We identified the monogenic recessive maize (Zea mays L.) mutant dizzy1 in a segregating F2-family by a forward genetic screen of the BonnMu population, a sequence-indexed collection of Mutator transposon-induced mutants. dizzy1 exhibits a dwarf phenotype with pronounced twisting of leaves and roots. Histological analyses revealed irregular cell organization in dizzy1, including enlarged upper epidermal cells in leaves and disorganized cortical cell architecture in roots. Physiological analysis of primary roots indicated reduced cell viability, reflected by increased membrane permeability and altered metabolic activity. Hormone response assays further showed that dizzy1 is insensitive to brassinolide, exhibits a delayed auxin-promoted shoot response, and displays altered gibberellin effects on lateral root development. Bulked segregant RNA sequencing mapped the dizzy1 locus to chromosome 2. Comparative transcriptome profiling of primary roots identified 4,378 differentially expressed genes between wild type and dizzy1, revealing widespread transcriptional reprogramming. Consistent with functional enrichment analyses, histochemical and spectrophotometric assays indicated elevated reactive oxygen species and increased lignin in diz1 primary roots. These findings define dizzy1 as a pleiotropic developmental mutant linking hormone signaling, redox homeostasis, and cell wall regulation in maize growth.

Background:The Critical Assessment of Functional Annotation (CAFA) is a community effort held to understand the field of computational protein function prediction. Every three years, since 2010, the organizers initiate an experiment to collect function predictions on a large set of proteins and then evaluate the performance of predicting methods on a subset of proteins that have accumulated experimental annotations between the submission deadline and the evaluation time. CAFA provides an independent and rigorous assessment of the current state of the art, thus leveling the playing field, highlighting successes, revealing bottlenecks, and offering a forum for the exchange of ideas in protein science. Here, we report the results of the fourth CAFA experiment (CAFA4). Results:CAFA4 featured the participation of 148 methods from 70 research groups on a total of 46,205 unique proteins over a 5-year annotation accumulation phase, the longest in any CAFA. In a comparison across CAFA2-CAFA4 methods, the prediction of Gene Ontology (GO) terms has clearly improved across all three GO aspects and traditional evaluation settings. While not achieving the first rank, several CAFA2 and CAFA3 methods featured in the top ten methods in many evaluations, suggesting that earlier methods still hold relevance. The performance is weaker in the newly introduced "partial knowledge" evaluation category (proteins with experimental annotations before submission deadline that gained additional annotations in the same GO aspect during the annotation accumulation phase), highlighting the need for a new class of methods. The rankings of the methods were stable over the years in traditional evaluation settings, but less so in the new partial knowledge evaluation. Overall, the field continues to progress with some influx of new participants. Sustained efforts will be necessary to substantially advance it.

Abstract Biological sequences are known to be not random. Thus, the comparison of in silico restriction fragment distributions of random and biological sequences may be an indicator of this non-randomness. Our analyses show that for most of the tested combinations of restriction enzyme and genome sequence the fragments per Megabase of the biological sequence deviate at least more then 10% from the corresponding random sequence. This deviation goes into both directions, i.e. clearly increased values are as common as clearly decreased values. Although there is no species- or restriction-enzyme-specific effect, a clear impact of the GC content both of the restriction site and of the genome sequence can be seen. In contrast to the random sequences, the genome sequences show distinct peaks in their fragment length distributions, hinting to repetitive elements such as transposons.

TheBonnMuresource represents a tagged collection of maize (Zea maysL.)Mutator(Mu) transposon-induced mutants, designed for functional genomics studies. Here, we describe the use of theBonnMucollection for identifying and characterizing mutations. Specifically, we describe workflows for use in both reverse and forward genetics strategies in maize. For reverse genetics, users first acquire aBonnMuF2stock of interest based on data accessible at the Maize Genetics and Genomics Database (MaizeGDB). We provide details here for their subsequent propagation and for the confirmation ofMuinsertions by genotyping via PCR, with the ultimate goal of establishing genotype–phenotype relationships of interest. For forward genetics studies, we describe a workflow that involves a combined approach of Mutant-Seq (Mu-Seq) and bulked segregant RNA-seq (BSR-Seq), to identify the causal gene underlying a mutant phenotype of interest.

Mutator(Mu) transposons facilitate untargeted insertional mutagenesis in maize by moving within the genome and disrupting genes. Such an approach has been used to generate collections such as theBonnMuresource, aMu-tagged maize population for functional genomics studies. Mutant-Seq (Mu-Seq) is a sequencing-based method for the high-throughput identification and mapping ofMuinsertion sites. The approach involves the construction of multiplexed sequencing libraries (known as Mu-Seq libraries) fromMu-tagged populations, followed by high-throughput sequencing and data processing using the Mu-Seq Workflow Utility (MuWU) tool, to determine the location ofMuinsertions. Here, we provide a detailed protocol for Mu-Seq, from the generation of the maizeMu-tagged mutant population to data analysis. Researchers can use this approach to develop mutant collections customized to specific genetic backgrounds of interest, which can aid in characterizing genotype-specific mutations and identifying candidate genes linked to visible mutant phenotypes.

BACKGROUND:In order to prioritize candidate genes for drought adaptation, we analyze genomic data of the more drought-resistant Brassicaceae species Eutrema salsugineum and Arabidopsis lyrata compared to more drought-sensitive species Arabidopsis thaliana and Brassica napus. We combine gene family expansion, which is an important driver of evolution in plants, unique to the drought-resistant species with differential expression under drought (DE). RESULTS:We show that combining trait-specific gene family expansion with differential expression identifies a concise set of candidate genes. To demonstrate that these are relevant for drought adaptation in tolerant species, we show enrichment of DE conserved between both tolerant species, DE unique to the tolerant species, and up-regulation. We show that specific functions are enriched, and that the set contains genes with functions such as root development in line with drought adaptation based on evidence from other species, while the background of all differentially expressed genes (DEGs) contains many general stress response genes. Whereas DEGs in general are rarely under diversifying selection, signatures of diversifying selection are slightly enriched in the candidate gene families, highly significantly enriched in DEGs in tolerant species-specific expanded gene families, and, in contrast, not enriched in DEGs in sensitive species-specific expanded gene families. CONCLUSIONS:Our approach identifies a concise and functionally relevant set of candidate genes for drought adaptation with promising targets for functional studies and crop improvement for drought tolerance. We propose that our method can also be used to prioritize candidate genes for adaptation to other environmental factors.

Inositol pyrophosphates (PP-InsPs) are important signaling molecules that regulate diverse cellular processes in eukaryotes, including energy homeostasis, phosphate (Pi) signaling, and phytohormone perception. Yet, in plants, the enzymes responsible for their turnover remain largely unknown. Using a non-hydrolysable PP-InsP analog in a pull-down approach, we identified a family of Arabidopsis NUDIX-type hydrolases (NUDTs) that group into two closely related subclades. Through in vitro assays, heterologous expression systems, and higher order gene-edited mutants, we explored the substrate specificities and physiological roles of these hydrolases. Using a combination of strong anion exchange high-performance liquid chromatography (SAX-HPLC), polyacrylamide gel electrophoresis (PAGE), and capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that their PP-InsP pyrophosphatase activity is enantiomer selective and Mg2+ dependent. Specifically, Subclade I NUDTs preferentially hydrolyze 4-InsP7, while Subclade II NUDTs target 3-InsP7, with minor activity against other PP-InsPs, including 5-InsP7. In higher order mutants of Subclade II NUDTs, we observed defects in both Pi and iron homeostasis, accompanied by increased levels of 1/3-InsP7 and 5-InsP7, with a markedly larger increase in 1/3-InsP7. Ectopic expression of NUDTs from both subclades induced local Pi starvation responses (PSRs), while RNA-seq analysis comparing wild-type (WT) and Subclade II nudt12/13/16 loss-of-function plants indicates additional PSR-independent roles, potentially involving 1/3-InsP7 in the regulation of plant defense. Consistently, nudt12/13/16 mutants displayed enhanced resistance to Pseudomonas syringae infection, indicating a role in bacterial pathogen susceptibility. Expanding beyond Subclade II NUDTs, we demonstrated susceptibility of the 3PP-position of PP-InsPs to enzymatic activities unrelated to NUDTs, and found that such activities are conserved across plants and humans. Additionally, we observed that NUDT effectors from pathogenic ascomycete fungi exhibit a substrate specificity similar to Subclade I NUDTs. Collectively, our findings reveal new roles for NUDTs in PP-InsP signaling, plant nutrient and immune responses, and highlight a cross-kingdom conservation of PP-InsP-metabolizing enzymes.

Classical genetic concepts to explain heterosis attribute the superiority of F1-hybrids over their homozygous parents to the complementation of unfavorable by beneficial alleles (dominance) or to heterozygote advantage (overdominance). Here we analyze 112 intermated B73xMo17 recombinant inbred lines of maize and their backcrosses to their original parents B73 and Mo17 to obtain hybrids with an average heterozygosity of 50

The precise functions of suberized apoplastic barriers in root water and nutrient transport physiology have not fully been elucidated. While lots of research has been performed with mutants of Arabidopsis, little to no data are available for mutants of agricultural crop or tree species. By employing a combined set of physiological, histochemical, analytical, and transport physiological methods as well as RNA-sequencing, this study investigated the implications of remarkable CRISPR/Cas9-induced suberization defects in young roots of the economically important gray poplar. While barely affecting overall plant development, contrary to literature-based expectations significant root suberin reductions of up to 80-95% in four independent mutants were shown to not evidently affect the root hydraulic conductivity during non-stress conditions. In addition, subliminal iron deficiency symptoms and increased translocation of a photosynthesis inhibitor as well as NaCl highlight the involvement of suberin in nutrient transport physiology. The multifaceted nature of the root hydraulic conductivity does not allow drawing simplified conclusions such as that the suberin amount must always be correlated with the water transport properties of roots. However, the decreased masking of plasma membrane surface area could facilitate the uptake but also leakage of beneficial and harmful solutes.

The BonnMu resource is a transposon tagged mutant collection designed for functional genomics studies in maize. To expand this resource, we crossed an active Mutator (Mu) stock with dent (B73, Co125) and flint (DK105, EP1, and F7) germplasm, resulting in the generation of 8064 mutagenized BonnMu F2-families. Sequencing of these Mu-tagged families revealed 425 924 presumptive heritable Mu insertions affecting 36 612 (83%) of the 44 303 high-confidence gene models of maize (B73v5). On average, we observed 12 Mu insertions per gene (425 924 total insertions/36 612 affected genes) and 53 insertions per BonnMu F2-family (425 924 total insertions/8064 families). Mu insertions and photos of seedling phenotypes from segregating BonnMu F2-families can be accessed through the Maize Genetics and Genomics Database (MaizeGDB). Downstream examination via the automated Mutant-seq Workflow Utility (MuWU) identified 94% of the presumptive germinal insertion sites in genic regions and only a small fraction of 6% inserting in non-coding intergenic sequences of the genome. Consistently, Mu insertions aligned with gene-dense chromosomal arms. In total, 42% of all BonnMu insertions were located in the 5' untranslated region of genes, corresponding to accessible chromatin. Furthermore, for 38% of the insertions (163 843 of 425 924 total insertions) Mu1, Mu8 and MuDR were confirmed to be the causal Mu elements. Our publicly accessible European BonnMu resource has archived insertions covering two major germplasm groups, thus facilitating both forward and reverse genetics studies.

The dominance model of heterosis explains the superiority of F1-hybrids via the complementation of unfavorable by beneficial alleles in many genes. Consistent with this model, genes active in only one parent and the hybrid display single-parent expression (SPE) complementation. Here we demonstrated, that SPE can explain up to 29% of heterotic variance in maize (Zea mays L.). Moreover, expression quantitative trait loci (eQTL) revealed, that the genotype of the active parent determines the regulation mode of SPE patterns in cis or trans, which might explain how phylogenetic distance translates to transcriptomic diversity and heterosis in hybrids. Furthermore, we showed that most eQTL are located in heterozygous regions of the genome and that a trans eQTL controls the activity of a SPE gene which regulates lateral root density in hybrids. We anticipate that our study will stimulate further research elucidating the regulation and molecular mechanisms underlying heterosis. ### Competing Interest Statement The authors have declared no competing interest.

Cold stress during early development limits maize (Zea mays L.) production in temperate zones. Low temperatures restrict root growth and reprogram gene expression. Here, we provide a systematic transcriptomic landscape of maize primary roots, their tissues, and cell types in response to cold stress. The epidermis exhibited a unique transcriptomic cold response, and genes involved in root hair formation were dynamically regulated in this cell type by cold. Consequently, activation of genes involved in root hair tip growth contributed to root hair recovery under moderate cold conditions. The maize root hair defective mutants roothair defective 5 (rth5) and roothair defective 6 (rth6) displayed enhanced cold tolerance with respect to primary root elongation. Furthermore, DEHYDRATION RESPONSE ELEMENT-BINDING PROTEIN 2.1 (DREB2.1) was the only member of the dreb subfamily of AP2/EREB transcription factor genes upregulated in primary root tissues and cell types but exclusively downregulated in root hairs upon cold stress. Plants overexpressing dreb2.1 significantly suppressed root hair elongation after moderate cold stress. Finally, the expression of rth3 was regulated by dreb2.1 under cold conditions, while rth6 transcription was regulated by DREB2.1 irrespective of the temperature regime. We demonstrated that dreb2.1 negatively regulates root hair plasticity at low temperatures by coordinating the expression of root hair defective genes in maize.

Background The transition from vegetative to floral phase is the result of complex crosstalk of exogenous and endogenous floral integrators. This critical physiological event is the response to environmental interaction, which causes biochemical cascades of reactions at different internal tissues, organs, and releases signals that make the plant moves from vegetative status to a reproductive phase. This network controlling flowering time is not deciphered largely in bread wheat. In this study, a comparative transcriptome analysis at a transition time in combination with genetic mapping was used to identify responsible genes in a stage and tissue-specific manner. For this reason, two winter cultivars that have been bred in Germany showing contrasting and stable heading time in different environments were selected for the analysis.Results In total, 670 and 1075 differentially expressed genes in the shoot apical meristem and leaf tissue, respectively, could be identified in 23 QTL intervals for the heading date. In the transition apex, Histone methylation H3-K36 and regulation of circadian rhythm are both controlled by the same homoeolog genes mapped in QTL TaHd112, TaHd124, and TaHd137. TaAGL14 gene that identifies the floral meristem was mapped in TaHd054 in the double ridge. In the same stage, the homoeolog located on chromosome 7D of FLOWERING TIME LOCUS T mapped on chr 7B, which evolved an antagonist function and acts as a flowering repressor was uncovered. The wheat orthologue of transcription factor ASYMMETRIC LEAVES 1 (AS1) was identified in the late reproductive stage and was mapped in TaHd102, which is strongly associated with heading date. Deletion of eight nucleotides in the AS1 promoter could be identified in the binding site of the SUPPRESSOR OF CONSTANS OVEREXPRESSION 1 (SOC1) gene in the late flowering cultivar. Both proteins AS1 and SOC1 are inducing flowering time in response to gibberellin biosynthesis.Conclusion The global transcriptomic at the transition phase uncovered stage and tissue-specific genes mapped in QTL of heading date in winter wheat. In response to Gibberellin signaling, wheat orthologous transcription factor AS1 is expressed in the late reproductive phase of the floral transition. The locus harboring this gene is the strongest QTL associated with the heading date trait in the German cultivars. Consequently, we conclude that this is another indication of the Gibberellin biosynthesis as the mechanism behind the heading variation in wheat.

The cuticle is a protective layer covering aerial plant organs. We studied the function of waxes for the establishment of the cuticular barrier in barley (Hordeum vulgare). The barley eceriferum mutants cer-za.227 and cer-ye.267 display reduced wax loads, but the genes affected, and the consequences of the wax changes for the barrier function remained unknown. Cuticular waxes and permeabilities were measured in cer-za.227 and cer-ye.267. The mutant loci were isolated by bulked segregant RNA sequencing. New cer-za alleles were generated by genome editing. The CER-ZA protein was characterized after expression in yeast and Arabidopsis cer4-3. Cer-za.227 carries a mutation in HORVU5Hr1G089230 encoding acyl-CoA reductase (FAR1). The cer-ye.267 mutation is located to HORVU4Hr1G063420 encoding β-ketoacyl-CoA synthase (KAS1) and is allelic to cer-zh.54. The amounts of intracuticular waxes were strongly decreased in cer-ye.267. The cuticular water loss and permeability of cer-za.227 were similar to wild-type (WT), but were increased in cer-ye.267. Removal of epicuticular waxes revealed that intracuticular, but not epicuticular waxes are required to regulate cuticular transpiration. The differential decrease in intracuticular waxes between cer-za.227 and cer-ye.267, and the removal of epicuticular waxes indicate that the cuticular barrier function mostly depends on the presence of intracuticular waxes.

Background: Plants differ in their ability to cope with external stresses (e.g., drought tolerance). Genome duplications are an important mechanism to enable plant adaptation. This leads to characteristic footprints in the genome, such as protein family expansion. We explore genetic diversity and uncover evolutionary adaptation to stresses by exploiting genome comparisons between stress tolerant and sensitive species and RNA-Seq data sets from stress experiments. Expanded gene families that are stress-responsive based on differential expression analysis could hint at species or clade-specific adaptation, making these gene families exciting candidates for follow-up tolerance studies and crop improvement. Software: Integration of such cross-species omics data is a challenging task, requiring various steps of transformation and filtering. Ultimately, visualization is crucial for quality control and interpretation. To address this, we developed A2TEA: Automated Assessment of Trait-specific Evolutionary Adaptations, a Snakemake workflow for detecting adaptation footprints in silico. It functions as a one-stop processing pipeline, integrating protein family, phylogeny, expression, and protein function analyses. The pipeline is accompanied by an R Shiny web application that allows exploring, highlighting, and exporting the results interactively. This allows the user to formulate hypotheses regarding the genomic adaptations of one or a subset of the investigated species to a given stress. Conclusions: While our research focus is on crops, the pipeline is entirely independent of the underlying species and can be used with any set of species. We demonstrate pipeline efficiency on real-world datasets and discuss the implementation and limits of our analysis workflow as well as planned extensions to its current state. The A2TEA workflow and web application are publicly available at: https://github.com/tgstoecker/A2TEA.Workflow and https://github.com/tgstoecker/A2TEA.WebApp, respectively.

The average sowing date of crops in temperate climate zones has been shifted forwards by several days, resulting in a changed photoperiod regime at the emergence stage. In the present study, we performed a global transcriptome profiling of plant development genes in the seedling stage of root and shoot apical meristems of a photoperiod-sensitive species (barley) and a photoperiod insensitive species (tomato) in short-day conditions (8h). Variant expression indicated differences in physiological development under this short day-length regime between species and tissues. The barley tissue transcriptome revealed reduced differentiation compared to tomato. In addition, decreased photosynthetic activity was observed in barley transcriptome and leaf chlorophyll content under 8h conditions, indicating a slower physiological development of shoot meristems than in tomatoes. The photomorphogenesis controlling cryptochrome gene cry1, with an effect on physiological differentiation, showed an underexpression in barley compared to tomato shoot meristems. This might lead to a cascade of suspended sink-source activities, which ultimately delay organ development and differentiation in barley shoot meristems under short photoperiods.

Abstract 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.

Populus is a valuable and fast-growing tree species commonly cultivated for economic and scientific purposes. But most of the poplar species are sensitive to drought and salt stress. Thus, we compared the physiological effects of osmotic stress (PEG8000) and salt treatment (NaCl) on poplar roots to identify potential strategies for future breeding or genetic engineering approaches. We investigated root anatomy using epifluorescence microscopy, changes in root suberin composition and amount using gas chromatography, transcriptional reprogramming using RNA sequencing, and modifications of root transport physiology using a pressure chamber. Poplar roots reacted to the imposed stress conditions, especially in the developing younger root tip region, with remarkable differences between both types of stress. Overall, the increase in suberin content was surprisingly small, but the expression of key suberin biosynthesis genes was strongly induced. Significant reductions of the radial water transport in roots were only observed for the osmotic and not the hydrostatic hydraulic conductivity. Our data indicate that the genetic enhancement of root suberization processes in poplar might be a promising target to convey increased tolerance, especially against toxic sodium chloride.

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.

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.