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.

The Ycf54 protein is part of the membrane component of Mg-protoporphyrin IX monomethyl ester cyclase from barley (Hordeum vulgare L.).

The biosynthesis of chlorophyll has been demonstrated to require an extensive set of enzymes, the initial stages of which are shared with the synthesis of heme. Of these enzymes, the most enigmatic is the Mg-protoporphyrin IX monomethyl ester cyclase (EC 1.14.13.81). This enzyme requires components found associated with the plastid membrane and the plastid soluble fraction. One of the components, XanL, is found associated with the membrane and another protein, Ycf54, has recently been identified based upon association with XanL. This study describes a deeper analysis of the role of Ycf54 in the enzyme and the localization of the protein in barley plastids. The results clearly demonstrate a strong association of Ycf54 with XanL, absence of Ycf54 from soluble fractions necessary for activity and more evidence for a third membrane localized component of the enzyme.

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.

Methods for the preparation of chlorophyllide a: an intermediate of the chlorophyll biosynthetic pathway.

Chlorophyllide a is a metabolite late in the biosynthesis of chlorophylls and bacteriochlorophylls. Isolation procedures for chlorophyllide a from Rhodobacter capsulatus CB1200 and barley (Hordeum vulgare L.) are described and compared. R. capsulatus CB1200 is a double mutant in the bacteriochlorophyllide a biosynthetic pathway, and chlorophyllide a is excreted by the cells when grown in Tween 80-containing liquid medium. It was purified by liquid or solid phase extraction, yielding 7 mg of chlorophyllide a from 1 L of culture. In a second approach, intrinsic chlorophyllase activity was used to dephytylate chlorophyll in an acetonic preparation of leaves of wild-type or chlorophyll b-deficient barley. Purification was achieved by liquid phase extraction, yielding 14 µg of chlorophyllide a per gram of barley leaves. Chlorophyllide a was identified by thin layer chromatography, absorption spectroscopy, and mass spectrometry.

A novel proteomic approach reveals a role for Mg-protoporphyrin IX in response to oxidative stress.

The presence of genes encoding organellar proteins in different cellular compartments necessitates a tight coordination of expression by the different genomes of the eukaryotic cell. This coordination of gene expression is achieved by organelle-to-nucleus communication. Stress-induced perturbations of the tetrapyrrole pathway trigger large changes in nuclear gene expression. In order to investigate whether the tetrapyrrole Mg-ProtoIX itself is an important part of plastid-to-nucleus communication, we used an affinity column containing Mg-ProtoIX covalently linked to an Affi-Gel matrix. The proteins that bound to Mg-ProtoIX were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis combined with nano liquid chromatography-mass spectrometry (MS)/MS. Thus, we present a novel proteomic approach to address the mechanisms involved in cellular signaling and we identified interactions between Mg-ProtoIX and a large number of proteins associated with oxidative stress responses. Our approach revealed an interaction between Mg-ProtoIX and the heat shock protein 90-type protein, HSP81-2 suggesting that a regulatory complex including HSP90 proteins and tetrapyrroles controlling gene expression is evolutionarily conserved between yeast and plants. In addition, our list of putative Mg-ProtoIX-binding proteins demonstrated that binding of tetrapyrroles does not depend on a specific amino acid motif but possibly on a specific fold of the protein.

A new method for isolating physiologically active Mg-protoporphyrin monomethyl ester, the substrate of the cyclase enzyme of the chlorophyll biosynthetic pathway.

Mg-protoporphyrin monomethyl ester (MPE) is a biosynthetic intermediate of chlorophyll and converted by MPE cyclase to protochlorophyllide. Limited availability of MPE has so far hampered cyclase research. In a new, simplified, method MPE was prepared from freeze dried bchE mutant Rhodobacter capsulatus DB575 cells by extraction with acetone/H(2)O/25% NH(3). Isolated MPE was identified by absorption and fluorescence spectroscopy, and its purity was analyzed by HPLC. The extracted MPE was dried and redissolved in buffered DMSO and its substrate activity is shown by enzymatic cyclase assays. A linear time course was observed for MPE conversion to protochlorophyllide by enzymes from barley etioplasts. Our innovation of freeze drying the R. capsulatus cells before extraction provides a high yield method for MPE, which is significantly faster and more reproducible than previous extraction methods.

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.

Xantha-l encodes a membrane subunit of the aerobic Mg-protoporphyrin IX monomethyl ester cyclase involved in chlorophyll biosynthesis.

Chlorophyll biosynthesis is a process involving approximately 20 different enzymatic steps. Half of these steps are common to the biosynthesis of other tetrapyrroles, such as heme. One of the least understood enzymatic steps is formation of the isocyclic ring, which is a characteristic feature of all (bacterio)chlorophyll molecules. In chloroplasts, formation of the isocyclic ring is an aerobic reaction catalyzed by Mg-protoporphyrin IX monomethyl ester cyclase. An in vitro assay for the aerobic cyclase reaction required membrane-bound and soluble components from the chloroplasts. Extracts from barley (Hordeum vulgare L.) mutants at the Xantha-l and Viridis-k loci showed no cyclase activity. Fractionation of isolated plastids by Percoll gradient centrifugation showed that xantha-l and viridis-k mutants are defective in components associated with chloroplast membranes. The Xantha-l gene, corresponding to Arabidopsis thaliana CHL27, Rubrivivax gelatinosus acsF, Chlamydomonas reinhardtii CRD1, and CTH1 and situated at the short arm of barley chromosome 3 (3H), was cloned, and the mutations in xantha-l(35), xantha-l(81), and xantha-l(82) were characterized. This finding connected biochemical and genetic data because it demonstrated that Xantha-l encodes a membrane-bound cyclase subunit. The evidence suggests that the aerobic cyclase requires at least one soluble and two membrane-bound components.