A pipeline for identification of causal mutations in barley identifies Xantha-j as the chlorophyll synthase gene.

Thousands of barley (Hordeum vulgare L.) mutants have been isolated over the last century, and many are stored in gene banks across various countries. In the present work, we developed a pipeline to efficiently identify causal mutations in barley. The pipeline is also efficient for mutations located in centromeric regions. Through bulked segregant analyses using whole genome sequencing of pooled F2 seedlings, we mapped 2 mutations and identified a limited number of candidate genes. We applied the pipeline on F2 mapping populations made from xan-j.59 (unknown mutation) and xan-l.82 (previously known). The Xantha-j (xan-j) gene was identified as encoding chlorophyll synthase, which catalyzes the last step in the chlorophyll biosynthetic pathway: the addition of a phytol moiety to the propionate side chain of chlorophyllide. Key amino acid residues in the active site, including the binding sites of the isoprenoid and chlorophyllide substrates, were analyzed in an AlphaFold2-generated structural model of the barley chlorophyll synthase. Three allelic mutants, xan-j.19, xan-j.59, and xan-j.64, were characterized. While xan-j.19 is a 1 base pair deletion and xan-j.59 is a nonsense mutation, xan-j.64 causes an S212F substitution in chlorophyll synthase. Our analyses of xan-j.64 and treatment of growing barley with clomazone, an inhibitor of chloroplastic isoprenoid biosynthesis, suggest that binding of the isoprenoid substrate is a prerequisite for the stable maintenance of chlorophyll synthase in the plastid. We further suggest that chlorophyll synthase is a sensor for coordinating chlorophyll and isoprenoid biosynthesis.

Barley Viridis-k links an evolutionarily conserved C-type ferredoxin to chlorophyll biosynthesis.

Ferredoxins are single-electron carrier proteins involved in various cellular reactions. In chloroplasts, the most abundant ferredoxin accepts electrons from photosystem I and shuttles electrons via ferredoxin NADP+ oxidoreductase to generate NADPH or directly to ferredoxin dependent enzymes. In addition, plants contain other isoforms of ferredoxins. Two of these, named FdC1 and FdC2 in Arabidopsis thaliana, have C-terminal extensions and functions that are poorly understood. Here we identified disruption of the orthologous FdC2 gene in barley (Hordeum vulgare L.) mutants at the Viridis-k locus; these mutants are deficient in the aerobic cyclase reaction of chlorophyll biosynthesis. The magnesium-protoporphyrin IX monomethyl ester cyclase is one of the least characterized enzymes of the chlorophyll biosynthetic pathway and its electron donor has long been sought. Agroinfiltrations showed that the viridis-k phenotype could be complemented in vivo by Viridis-k but not by canonical ferredoxin. VirK could drive the cyclase reaction in vitro and analysis of cyclase mutants showed that in vivo accumulation of VirK is dependent on cyclase enzyme levels. The chlorophyll deficient phenotype of viridis-k mutants suggests that VirK plays an essential role in chlorophyll biosynthesis that cannot be replaced by other ferredoxins, thus assigning a specific function to this isoform of C-type ferredoxins.

A guide to barley mutants.

BACKGROUND: Mutants have had a fundamental impact upon scientific and applied genetics. They have paved the way for the molecular and genomic era, and most of today's crop plants are derived from breeding programs involving mutagenic treatments. RESULTS: Barley (Hordeum vulgare L.) is one of the most widely grown cereals in the world and has a long history as a crop plant. Barley breeding started more than 100 years ago and large breeding programs have collected and generated a wide range of natural and induced mutants, which often were deposited in genebanks around the world. In recent years, an increased interest in genetic diversity has brought many historic mutants into focus because the collections are regarded as valuable resources for understanding the genetic control of barley biology and barley breeding. The increased interest has been fueled also by recent advances in genomic research, which provided new tools and possibilities to analyze and reveal the genetic diversity of mutant collections. CONCLUSION: Since detailed knowledge about phenotypic characters of the mutants is the key to success of genetic and genomic studies, we here provide a comprehensive description of mostly morphological barley mutants. The review is closely linked to the International Database for Barley Genes and Barley Genetic Stocks ( bgs.nordgen.org ) where further details and additional images of each mutant described in this review can be found.

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.

Analysis of early-flowering genes at barley chromosome 2H expands the repertoire of mutant alleles at the Mat-c locus.

KEY MESSAGE: Analyses of barley mat-c loss of function mutants reveal deletions, splice-site mutations and nonsynonymous substitutions in a key gene regulating early flowering. Optimal timing of flowering is critical for reproductive success and crop yield improvement. Several major quantitative trait loci for flowering time variation have been identified in barley. In the present study, we analyzed two near-isogenic lines, BW507 and BW508, which were reported to carry two independent early-flowering mutant loci, mat-b.7 and mat-c.19, respectively. Both introgression segments are co-localized in the pericentromeric region of chromosome 2H. We mapped the mutation in BW507 to a 31 Mbp interval on chromosome 2HL and concluded that BW507 has a deletion of Mat-c, which is an ortholog of Antirrhinum majus CENTRORADIALIS (AmCEN) and Arabidopsis thaliana TERMINAL FLOWER1 (AtTFL1). Contrary to previous reports, our data showed that both BW507 and BW508 are Mat-c deficient and none of them are mat-b.7 derived. This work complements previous studies by identifying the uncharacterized mat-c.19 mutant and seven additional mat-c mutants. Moreover, we explored the X-ray structure of AtTFL1 for prediction of the functional effects of nonsynonymous substitutions caused by mutations in Mat-c.

Mutations in the gene of the Gα subunit of the heterotrimeric G protein are the cause for the brachytic1 semi-dwarf phenotype in barley and applicable for practical breeding.

BACKGROUND: Short-culm mutants have been widely used in breeding programs to increase lodging resistance. In barley (Hordeum vulgare L.), several hundreds of short-culm mutants have been isolated over the years. The objective of the present study was to identify the Brachytic1 (Brh1) semi-dwarfing gene and to test its effect on yield and malting quality. RESULTS: Double-haploid lines generated through a cross between a brh1.a mutant and the European elite malting cultivar Quench, showed good malting quality but a decrease in yield. Especially the activities of the starch degrading enzymes ß-amylase and free limit dextrinase were high. A syntenic approach comparing markers in barley to those in rice (Oryza sativa L.), sorghum (Sorghum bicolor Moench) and brachypodium (Brachypodium distachyon P. Beauv) helped us to identify Brh1 as an orthologue of rice D1 encoding the Gα subunit of a heterotrimeric G protein. We demonstrated that Brh1 is allelic to Ari-m. Sixteen different mutant alleles were described at the DNA level. CONCLUSIONS: Mutants in the Brh1 locus are deficient in the Gα subunit of a heterotrimeric G protein, which shows that heterotrimeric G proteins are important regulators of culm length in barley. Mutant alleles do not have any major negative effects on malting quality.

Genetic linkage facilitates cloning of Ert-m regulating plant architecture in barley and identified a strong candidate of Ant1 involved in anthocyanin biosynthesis.

The erectoides-m anthocyanin-less 1 (ert-m ant1) double mutants are among the very few examples of induced double mutants in barley. From phenotypic observations of mutant plants it is known that the Ert-m gene product regulates plant architecture whereas the Ant1 gene product is involved in anthocyanin biosynthesis. We used a near-isogenic line of the cultivar Bowman, BW316 (ert-m.34), to create four F2-mapping populations by crosses to the barley cultivars Barke, Morex, Bowman and Quench. We phenotyped and genotyped 460 plants, allowing the ert-m mutation to be mapped to an interval of 4.7 cM on the short arm of barley chromosome 7H. Bioinformatic searches identified 21 candidate gene models in the mapped region. One gene was orthologous to a regulator of Arabidopsis thaliana plant architecture, ERECTA, encoding a leucine-rich repeat receptor-like kinase. Sequencing of HvERECTA in barley ert-m mutant accessions identified severe DNA changes in 15 mutants, including full gene deletions in ert-m.40 and ert-m.64. Both deletions, additionally causing anthocyanin deficiency, were found to stretch over a large region including two putative candidate genes for the anthocyanin biosynthesis locus Ant1. Analyses of ert-m and ant1 single- and double-deletion mutants suggest Ant1 as a closely linked gene encoding a R2R3 myeloblastosis transcription factor.

Induced variations in brassinosteroid genes define barley height and sturdiness, and expand the green revolution genetic toolkit.

Reduced plant height and culm robustness are quantitative characteristics important for assuring cereal crop yield and quality under adverse weather conditions. A very limited number of short-culm mutant alleles were introduced into commercial crop cultivars during the Green Revolution. We identified phenotypic traits, including sturdy culm, specific for deficiencies in brassinosteroid biosynthesis and signaling in semidwarf mutants of barley (Hordeum vulgare). This set of characteristic traits was explored to perform a phenotypic screen of near-isogenic short-culm mutant lines from the brachytic, breviaristatum, dense spike, erectoides, semibrachytic, semidwarf, and slender dwarf mutant groups. In silico mapping of brassinosteroid-related genes in the barley genome in combination with sequencing of barley mutant lines assigned more than 20 historic mutants to three brassinosteroid-biosynthesis genes (BRASSINOSTEROID-6-OXIDASE, CONSTITUTIVE PHOTOMORPHOGENIC DWARF, and DIMINUTO) and one brassinosteroid-signaling gene (BRASSINOSTEROID-INSENSITIVE1 [HvBRI1]). Analyses of F2 and M2 populations, allelic crosses, and modeling of nonsynonymous amino acid exchanges in protein crystal structures gave a further understanding of the control of barley plant architecture and sturdiness by brassinosteroid-related genes. Alternatives to the widely used but highly temperature-sensitive uzu1.a allele of HvBRI1 represent potential genetic building blocks for breeding strategies with sturdy and climate-tolerant barley cultivars.

Induced mutations in circadian clock regulator Mat-a facilitated short-season adaptation and range extension in cultivated barley.

Time to flowering has an important impact on yield and has been a key trait in the domestication of crop plants and the spread of agriculture. In 1961, the cultivar Mari (mat-a.8) was the very first induced early barley (Hordeum vulgare L.) mutant to be released into commercial production. Mari extended the range of two-row spring barley cultivation as a result of its photoperiod insensitivity. Since its release, Mari or its derivatives have been used extensively across the world to facilitate short-season adaptation and further geographic range extension. By exploiting an extended historical collection of early-flowering mutants of barley, we identified Praematurum-a (Mat-a), the gene responsible for this key adaptive phenotype, as a homolog of the Arabidopsis thaliana circadian clock regulator Early Flowering 3 (Elf3). We characterized 87 induced mat-a mutant lines and identified >20 different mat-a alleles that had clear mutations leading to a defective putative ELF3 protein. Expression analysis of HvElf3 and Gigantea in mutant and wild-type plants demonstrated that mat-a mutations disturb the flowering pathway, leading to the early phenotype. Alleles of Mat-a therefore have important and demonstrated breeding value in barley but probably also in many other day-length-sensitive crop plants, where they may tune adaptation to different geographic regions and climatic conditions, a critical issue in times of global warming.

Ant13 Encodes Regulatory Factor WD40 Controlling Anthocyanin and Proanthocyanidin Synthesis in Barley (Hordeum vulgare L.).

Flavonoid compounds like anthocyanins and proanthocyanidins are important plant secondary metabolites having wide biological activities for humans. In this study, the molecular function of the Ant13 locus, which is one of the key loci governing flavonoid synthesis in barley, was determined. It was found that Ant13 encodes a WD40-type regulatory protein, which is required for transcriptional activation of a set of structural genes encoding enzymes of flavonoid biosynthesis at the leaf sheath base (colored by anthocyanins) and in grains (which accumulate proanthocyanidins). Besides its role in flavonoid biosynthesis, pleiotropic effects of this gene in plant growth were revealed. The mutants deficient in the Ant13 locus showed similar germination rates but a decreased rate of root and shoot growth and yield-related parameters in comparison to the parental cultivars. This is the seventh Ant locus (among 30) for which molecular functions in flavonoid biosynthesis regulation have been determined.

Identification of a candidate dwarfing gene in Pallas, the first commercial barley cultivar generated through mutational breeding.

Many induced mutants are available in barley (Hordeum vulgare L.). One of the largest groups of induced mutants is the Erectoides (ert) mutants, which is characterized by a compact and upright spike and a shortened culm. One isolated mutant, ert-k.32, generated by X-ray treatment and registered in 1958 under the named "Pallas", was the first ever induced barley mutant to be released on the market. Its value was improved culm strength and enhanced lodging resistance. In this study, we aimed to identify the casual gene of the ert-k.32 mutant by whole genome sequencing of allelic ert-k mutants. The suggested Ert-k candidate gene, HORVU.MOREX.r3.6HG0574880, is located in the centromeric region of chromosome 6H. The gene product is an alpha/beta hydrolase with a catalytic triad in the active site composed of Ser-167, His-261 and Asp-232. In comparison to proteins derived from the Arabidopsis genome, ErtK is most similar to a thioesterase with de-S-acylation activity. This suggests that ErtK catalyzes post-translational modifications by removing fatty acids that are covalently attached to cysteine residues of target proteins involved in regulation of plant architecture and important commercial traits such as culm stability and lodging resistance.

Analyses of MADS-box Genes Suggest HvMADS56 to Regulate Lateral Spikelet Development in Barley.

MADS-box transcription factors are crucial regulators of inflorescence and flower development in plants. Therefore, the recent interest in this family has received much attention in plant breeding programs due to their impact on plant development and inflorescence architecture. The aim of this study was to investigate the role of HvMADS-box genes in lateral spikelet development in barley (Hordeum vulgare L.). A set of 30 spike-contrasting barley lines were phenotypically and genotypically investigated under controlled conditions. We detected clear variations in the spike and spikelet development during the developmental stages among the tested lines. The lateral florets in the deficiens and semi-deficiens lines were more reduced than in two-rowed cultivars except cv. Kristina. Interestingly, cv. Kristina, int-h.43 and int-i.39 exhibited the same behavior as def.5, def.6, semi-def.1, semi-def.8 regarding development and showed reduced lateral florets size. In HOR1555, HOR7191 and HOR7041, the lateral florets continued their development, eventually setting seeds. In contrast, lateral florets in two-rowed barley stopped differentiating after the awn primordia stage giving rise to lateral floret sterility. At harvest, the lines tested showed large variation for all central and lateral spikelet-related traits. Phylogenetic analysis showed that more than half of the 108 MADS-box genes identified are highly conserved and are expressed in different barley tissues. Re-sequence analysis of a subset of these genes showed clear polymorphism in either SNPs or in/del. Variation in HvMADS56 correlated with altered lateral spikelet morphology. This suggests that HvMADS56 plays an important role in lateral spikelet development in barley.

Aerobic Barley Mg-protoporphyrin IX Monomethyl Ester Cyclase is Powered by Electrons from Ferredoxin.

Chlorophyll is the light-harvesting molecule central to the process of photosynthesis. Chlorophyll is synthesized through 15 enzymatic steps. Most of the reactions have been characterized using recombinant proteins. One exception is the formation of the isocyclic E-ring characteristic of chlorophylls. This reaction is catalyzed by the Mg-protoporphyrin IX monomethyl ester cyclase encoded by Xantha-l in barley (Hordeum vulgare L.). The Xantha-l gene product (XanL) is a membrane-bound diiron monooxygenase, which requires additional soluble and membrane-bound components for its activity. XanL has so far been impossible to produce as an active recombinant protein for in vitro assays, which is required for deeper biochemical and structural analyses. In the present work, we performed cyclase assays with soluble and membrane-bound fractions of barley etioplasts. Addition of antibodies raised against ferredoxin or ferredoxin-NADPH oxidoreductase (FNR) inhibited assays, strongly suggesting that reducing electrons for the cyclase reaction involves ferredoxin and FNR. We further developed a completely recombinant cyclase assay. Expression of active XanL required co-expression with an additional protein, Ycf54. In vitro cyclase activity was obtained with recombinant XanL in combination with ferredoxin and FNR. Our experiment demonstrates that the cyclase is a ferredoxin-dependent enzyme. Ferredoxin is part of the photosynthetic electron-transport chain, which suggests that the cyclase reaction might be connected to photosynthesis under light conditions.

Comparing two microarray platforms for identifying mutated genes in barley (Hordeum vulgare L.).

We have previously described the evaluation of a cDNA microarray platform to identify and clone mutated barley (Hordeum vulgare L.) genes, using their transcriptionally defective mutant alleles (S. Zakhrabekova, C.G. Kannangara, D. von Wettstein, M. Hansson, A microarray approach for identification of mutated genes, Plant Physiol. Biochem. 40 (2002) 189-197). It was concluded that competitive hybridization between phenotypically similar mutants could specifically highlight an arrayed clone, corresponding to the mutated gene. In this study we evaluate whether the Affymetrix microarray platform can be used for the same purpose. The Affymetrix barley microarray contains a large number of clones (22,792 probe sets). In this and the previous study we used two barley mutant strains, xantha-h.57 and xantha-f.27, with known mutations in different subunit genes of the chlorophyll biosynthetic enzyme magnesium chelatase (EC 6.6.1.1). Mutant xantha-h.57 produces no Xantha-h mRNA whereas in xantha-f.27 the nonsense mutation in the last exon of the gene, results in nonsense-mediated decay of Xantha-f mRNA. We conclude that the Affymetrix platform meets our requirements and that our approach successfully highlighted the arrayed Xantha-h clone and that Xantha-f was among the top fourteen candidates.

Generation and analysis of 9792 EST sequences from cold acclimated oat, Avena sativa.

BACKGROUND: Oat is an important crop in North America and northern Europe. In Scandinavia, yields are limited by the fact that oat cannot be used as a winter crop. In order to develop such a crop, more knowledge about mechanisms of cold tolerance in oat is required. RESULTS: From an oat cDNA library 9792 single-pass EST sequences were obtained. The library was prepared from pooled RNA samples isolated from leaves of four-week old Avena sativa (oat) plants incubated at +4 degrees C for 4, 8, 16 and 32 hours. Exclusion of sequences shorter than 100 bp resulted in 8508 high-quality ESTs with a mean length of 710.7 bp. Clustering and assembly identified a set of 2800 different transcripts denoted the Avena sativa cold induced UniGene set (AsCIUniGene set). Taking advantage of various tools and databases, putative functions were assigned to 1620 (58%) of these genes. Of the remaining 1180 unclassified sequences, 427 appeared to be oat-specific since they lacked any significant sequence similarity (Blast E values > 10(-10)) to any sequence available in the public databases. Of the 2800 UniGene sequences, 398 displayed significant homology (BlastX E values < or = 10(-10)) to genes previously reported to be involved in cold stress related processes. 107 novel oat transcription factors were also identified, out of which 51 were similar to genes previously shown to be cold induced. The CBF transcription factors have a major role in regulating cold acclimation. Four oat CBF sequences were found, belonging to the monocot cluster of DREB family ERF/AP2 domain proteins. Finally in the total EST sequence data (5.3 Mbp) approximately 400 potential SSRs were found, a frequency similar to what has previously been identified in Arabidopsis ESTs. CONCLUSION: The AsCIUniGene set will now be used to fabricate an oat biochip, to perform various expression studies with different oat cultivars incubated at varying temperatures, to generate molecular markers and provide tools for various genetic transformation experiments in oat. This will lead to a better understanding of the cellular biology of this important crop and will open up new ways to improve its agronomical properties.