient gene activation by artificial transcription factors for resolving gene regulation in maize.

Understanding gene regulatory networks is essential to elucidate developmental processes and environmental responses. Here, we studied regulation of a maize (Zea mays) transcription factor gene using designer transcription activator-like effectors (dTALes), which are synthetic Type III TALes of the bacterial genus Xanthomonas and serve as inducers of disease susceptibility gene transcription in host cells. The maize pathogen Xanthomonas vasicola pv. vasculorum was used to introduce 2 independent dTALes into maize cells to induced expression of the gene glossy3 (gl3), which encodes a MYB transcription factor involved in biosynthesis of cuticular wax. RNA-seq analysis of leaf samples identified, in addition to gl3, 146 genes altered in expression by the 2 dTALes. Nine of the 10 genes known to be involved in cuticular wax biosynthesis were upregulated by at least 1 of the 2 dTALes. A gene previously unknown to be associated with gl3, Zm00001d017418, which encodes aldehyde dehydrogenase, was also expressed in a dTALe-dependent manner. A chemically induced mutant and a CRISPR-Cas9 mutant of Zm00001d017418 both exhibited glossy leaf phenotypes, indicating that Zm00001d017418 is involved in biosynthesis of cuticular waxes. Bacterial protein delivery of dTALes proved to be a straightforward and practical approach for the analysis and discovery of pathway-specific genes in maize.

ENHANCED GRAVITROPISM 2 coordinates molecular adaptations to gravistimulation in the elongation zone of barley roots.

Root gravitropism includes gravity perception in the root cap, signal transduction between root cap and elongation zone, and curvature response in the elongation zone. The barley (Hordeum vulgare) mutant enhanced gravitropism 2 (egt2) displays a hypergravitropic root phenotype. We compared the transcriptomic reprogramming of the root cap, the meristem, and the elongation zone of wild-type (WT) and egt2 seminal roots upon gravistimulation in a time-course experiment and identified direct interaction partners of EGT2 by yeast-two-hybrid screening and bimolecular fluorescence complementation validation. We demonstrated that the elongation zone is subjected to most transcriptomic changes after gravistimulation. Here, 33% of graviregulated genes are also transcriptionally controlled by EGT2, suggesting a central role of this gene in controlling the molecular networks associated with gravitropic bending. Gene co-expression analyses suggested a role of EGT2 in cell wall and reactive oxygen species-related processes, in which direct interaction partners of EGT2 regulated by EGT2 and gravity might be involved. Taken together, this study demonstrated the central role of EGT2 and its interaction partners in the networks controlling root zone-specific transcriptomic reprogramming of barley roots upon gravistimulation. These findings can contribute to the development of novel root idiotypes leading to improved crop performance.

Maize lateral rootless 1 encodes a homolog of the DCAF protein subunit of the CUL4-based E3 ubiquitin ligase complex.

In maize (Zea mays L.), lateral roots are formed in the differentiation zone of all root types in a multi-step process. The maize mutant lateral rootless 1 (lrt1) is defective in lateral root formation in primary and seminal roots but not in shoot-borne roots. We cloned the lrt1 gene by mapping in combination with BSA-seq and subsequent validation via CRISPR/Cas9. The lrt1 gene encodes a 209 kDa homolog of the DDB1-CUL4-ASSOCIATED FACTOR (DCAF) subunit of the CUL4-based E3 ubiquitin ligase (CRL4) complex localized in the nucleus. DDB1-CUL4-ASSOCIATED FACTOR proteins are encoded by an evolutionary old gene family already present in nonseed plants. They are adaptors that bind substrate proteins and promote their ubiquitylation, thus typically marking them for subsequent degradation in the 26S proteasome. Gene expression studies demonstrated that lrt1 transcripts are expressed preferentially in the meristematic zone of all root types of maize. Downregulation of the rum1 gene in lrt1 mutants suggests that lrt1 acts upstream of the lateral root regulator rum1. Our results demonstrate that DCAF proteins play a key role in root-type-specific lateral root formation in maize. Together with its role in nitrogen acquisition in nitrogen-poor soil, lrt1 could be a promising target for maize improvement.

Root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism.

Root angle in crops represents a key trait for efficient capture of soil resources. Root angle is determined by competing gravitropic versus antigravitropic offset (AGO) mechanisms. Here we report a root angle regulatory gene termed ENHANCED GRAVITROPISM1 (EGT1) that encodes a putative AGO component, whose loss-of-function enhances root gravitropism. Mutations in barley and wheat EGT1 genes confer a striking root phenotype, where every root class adopts a steeper growth angle. EGT1 encodes an F-box and Tubby domain-containing protein that is highly conserved across plant species. Haplotype analysis found that natural allelic variation at the barley EGT1 locus impacts root angle. Gravitropic assays indicated that Hvegt1 roots bend more rapidly than wild-type. Transcript profiling revealed Hvegt1 roots deregulate reactive oxygen species (ROS) homeostasis and cell wall-loosening enzymes and cofactors. ROS imaging shows that Hvegt1 root basal meristem and elongation zone tissues have reduced levels. Atomic force microscopy measurements detected elongating Hvegt1 root cortical cell walls are significantly less stiff than wild-type. In situ analysis identified HvEGT1 is expressed in elongating cortical and stele tissues, which are distinct from known root gravitropic perception and response tissues in the columella and epidermis, respectively. We propose that EGT1 controls root angle by regulating cell wall stiffness in elongating root cortical tissue, counteracting the gravitropic machinery's known ability to bend the root via its outermost tissues. We conclude that root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism.

Cuticular transpiration is not affected by enhanced wax and cutin amounts in response to osmotic stress in barley.

The plant cuticle, which covers all aerial parts of plants in their primary developmental stage, is the major barrier against water loss from leaves. Accumulation of cutin and waxes has often been linked to drought tolerance. Here we investigated whether cutin and waxes play a role in the drought adaption of barley mimicked by osmotic stress acting on roots. We compared the cuticle properties of cultivated barley (Hordeum vulgare spp. vulgare) with wild barley (Hordeum vulgare spp. spontaneum), and tested whether wax and cutin composition or amount and cuticular transpiration could be future breeding targets for more drought-tolerant barley lines. In response to osmotic stress, accumulation of wax crystals was observed. This coincides with an increased wax and cutin gene expression and a total increase of wax and cutin amounts in leaves, which seems to be a general response triggered through root shoot signalling. Stomatal conductance decreased fast and significantly, whereas cuticular conductance remained unaffected in both wild and cultivated barley. The often-made conclusion that higher amounts of wax and cutin necessarily reduce cuticular transpiration and thus enhance drought tolerance is not always straightforward. To prevent water loss, stomatal regulation under water stress is much more important than regulation or adaptation of cuticular transpiration in response to drought.

Single-parent expression complementation contributes to phenotypic heterosis in maize hybrids.

The dominance model of heterosis explains the superior performance of F1-hybrids via the complementation of deleterious alleles by beneficial alleles in many genes. Genes active in one parent but inactive in the second lead to single-parent expression (SPE) complementation in maize (Zea mays L.) hybrids. In this study, SPE complementation resulted in approximately 700 additionally active genes in different tissues of genetically diverse maize hybrids on average. We established that the number of SPE genes is significantly associated with mid-parent heterosis (MPH) for all surveyed phenotypic traits. In addition, we highlighted that maternally (SPE_B) and paternally (SPE_X) active SPE genes enriched in gene co-expression modules are highly correlated within each SPE type but separated between these two SPE types. While SPE_B-enriched co-expression modules are positively correlated with phenotypic traits, SPE_X-enriched modules displayed a negative correlation. Gene ontology term enrichment analyses indicated that SPE_B patterns are associated with growth and development, whereas SPE_X patterns are enriched in defense and stress response. In summary, these results link the degree of phenotypic MPH to the prevalence of gene expression complementation observed by SPE, supporting the notion that hybrids benefit from SPE complementation via its role in coordinating maize development in fluctuating environments.

MuWU: Mutant-seq library analysis and annotation.

MOTIVATION: Insertional mutagenesis allows for the creation of loss-of-function mutations on a genome-wide scale. In theory, every gene can be 'knocked out' via the insertion of an additional DNA sequence. Resources of sequence-indexed mutants of plant and animal model organisms are instrumental for functional genomics studies. Such repositories significantly speed up the acquisition of interesting genotypes and allow for the validation of hypotheses regarding phenotypic consequences in reverse genetics. To create such resources, comprehensive sequencing of flanking sequence tags using protocols such as Mutant-seq requires various downstream computational tasks, and these need to be performed in an efficient and reproducible manner. RESULTS: Here, we present MuWU, an automated Mutant-seq workflow utility initially created for the identification of Mutator insertion sites of the BonnMu resource, representing a reverse genetics mutant collection for functional genetics in maize (Zea mays). MuWU functions as a fast, one-stop downstream processing pipeline of Mutant-seq reads. It takes care of all complex bioinformatic tasks, such as identifying tagged genes and differentiating between germinal and somatic mutations/insertions. Furthermore, MuWU automatically assigns insertions to the corresponding mutated seed stocks. We discuss the implementation and how parameters can easily be adapted to use MuWU for other species/transposable elements. AVAILABILITY AND IMPLEMENTATION: MuWU is a Snakemake-based workflow and freely available at https://github.com/tgstoecker/MuWU. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

ENHANCED GRAVITROPISM 2 encodes a STERILE ALPHA MOTIF-containing protein that controls root growth angle in barley and wheat.

The root growth angle defines how roots grow toward the gravity vector and is among the most important determinants of root system architecture. It controls water uptake capacity, nutrient use efficiency, stress resilience, and, as a consequence, yield of crop plants. We demonstrated that the egt2 (enhanced gravitropism 2) mutant of barley exhibits steeper root growth of seminal and lateral roots and an auxin-independent higher responsiveness to gravity compared to wild-type plants. We cloned the EGT2 gene by a combination of bulked-segregant analysis and whole genome sequencing. Subsequent validation experiments by an independent CRISPR/Cas9 mutant allele demonstrated that egt2 encodes a STERILE ALPHA MOTIF domain-containing protein. In situ hybridization experiments illustrated that EGT2 is expressed from the root cap to the elongation zone. We demonstrated the evolutionary conserved role of EGT2 in root growth angle control between barley and wheat by knocking out the EGT2 orthologs in the A and B genomes of tetraploid durum wheat. By combining laser capture microdissection with RNA sequencing, we observed that seven expansin genes were transcriptionally down-regulated in the elongation zone. This is consistent with a role of EGT2 in this region of the root where the effect of gravity sensing is executed by differential cell elongation. Our findings suggest that EGT2 is an evolutionary conserved regulator of root growth angle in barley and wheat that could be a valuable target for root-based crop improvement strategies in cereals.

Cold response and tolerance in cereal roots.

Cold stress adversely affects plant growth and is a limiting factor in crop productivity. Temperature volatility as a consequence of climate change will increase the effects of cold stress on crop cultivation. Low temperatures frequently occur early after planting in temperate climates and severely affect root development in cereals. In this review we address the question how cereal root systems respond to cold on different scales. First, we summarize the morphological, physiological and cellular responses of cereal roots to cold stress and how these processes are regulated by phytohormones. Subsequently, we highlight the status of the genetic and molecular dissection of cold tolerance with emphasis on the role of cold-responsive genes in improving cold tolerance in cereal roots. Finally, we discuss the role of beneficial microorganisms and mineral nutrients in ameliorating the effects of cold stress in cereal roots. A comprehensive knowledge of the molecular mechanisms underlying cold tolerance will ensure yield stability by enabling the generation of cold-tolerant crop genotypes.

Spatiotemporal Dynamics of Maize (Zea mays L.) Root Growth and Its Potential Consequences for the Assembly of the Rhizosphere Microbiota.

Numerous studies have shown that plants selectively recruit microbes from the soil to establish a complex, yet stable and quite predictable microbial community on their roots - their "microbiome." Microbiome assembly is considered as a key process in the self-organization of root systems. A fundamental question for understanding plant-microbe relationships is where a predictable microbiome is formed along the root axis and through which microbial dynamics the stable formation of a microbiome is challenged. Using maize as a model species for which numerous data on dynamic root traits are available, this mini-review aims to give an integrative overview on the dynamic nature of root growth and its consequences for microbiome assembly based on theoretical considerations from microbial community ecology.

Plant flavones enrich rhizosphere Oxalobacteraceae to improve maize performance under nitrogen deprivation.

Beneficial interactions between plant roots and rhizosphere microorganisms are pivotal for plant fitness. Nevertheless, the molecular mechanisms controlling the feedback between root architecture and microbial community structure remain elusive in maize. Here, we demonstrate that transcriptomic gradients along the longitudinal root axis associate with specific shifts in rhizosphere microbial diversity. Moreover, we have established that root-derived flavones predominantly promote the enrichment of bacteria of the taxa Oxalobacteraceae in the rhizosphere, which in turn promote maize growth and nitrogen acquisition. Genetic experiments demonstrate that LRT1-mediated lateral root development coordinates the interactions of the root system with flavone-dependent Oxalobacteraceae under nitrogen deprivation. In summary, these experiments reveal the genetic basis of the reciprocal interactions between root architecture and the composition and diversity of specific microbial taxa in the rhizosphere resulting in improved plant performance. These findings may open new avenues towards the breeding of high-yielding and nutrient-efficient crops by exploiting their interaction with beneficial soil microorganisms.

Assembly Patterns of the Rhizosphere Microbiome Along the Longitudinal Root Axis of Maize (Zea mays L.).

It is by now well proven that different plant species within their specific root systems select for distinct subsets of microbiota from bulk soil - their individual rhizosphere microbiomes. In maize, root growth advances several centimeters each day, with the locations, quality and quantity of rhizodeposition changing. We investigated the assembly of communities of prokaryotes (archaea and bacteria) and their protistan predators (Cercozoa, Rhizaria) along the longitudinal root axis of maize (Zea mays L.). We grew maize plants in an agricultural loamy soil and sampled rhizosphere soil at distinct locations along maize roots. We applied high-throughput sequencing, followed by diversity and network analyses in order to track changes in relative abundances, diversity and co-occurrence of rhizosphere microbiota along the root axis. Apart from a reduction of operational taxonomic unit (OTU) richness and a strong shift in community composition between bulk soil and root tips, patterns of microbial community assembly along maize-roots were more complex than expected. High variation in beta diversity at root tips and the root hair zone indicated substantial randomness of community assembly. Root hair zone communities were characterized by massive co-occurrence of microbial taxa, likely fueled by abundant resource supply from rhizodeposition. Further up the root where lateral roots emerged processes of community assembly appeared to be more deterministic (e.g., through competition and predation). This shift toward significance of deterministic processes was revealed by low variability of beta diversity, changes in network topology, and the appearance of regular phylogenetic co-occurrence patterns in bipartite networks between prokaryotes and their potential protistan predators. Such patterns were strongest in regions with fully developed laterals, suggesting that a consistent rhizosphere microbiome finally assembled. For the targeted improvement of microbiome function, such knowledge on the processes of microbiome assembly on roots and its temporal and spatial variability is crucially important.

BonnMu: A Sequence-Indexed Resource of Transposon-Induced Maize Mutations for Functional Genomics Studies.

Sequence-indexed insertional libraries in maize (Zea mays) are fundamental resources for functional genetics studies. Here, we constructed a Mutator (Mu) insertional library in the B73 inbred background designated BonnMu A total of 1,152 Mu-tagged F2-families were sequenced using the Mu-seq approach. We detected 225,936 genomic Mu insertion sites and 41,086 high quality germinal Mu insertions covering 16,392 of the annotated maize genes (37% of the B73v4 genome). On average, each F2-family of the BonnMu libraries captured 37 germinal Mu insertions in genes of the Filtered Gene Set (FGS). All BonnMu insertions and phenotypic seedling photographs of Mu-tagged F2-families can be accessed via MaizeGDB.org Downstream examination of 137,410 somatic and germinal insertion sites revealed that 50% of the tagged genes have a single hotspot, targeted by Mu By comparing our BonnMu (B73) data to the UniformMu (W22) library, we identified conserved insertion hotspots between different genetic backgrounds. Finally, the vast majority of BonnMu and UniformMu transposons was inserted near the transcription start site of genes. Remarkably, 75% of all BonnMu insertions were in closer proximity to the transcription start site (distance: 542 bp) than to the start codon (distance: 704 bp), which corresponds to open chromatin, especially in the 5' region of genes. Our European sequence-indexed library of Mu insertions provides an important resource for functional genetics studies of maize.