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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 mata 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 daylength-sensitive crop plants, where they may tune adaptation to different geographic regions and climatic conditions, a critical issue in times of global warming.earliness | food security | timing of flowering | molecular breeding | synteny

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 brassinosteroidrelated 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.

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A molecule of chlorophyll is synthesized from eight molecules of delta-aminolevulinate (DALA), the universal precursor of porphyrins. The light-regulated conversion of glutamate to delta-aminolevulinate in the stroma of greening plastids involves the reduction of glutamate to glutamate-1-semialdehyde and its subsequent transamination. The components performing this conversion have been isolated from barley and Chlamydomonas and separated into three fractions by serial affinity chromatography on Blue Sepharose and haem- or chlorophyllin-Sepharose. The complete reaction can be performed in vitro in a reconstituted assay by combining all three fractions. An RNA is the essential component of the chlorophyllin-Sepharose-bound fraction. By nucleotide sequence analysis, we have now identified this RNA as a chloroplast glutamate acceptor RNA. Glutamate attached by an aminoacyl bond to the 3'-terminal adenosine of this RNA is a substrate for the enzyme(s) which perform the subsequent reactions. This reaction represents a novel role for transfer RNA: participation in the metabolic conversion of its cognate amino acid into another metabolite of low relative molecular mass which subsequently is not used in peptide bond synthesis.

The isocyclic ring of bacteriochlorophyll (BChl) is formed by the conversion of Mg-protoporphyrin monomethyl ester (MPE) to protochlorophyllide (PChlide). Similarities revealed by BLAST searches with the putative anaerobic MPE-cyclase BchE suggested to us that this protein also uses a cobalamin cofactor. We found that vitamin B12 (B12)-requiring mutants of the bluE and bluB genes of Rhodobacter capsulatus, grown without B 12, accumulated Mgporphyrins. Laser desorption͞ionization time-of-flight (LDI-TOF) MS and NMR spectroscopy identified them as MPE and its 3-vinyl-8-ethyl (mvMPE) derivative. An in vivo assay was devised for the cyclase converting MPE to PChlide. Cyclase activity in the B12-dependent mutants required B12 but not protein synthesis. The following reaction mechanism is proposed for this MPE-cyclase reaction. Adenosylcobalamin forms the adenosyl radical, which leads to withdrawal of a hydrogen atom and formation of the benzylic-type 13 1 -radical of MPE. Withdrawal of an electron gives the 13 1 -cation of MPE. Hydroxyl ion attack on the cation gives 13 1 -hydroxy-MPE. Withdrawal of three hydrogen atoms leads successively to 13 1 -keto-MPE, its 13 2 -radical, and cyclization to PChlide. The Mg-protoporphyrin monomethyl ester (MPE)-cyclase is an enzyme activity converting MPE to protochlorophyllide (PChlide) in the Mg branch of the tetrapyrrole biosynthetic pathway leading to chlorophyll and bacteriochlorophyll (BChl) (Fig. 1). Cobalamin is formed from uroporphyrinogen, the first tetrapyrrole intermediate. During evolution cobalt tetrapyrrole may thus have preceded the formation of chlorophyll and an oxygen atmosphere. The cobalt tetrapyrroles still play an essential role in anaerobic reactions involved in energy production. Many photosynthetic bacteria form their BChl only under anaerobic conditions. Consequently, as shown in ref. 1, the 13 1 -oxo group of the isocyclic ring in Rhodobacter sphaeroides is derived from water and not molecular oxygen, as it is in higher plants (2). Aerobic cyclase activity has been demonstrated in higher plants (3) and Synechocystis (4). In both cases a soluble and a membrane fraction were shown to be required, but purification and identification of the proteins involved were not reported. In vitro anaerobic cyclase activity has not been reported.MPE accumulates in transposon mutants of the Rhodobacter capsulatus bchE gene, suggesting that the encoded protein is required for MPE-cyclase activity (5). Sequence similarities of BchE to P-methylase from Streptomyces hygroscopicus suggested to us a cobalamin requirement for the MPE-cyclase. Disruption of the vitamin B 12 (B 12 ) biosynthetic genes bluE and bluB in R. capsulatus strongly inhibited formation of BChl and proteins of the photosynthetic apparatus (6, 7).We show here that MPE, an intermediate of the BChl biosynthetic pathway, accumulates in B 12 -requiring mutants carrying bluE and bluB genes. Furthermore, MPE can be efficiently converted to PChlide by these mutants, but only when a form of cobalamin is provided. ...

SummaryThe N-terminal region of a 60 kDa, jasmonate-induced protein of barley leaves (JIP60) is shown to be homologous to the catalytic domains of plant ribosomeinactivating proteins (RIP). Western blotting of leaf extracts and in vitro reconstitution experiments indicate that JIP60 is synthesized as a precursor which is processed in vivo. This is in keeping with in vitro translation experiments indicating that a deletion derivative of the N-terminal region, but not the putative precursor, strongly inhibits protein synthesis on reticuIocyte ribosomes. The inhibition of ribosome function is associated with depurination of 26S rRNA, characteristic of plant RIPs. This indicates that JIP60 is a novel ribosome-inactivating protein requiring at least two processing events for full activation. JIP60 derivatives do not significantly Inhibit in vitro protein synthesis on wheat germ ribosomes. These and other results suggest that JIP60 may be involved in plant defence.

Chlorophyll biosynthesis is a process involving Ϸ20 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 membranebound 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.barley ͉ acsF ͉ CHL27 ͉ CRD1 ͉ mutant