Phenotypic characterization and candidate gene analysis of a short kernel and brassinosteroid insensitive mutant from hexaploid oat (Avena sativa).

In an ethyl methanesulfonate oat (Avena sativa) mutant population we have found a mutant with striking differences to the wild-type (WT) cv. Belinda. We phenotyped the mutant and compared it to the WT. The mutant was crossed to the WT and mapping-by-sequencing was performed on a pool of F2 individuals sharing the mutant phenotype, and variants were called. The impacts of the variants on genes present in the reference genome annotation were estimated. The mutant allele frequency distribution was combined with expression data to identify which among the affected genes was likely to cause the observed phenotype. A brassinosteroid sensitivity assay was performed to validate one of the identified candidates. A literature search was performed to identify homologs of genes known to be involved in seed shape from other species. The mutant had short kernels, compact spikelets, altered plant architecture, and was found to be insensitive to brassinosteroids when compared to the WT. The segregation of WT and mutant phenotypes in the F2 population was indicative of a recessive mutation of a single locus. The causal mutation was found to be one of 123 single-nucleotide polymorphisms (SNPs) spanning the entire chromosome 3A, with further filtering narrowing this down to six candidate genes. In-depth analysis of these candidate genes and the brassinosteroid sensitivity assay suggest that a Pro303Leu substitution in AVESA.00010b.r2.3AG0419820.1 could be the causal mutation of the short kernel mutant phenotype. We identified 298 oat proteins belonging to orthogroups of previously published seed shape genes, with AVESA.00010b.r2.3AG0419820.1 being the only of these affected by a SNP in the mutant. The AVESA.00010b.r2.3AG0419820.1 candidate is functionally annotated as a GSK3/SHAGGY-like kinase with homologs in Arabidopsis, wheat, barley, rice, and maize, with several of these proteins having known mutants giving rise to brassinosteroid insensitivity and shorter seeds. The substitution in AVESA.00010b.r2.3AG0419820.1 affects a residue with a known gain-of function substitution in Arabidopsis BRASSINOSTEROID-INSENSITIVE2. We propose a gain-of-function mutation in AVESA.00010b.r2.3AG0419820.1 as the most likely cause of the observed phenotype, and name the gene AsGSK2.1. The findings presented here provide potential targets for oat breeders, and a step on the way towards understanding brassinosteroid signaling, seed shape and nutrition in oats.

The dynamics of touch-responsive gene expression in cereals.

Wind, rain, herbivores, obstacles, neighbouring plants, etc. provide important mechanical cues to steer plant growth and survival. Mechanostimulation to stimulate yield and stress resistance of crops is of significant research interest, yet a molecular understanding of transcriptional responses to touch is largely absent in cereals. To address this, we performed whole-genome transcriptomics following mechanostimulation of wheat, barley, and the recent genome-sequenced oat. The largest transcriptome changes occurred ±25 min after touching, with most of the genes being upregulated. While most genes returned to basal expression level by 1-2 h in oat, many genes retained high expression even 4 h post-treatment in barley and wheat. Functional categories such as transcription factors, kinases, phytohormones, and Ca2+ regulation were affected. In addition, cell wall-related genes involved in (hemi)cellulose, lignin, suberin, and callose biosynthesis were touch-responsive, providing molecular insight into mechanically induced changes in cell wall composition. Furthermore, several cereal-specific transcriptomic footprints were identified that were not observed in Arabidopsis. In oat and barley, we found evidence for systemic spreading of touch-induced signalling. Finally, we provide evidence that both the jasmonic acid-dependent and the jasmonic acid-independent pathways underlie touch-signalling in cereals, providing a detailed framework and marker genes for further study of (a)biotic stress responses in cereals.

Genome analysis in Avena sativa reveals hidden breeding barriers and opportunities for oat improvement.

Oat (Avena sativa L.) is an important and nutritious cereal crop, and there is a growing need to identify genes that contribute to improved oat varieties. Here we utilize a newly sequenced and annotated oat reference genome to locate and characterize quantitative trait loci (QTLs) affecting agronomic and grain-quality traits in five oat populations. We find strong and significant associations between the positions of candidate genes and QTL that affect heading date, as well as those that influence the concentrations of oil and ß-glucan in the grain. We examine genome-wide recombination profiles to confirm the presence of a large, unbalanced translocation from chromosome 1 C to 1 A, and a possible inversion on chromosome 7D. Such chromosome rearrangements appear to be common in oat, where they cause pseudo-linkage and recombination suppression, affecting the segregation, localization, and deployment of QTLs in breeding programs.

The mosaic oat genome gives insights into a uniquely healthy cereal crop.

Cultivated oat (Avena sativa L.) is an allohexaploid (AACCDD, 2n = 6x = 42) thought to have been domesticated more than 3,000 years ago while growing as a weed in wheat, emmer and barley fields in Anatolia1,2. Oat has a low carbon footprint, substantial health benefits and the potential to replace animal-based food products. However, the lack of a fully annotated reference genome has hampered efforts to deconvolute its complex evolutionary history and functional gene dynamics. Here we present a high-quality reference genome of A. sativa and close relatives of its diploid (Avena longiglumis, AA, 2n = 14) and tetraploid (Avena insularis, CCDD, 2n = 4x = 28) progenitors. We reveal the mosaic structure of the oat genome, trace large-scale genomic reorganizations in the polyploidization history of oat and illustrate a breeding barrier associated with the genome architecture of oat. We showcase detailed analyses of gene families implicated in human health and nutrition, which adds to the evidence supporting oat safety in gluten-free diets, and we perform mapping-by-sequencing of an agronomic trait related to water-use efficiency. This resource for the Avena genus will help to leverage knowledge from other cereal genomes, improve understanding of basic oat biology and accelerate genomics-assisted breeding and reanalysis of quantitative trait studies.

Characterisation of Grains and Flour Fractions from Field Grown Transgenic Oil-Accumulating Wheat Expressing Oat WRI1.

Wheat (Triticum aestivum L.) is one of the major staple crops in the world and is used to prepare a range of foods. The development of new varieties with wider variation in grain composition could broaden their use. We characterized grains and flours from oil-accumulating transgenic wheat expressing the oat (Avena sativa L.) endosperm WRINKLED1 (AsWRI1) grown under field conditions. Lipid and starch accumulation was determined in developing caryopses of AsWRI1-wheat and X-ray microtomography was used to study grain morphology. The developing caryopses of AsWRI1-wheat grains had increased triacylglycerol content and decreased starch content compared to the control. Mature AsWRI1-wheat grains also had reduced weight, were wrinkled and had a shrunken endosperm and X-ray tomography revealed that the proportion of endosperm was decreased while that of the aleurone was increased. Grains were milled to produce two white flours and one bran fraction. Mineral and lipid analyses showed that the flour fractions from the AsWRI1-wheat were contaminated with bran, due to the effects of the changed morphology on milling. This study gives a detailed analysis of grains from field grown transgenic wheat that expresses a gene that plays a central regulatory role in carbon allocation and significantly affects grain composition.

Analysis of barley mutants ert-c.1 and ert-d.7 reveals two loci with additive effect on plant architecture.

MAIN CONCLUSION: Both mutant ert-c.1 and ert-d.7 carry T2-T3 translocations in the Ert-c gene. Principal coordinate analyses revealed the translocation types and translocation breakpoints. Mutant ert-d.7 is an Ert-c Ert-d double mutant. Mutations in the Ert-c and Ert-d loci are among the most common barley mutations affecting plant architecture. The mutants have various degrees of erect and compact spikes, often accompanied with short and stiff culms. In the current study, complementation tests, linkage mapping, principal coordinate analyses and fine mapping were conducted. We conclude that the original ert-d.7 mutant does not only carry an ert-d mutation but also an ert-c mutation. Combined, mutations in Ert-c and Ert-d cause a pyramid-dense spike phenotype, whereas mutations in only Ert-c or Ert-d give a pyramid and dense phenotype, respectively. Associations between the Ert-c gene and T2-T3 translocations were detected in both mutant ert-c.1 and ert-d.7. Different genetic association patterns indicate different translocation breakpoints in these two mutants. Principal coordinate analysis based on genetic distance and screening of recombinants from all four ends of polymorphic regions was an efficient way to narrow down the region of interest in translocation-involved populations. The Ert-c gene was mapped to the marker interval of 2_0801to1_0224 on 3HL near the centromere. The results illuminate a complex connection between two single genes having additive effects on barley spike architecture and will facilitate the identification of the Ert-c and Ert-d genes.

Development and characterization of an EMS-mutagenized wheat population and identification of salt-tolerant wheat lines.

BACKGROUND: Triticum aestivum (wheat) is one of the world's oldest crops and has been used for >8000 years as a food crop in North Africa, West Asia and Europe. Today, wheat is one of the most important sources of grain for humans, and is cultivated on greater areas of land than any other crop. As the human population increases and soil salinity becomes more prevalent, there is increased pressure on wheat breeders to develop salt-tolerant varieties in order to meet growing demands for yield and grain quality. Here we developed a mutant wheat population using the moderately salt-tolerant Bangladeshi variety BARI Gom-25, with the primary goal of further increasing salt tolerance. RESULTS: After titrating the optimal ethyl methanesulfonate (EMS) concentration, ca 30,000 seeds were treated with 1% EMS, and 1676 lines, all originating from single seeds, survived through the first four generations. Most mutagenized lines showed a similar phenotype to BARI Gom-25, although visual differences such as dwarfing, giant plants, early and late flowering and altered leaf morphology were seen in some lines. By developing an assay for salt tolerance, and by screening the mutagenized population, we identified 70 lines exhibiting increased salt tolerance. The selected lines typically showed a 70% germination rate on filter paper soaked in 200 mM NaCl, compared to 0-30% for BARI Gom-25. From two of the salt-tolerant OlsAro lines (OA42 and OA70), genomic DNA was sequenced to 15x times coverage. A comparative analysis against the BARI Gom-25 genomic sequence identified a total of 683,201 (OA42), and 768,954 (OA70) SNPs distributed throughout the three sub-genomes (A, B and D). The mutation frequency was determined to be approximately one per 20,000 bp. All the 70 selected salt-tolerant lines were tested for root growth in the laboratory, and under saline field conditions in Bangladesh. The results showed that all the lines selected for tolerance showed a better salt tolerance phenotype than both BARI Gom-25 and other local wheat varieties tested. CONCLUSION: The mutant wheat population developed here will be a valuable resource in the development of novel salt-tolerant varieties for the benefit of saline farming.

Exophiala macquariensis sp. nov., a cold adapted black yeast species recovered from a hydrocarbon contaminated sub-Antarctic soil.

A new black yeast species, Exophiala macquariensis is described that is a member of the ascomycete family Herpotrichiellaceae, order Chaetothyriales. The genus Exophiala is comprised of opportunistic pathogens isolated from clinical specimens as well as species recovered from hydrocarbon contaminated environments. Several species have been reported to be able to degrade benzene, toluene, ethylbenzene and xylenes. Here, a novel species of Exophiala (CZ06) previously isolated from a Sub-Antarctic, Macquarie Island soil that was spiked with Special Antarctic Blend diesel fuel (SAB) is described. This isolate has the capacity of toluene biodegradation at cold temperatures. Multilocus sequence typing showed that this fungus was closely related to the pathogenic species Exophiala salmonis and Exophiala equina. With the capacity to utilise hydrocarbons as a sole carbon source at 10 °C, this fungus has great potential for future bioremediation applications.

PCR-mediated deletion of plasmid DNA.

The PCR-mediated plasmid DNA deletion method is a simple approach to delete DNA sequences from plasmids using only one round of PCR, with two primers, and without ligation or purification prior to in vivo recombination. By using only PCR, the method is sequence independent and, as shown in this study, is applicable to various sizes of plasmids and deletions using minimal primer design.

Rhodobacter capsulatus magnesium chelatase subunit BchH contains an oxygen sensitive iron-sulfur cluster.

Magnesium chelatase is the first unique enzyme of the bacteriochlorophyll biosynthetic pathway. It consists of three subunits (BchI, BchD, and BchH). Amino acid sequence analysis of the Rhodobacter capsulatus BchH revealed a novel cysteine motif (393CX2CX3CX14C) that was found in only six other proteobacteria (CX2CX3CX11-14C). The cysteine motif is likely to coordinate an unprecedented [Fe-S] cluster. Purified BchH demonstrated absorbance in the 460 nm region. This absorbance was abolished in BchH proteins with alanine substitutions at positions Cys396 and Cys414. These modified proteins were also EPR silent. In contrast, wild type BchH protein in the reduced state showed EPR signals resembling those of a [4Fe-4S] cluster with rhombic symmetry and g values at 1.90, 1.93, and 2.09, superimposed with a [3Fe-4S] cluster centered at g = 2.02. The [3Fe-4S] signal was observed independently of the [4Fe-4S] signal under oxidizing conditions. Mg-chelatase activity assays showed that the cluster is not catalytic. We suggest that the [4Fe-4S] and [3Fe-4S] signals originate from a single coordination site on the monomeric BchH protein and that the [4Fe-4S] cluster is sensitive to oxidation. It is speculated that the cluster participates in the switching between aerobic and anaerobic life of the proteobacteria.

ATPase activity associated with the magnesium chelatase H-subunit of the chlorophyll biosynthetic pathway is an artefact.

Magnesium chelatase inserts Mg2+ into protoporphyrin IX and is the first unique enzyme of the chlorophyll biosynthetic pathway. It is a heterotrimeric enzyme, composed of I- (40 kDa), D- (70 kDa) and H- (140 kDa) subunits. The I- and D-proteins belong to the family of AAA+ (ATPases associated with various cellular activities), but only I-subunit hydrolyses ATP to ADP. The D-subunits provide a platform for the assembly of the I-subunits, which results in a two-tiered hexameric ring complex. However, the D-subunits are unstable in the chloroplast unless ATPase active I-subunits are present. The H-subunit binds protoporphyrin and is suggested to be the catalytic subunit. Previous studies have indicated that the H-subunit also has ATPase activity, which is in accordance with an earlier suggested two-stage mechanism of the reaction. In the present study, we demonstrate that gel filtration chromatography of affinity-purified Rhodobacter capsulatus H-subunit produced in Escherichia coli generates a high- and a low-molecular-mass fraction. Both fractions were dominated by the H-subunit, but the ATPase activity was only found in the high-molecular-mass fraction and magnesium chelatase activity was only associated with the low-molecular-mass fraction. We demonstrated that light converted monomeric low-molecular-mass H-subunit into high-molecular-mass aggregates. We conclude that ATP utilization by magnesium chelatase is solely connected to the I-subunit and suggest that a contaminating E. coli protein, which binds to aggregates of the H-subunit, caused the previously reported ATPase activity of the H-subunit.

Characterization of eight barley xantha-f mutants deficient in magnesium chelatase.

Magnesium chelatase (EC 6.6.1.1) catalyses the insertion of magnesium into protoporphyrin IX, the first unique step of the chlorophyll biosynthetic pathway. The enzyme is composed of three different subunits of approximately 40, 70 and 140 kDa. In barley (Hordeum vulgare L.) the subunits are encoded by the genes Xantha-h, Xantha-g and Xantha-f. In the 1950s, eight induced xantha-f mutants were isolated. In this work we characterized these mutations at the DNA level and provided explanations for their phenotypes. The xantha-f10 mutation is a 3 bp deletion, resulting in a polypeptide lacking the glutamate residue at position 424. The leaky mutation xantha-f26 has a missense mutation leading to a M632R exchange. The xantha-f27 and -f40 are deletions of 14 and 2 bp, respectively, resulting in truncated polypeptides of 1104 and 899 amino acid residues, respectively. Mutation xantha-f41 is an in-frame deletion that removes A439, L440, Q441 and V442 from the resulting protein. Mutation xantha-f58 is most likely a deletion of the whole Xantha-f gene, as no DNA fragments could be detected by PCR or southern blot experiments. The slightly leaky xantha-f60 and non-leaky -f68 mutations each have a missense mutation causing a P393L and G794E exchange in the polypeptide, respectively.