Diter von Wettstein

tRNAGlu as a cofactor in δ-aminolevulinate biosynthesis: steps that regulate chlorophyll synthesis

Abstract In plants δ-aminolevulinate is formed from the intact carbon skeleton of glutamate catalysed by the action of three enzymes. The first step of the pathway is activation of glutamate by ligation to δ-ALA-RNA, a reaction identical to that in protein synthesis. Intriguingly, this RNA has been identified as the chloroplast tRNA Glu .

Genetically engineered stem rust resistance in barley using the Rpg1 gene

The stem-rust-susceptible barley cv. Golden Promise was transformed by Agrobacterium-mediated transformation of immature zygotic embryos with the Rpg1 genomic clone of cv. Morex containing a 520-bp 5′ promoter region, 4,919-bp gene region, and 547-bp 3′ nontranscribed sequence. Representatives of 42 transgenic barley lines obtained were characterized for their seedling infection response to pathotype Pgt-MCC of the stem rust fungus Puccinia graminis f. sp. tritici. Golden Promise was converted from a highly susceptible cultivar into a highly resistant one by transformation with the dominant Rpg1 gene. A single copy of the gene was sufficient to confer resistance against stem rust, and progenies from several transformants segregated in a 3:1 ratio for resistance/susceptibility as expected for Mendelian inheritance. These results unequivocally demonstrate that the DNA segment isolated by map-based cloning is the functional Rpg1 gene for stem rust, resistance. One of the remarkable aspects about the transformants is that they exhibit a higher level of resistance than the original sources of Rpg1 (cvs. Chevron and Peatland). In most cases, the Golden Promise transformants exhibited a highly resistant reaction where no visible sign of infection was evident. Hypersensitive necrotic “fleck” reactions were also observed, but less frequently. With both infection types, pathogen sporulation was prevented. Southern blot and RT-PCR analysis revealed that neither Rpg1 gene copy number nor expression levels could account for the increased resistance observed in Golden Promise transformants. Nevertheless, this research demonstrates that stem-rust-susceptible barley can be made resistant by transformation with the cloned Rpg1 gene.

Transcriptome and metabolome profiling of field-grown transgenic barley lack induced differences but show cultivar-specific variances

The aim of the present study was to assess possible adverse effects of transgene expression in leaves of field-grown barley relative to the influence of genetic background and the effect of plant interaction with arbuscular mycorrhizal fungi. We conducted transcript profiling, metabolome profiling, and metabolic fingerprinting of wild-type accessions and barley transgenics with seed-specific expression of (1,3-1, 4)-β-glucanase (GluB) in Baronesse (B) as well as of transgenics in Golden Promise (GP) background with ubiquitous expression of codon-optimized Trichoderma harzianum endochitinase (ChGP). We found more than 1,600 differential transcripts between varieties GP and B, with defense genes being strongly overrepresented in B, indicating a divergent response to subclinical pathogen challenge in the field. In contrast, no statistically significant differences between ChGP and GP could be detected based on transcriptome or metabolome analysis, although 22 genes and 4 metabolites were differentially abundant when comparing GluB and B, leading to the distinction of these two genotypes in principle component analysis. The coregulation of most of these genes in GluB and GP, as well as simple sequence repeat-marker analysis, suggests that the distinctive alleles in GluB are inherited from GP. Thus, the effect of the two investigated transgenes on the global transcript profile is substantially lower than the effect of a minor number of alleles that differ as a consequence of crop breeding. Exposing roots to the spores of the mycorrhizal Glomus sp. had little effect on the leaf transcriptome, but central leaf metabolism was consistently altered in all genotypes.

Barley glutamyl tRNAGlu reductase: Mutations affecting haem inhibition and enzyme activity

Glutamyl tRNA(Glu) reductase converts glutamate molecules that are ligated at their alpha-carboxyl groups to tRNA(Glu) into glutamate 1-semialdehyde, an intermediate in the synthesis of 5-aminolevulinate, chlorophyll and haem. The mature plant enzymes contain a highly conserved extension of 31-34 amino acids at the N-terminus not present in bacterial enzymes. It is shown that barley glutamyl tRNAGlu reductases with a deletion of the 30 N-terminal amino acids have the same high specific activity as the untruncated enzymes, but are highly resistant to feed-back inhibition by haem. This peptide domain thus interacts directly or indirectly with haem and the toxicity of the 30 amino acid peptide for Escherichia coli experienced in mutant rescue and overexpression experiments can be explained by extensive haem removal from the metabolic pools that cannot be tolerated by the cell. Induced missense mutations identify nine amino acids in the 451 residue long C-terminal part of the barley glutamyl tRNA(Glu) reductase which upon substitution curtail drastically, but do not eliminate entirely the catalytic activity of the enzyme. These amino acids are thus important for the catalytic reaction or tRNA binding.

Macromolecular physiology of plastids XIV.Viridis mutants in barley: Genetic, fluoroscopic and ultrastructural characterisation

A sample of 42viridis mutants of barley has been localised by diallelic crosses to 32 nuclear genes. They have been grouped into five different categories on the basis of their fluorescence induction kinetics; this was used as a rapid method for the determination of their photosynthetic capacities. The mutants within each category were further characterised by low temperature fluorescence emission spectroscopy, chlorophyll content, visible fluorescence under UV light, viability, and chloroplast ultrastructure as seen by thin-section electron microscopy. The photosystem I-type mutants have a high initial fluorescence, variable fluorescence, but no fluorescence decline. They fluoresce under UV light and are seedling lethals, but in general have well-developed thylakoid systems. Photosystem II-type mutants, which have a high initial fluorescence, no variable fluorescence, and no fluorescence decline, fluoresce brightly under UV light and are all seedling lethals. Their chloroplast ultrastructure is characterised by giant grana, but as a group, their chlorophyll content is low. The nature of the photosynthetic defects of mutants in the third category is not known, but they are unusual in having a steady-state fluorescence lower than the initial fluorescence. Many of these mutants have a near-normal chloroplast ultrastructure, high chlorophyll content, and are viable under favourable conditions. The fourth group of mutants have fluorescence induction kinetics similar to those of the wild-type, and they are not considered to have major photosynthetic defects, as might be expected from the fact that many will survive under suitable growth conditions. The fifth category contained mutants with extremely low levels of chlorophyll under the growth conditions used.The low temperature fluorescence emission peak at long wavelengths is in part due to antennae chlorophyll of photosystem I. In the mutants deficient in photosystem I, this peak was present, but its wavelength was shifted from 739 nm (wild-type) to 729–731 nm. However, photosynthetically competent mutants with reduced amounts of chlorophyll also showed similar or greater wavelength shifts, and it is concluded that at present, fluorescence emission spectra can not be used to predict the nature of the photosynthetic defects. Similarly, it was not possible to discern a usable relationship between chloroplast ultrastructure and specific photosynthetic deficiencies.

Transgenerational Inheritance of Modified DNA Methylation Patterns and Enhanced Tolerance Induced by Heavy Metal Stress in Rice (Oryza sativa L.)

Background DNA methylation is sensitive and responsive to stressful environmental conditions. Nonetheless, the extent to which condition-induced somatic methylation modifications can impose transgenerational effects remains to be fully understood. Even less is known about the biological relevance of the induced epigenetic changes for potentially altered well-being of the organismal progenies regarding adaptation to the specific condition their progenitors experienced. Methodology/Principal Findings We analyzed DNA methylation pattern by gel-blotting at genomic loci representing transposable elements and protein-coding genes in leaf-tissue of heavy metal-treated rice (Oryza sativa) plants (S0), and its three successive organismal generations. We assessed expression of putative genes involved in establishing and/or maintaining DNA methylation patterns by reverse transcription (RT)-PCR. We measured growth of the stressed plants and their unstressed progenies vs. the control plants. We found (1) relative to control, DNA methylation patterns were modified in leaf-tissue of the immediately treated plants, and the modifications were exclusively confined to CHG hypomethylation; (2) the CHG-demethylated states were heritable via both maternal and paternal germline, albeit often accompanying further hypomethylation; (3) altered expression of genes encoding for DNA methyltransferases, DNA glycosylase and SWI/SNF chromatin remodeling factor (DDM1) were induced by the stress; (4) progenies of the stressed plants exhibited enhanced tolerance to the same stress their progenitor experienced, and this transgenerational inheritance of the effect of condition accompanying heritability of modified methylation patterns. Conclusions/Significance Our findings suggest that stressful environmental condition can produce transgenerational epigenetic modifications. Progenies of stressed plants may develop enhanced adaptability to the condition, and this acquired trait is inheritable and accord with transmission of the epigenetic modifications. We suggest that environmental induction of heritable modifications in DNA methylation provides a plausible molecular underpinning for the still contentious paradigm of inheritance of acquired traits originally put forward by Jean-Baptiste Lamarck more than 200 years ago.

Persistent whole-chromosome aneuploidy is generally associated with nascent allohexaploid wheat

Allopolyploidization has been a driving force in plant evolution. Formation of common wheat (Triticum aestivum L.) represents a classic example of successful speciation via allopolyploidy. Nevertheless, the immediate chromosomal consequences of allopolyploidization in wheat remain largely unexplored. We report here an in-depth investigation on transgenerational chromosomal variation in resynthesized allohexaploid wheats that are identical in genome constitution to common wheat. We deployed sequential FISH, genomic in situ hybridization (GISH), and homeolog-specific pyrosequencing, which enabled unequivocal identification of each of the 21 homologous chromosome pairs in each of >1,000 individual plants from 16 independent lines. We report that whole-chromosome aneuploidy occurred ubiquitously in early generations (from selfed generation S1 to >S20) of wheat allohexaploidy although at highly variable frequencies (20–100%). In contrast, other types of gross structural variations were scant. Aneuploidy included an unexpected hidden type, which had a euploid chromosome number of 2n = 42 but with simultaneous loss and gain of nonhomeologous chromosomes. Of the three constituent subgenomes, B showed the most lability for aneuploidy, followed by A, but the recently added D subgenome was largely stable in most of the studied lines. Chromosome loss and gain were also unequal across the 21 homologous chromosome pairs. Pedigree analysis showed no evidence for progressive karyotype stabilization even with multigenerational selection for euploidy. Profiling of two traits directly related to reproductive fitness showed that although pollen viability was generally reduced by aneuploidy, the adverse effect of aneuploidy on seed-set is dependent on both aneuploidy type and synthetic line.

Protein body formation in the developing barley endosperm

The ultrastructure of the pericarp, testa, aleurone and endosperm in a developing barley grain is presented in the form of a reconstructed section cut tangential to the dorsal surface of the grain and extending half way to the center of the endosperm.Protein body formation in the endosperm is examined in Carlsberg II and Bomi barley and two mutants defective in hordein synthesis. Protein bodies of complex morphology are deposited in large as well as small vacuoles. They comprise clusters of homogeneous components, embedded in a fibrillar matrix, associated with electron-dense spheres and numerous vesicles. The fibrillar matrix is interpreted to be a transient stage in the condensation of storage proteins into a homogeneous structure. The polypeptide composition of protein bodies determines their ultrastructure. The reduction in the synthesis of ‘B’ type hordein in mutant Risø 56 increases the proportion of the fibrillar matrix, while a more drastic alteration in storage protein condensation is observed in mutant Risø 1508, which is highly deficient in both ‘B’ and ‘C’ type hordein polypeptides.

Three thioredoxin targets in the inner envelope membrane of chloroplasts function in protein import and chlorophyll metabolism

Thioredoxins (Trxs) are ubiquitous small proteins with a redox-active disulfide bridge. In their reduced form, they constitute very efficient protein disulfide oxidoreductases. In chloroplasts, two types of Trxs (f and m) coexist and play central roles in the regulation of the Calvin cycle and other processes. Here, we identified a class of Trx targets in the inner plastid envelope membrane of chloroplasts that share a CxxC motif ≈73 aa from their carboxyl-terminal end. Members of this group belong to a superfamily of Rieske iron–sulfur proteins involved in protein translocation and chlorophyll metabolism. These proteins include the protein translocon protein TIC55, the precursor NADPH:protochlorophyllide oxidoreductase translocon protein PTC52, which operates as protochlorophyllide a-oxygenase, and the lethal leaf spot protein LLS1, which is identical with pheophorbide a oxygenase. The role of these proteins in dark/light regulation and oxidative control by the Trx system is discussed.

Physical and genetic mapping of barley (Hordeum vulgare) germin-like cDNAs

Germin with oxalate oxidase and superoxide dismutase activity is a homohexamer of six manganese-containing interlocked β-jellyroll monomers with extreme resistance to heat and proteolytic degradation [Woo, E.-J., Dunwell, J. M., Goodenough, P. W., Marvier, A. C. & Pickersill, R. W. (2000) Nat. Struct. Biol. 7, 1036–1038]. This structure is conserved in germin-like proteins (GLPs) with other enzymatic functions and characteristic for proteins deposited in plant cell walls in response to pathogen attack and abiotic stress. Comparative nucleotide and amino acid sequence analyses of 49,610 barley expressed sequence tags identified 124 germin and germin-like cDNAs, which distributed into five subfamilies designated HvGER-I to HvGER-V. Representative cDNAs for these subfamilies hybridized to 67 bacterial artificial chromosome (BAC) clones from a library containing 6.3 genomic equivalents. Twenty-six BAC clones hybridized to the subfamily IV probe and identified a gene-rich region including clone 418E1 of 96 kb encoding eight GLPs (i.e., 1 gene per 12 kb). This BAC clone lacked highly repeated sequences and mapped to the subtelomeric region of the long arm of chromosome 4(4H). Among the six genes of the contig expressed in leaves, one specifies a protein known to be associated with papilla formation in the epidermis upon powdery mildew infection. Three structural genes for oxalate oxidase are present in subfamily I and eight GLPs of various functions in the other subfamilies. These genes map at loci in chromosomes 1(7H), 2 (2H), 3(3H), 4(4H), and 7(5H). Some are present on a single BAC clone. The results are discussed in relation to cereal genome organization.