Suberin deficiency and its effect on the transport physiology of young poplar roots.

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.

Isolation and characterization of the gene HvFAR1 encoding acyl-CoA reductase from the cer-za.227 mutant of barley (Hordeum vulgare) and analysis of the cuticular barrier functions.

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 ß-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.

Transcriptome profiling at the transition to the reproductive stage uncovers stage and tissue-specific genes in wheat.

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.

Populus × canescens root suberization in reaction to osmotic and salt stress is limited to the developing younger root tip region.

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.

A2TEA: Identifying trait-specific evolutionary adaptations.

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.

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.

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.