D. von Wettstein

Structural genes for Mg-chelatase subunits in barley:Xantha-f, -g and-h

Barley mutants in the lociXantha-f, Xantha-g andXantha-h, when fed with 5-aminolevulinate in the dark, accumulate protoporphyrin IX. Mutant alleles at these loci that are completely blocked in protochlorophyllide synthesis are also blocked in development of prolamellar bodies in etioplasts. In contrast to wild type, thexan-f, -g and-h mutants had no detectable Mg-chelatase activity, whereas they all had methyltransferase activity for synthesis of Mg-protoporphyrin monomethyl ester. Antibodies recognising the CH42 protein ofArabidopsis thaliana and the OLIVE (OLI) protein ofAntirrhinum majus immunoreacted in wild-type barley with 42 and 150 kDa proteins, respectively. Thexan-h mutants lacked the protein reacting with antibodies raised against the CH42 protein. Twoxan-f mutants lacked the 150 kDa protein recognised by the anti-OLI antibody. Barley genes homologous to theA. majus olive and theA. thaliana Ch-42 genes were cloned using PCR and screening of cDNA and genomic libraries. Probes for these genes were applied to Northern blots of RNA from thexantha mutants and confirmed the results of the Western analysis. The mutantsxan-f27, -f40, -h56 and-h57 are defective in transcript accumulation while-h38 is defective in translation. Southern blot analysis established thath38 has a deletion of part of the gene. Mutantsxan-f10 and-f41 produce both transcript and protein and it is suggested that these mutations are in the catalytic sites of the protein. It is concluded thatXan-f and-h genes encode two subunits of the barley Mg-chelatase and thatXan-g is likely to encode a third subunit. The XAN-F protein displays 82% amino acid sequence identity to the OLI protein ofAntirrhinum, 66% to theSynechocystis homologue and 34% identity to theRhodobacter BchH subunit of Mg-chelatase. The XAN-H protein has 85% amino acid sequence identity to theArabidopsis CH42 protein, 69% identity to theEuglena CCS protein, 70% identity to theCryptomonas BchA andOlisthodiscus CssA proteins, as well as 49% identity to theRhodobacter BchI subunit of Mg-chelatase. Identification of the barleyXan-f andXan-h encoded proteins as subunits required for Mg-chelatase activity supports the notion that theAntirrhinum OLI protein and theArabidopsis CH42 protein are subunits of Mg-chelatase in these plants. The expression of both theXan-f and-h genes in wild-type barley is light induced in leaves of greening seedlings, and in green tissue the genes are under the control of a circadian clock.

Inheritance of bud-set and bud-flushing in Picea abies (L.) Karst.

SummaryDiallel crosses were made between clones of a French and a Swedish provenance of Picea abies. The former is characterized by a long critical night length for bud-set and a late flushing of the buds; the latter by a short critical night length for bud-set and an early flushing of the buds. The F1 hybrid seedlings and their French and Swedish intraprovenance half-sibs were tested over three growth periods in the phytotron at the College of Forestry, Stockholm. In comparison with intraprovenance half-sibs, the hybrid progenies gave, on average, an intermediate response for the photoperiodic control over budset and for the temperature requirements for bud-flushing. This indicates the prevalence of additive action of multiple factors in the determination of the photoperiodic and temperature response. However, individual interprovenance hybrid progenies revealed a range of responses, and in certain combinations the response of these hybrids and their intraprovenance half-sibs coincided. By selection of suitable parents, interprovenance crosses can be used to produce hybrids with desired photoperiodic characteristics and temperature requirements.

Magnesium chelatase: association with ribosomes and mutant complementation studies identify barley subunit Xantha-G as a functional counterpart of Rhodobacter subunit BchD

Abstract Magnesium chelatase catalyses the insertion of Mg2+ into protoporphyrin and is found exclusively in organisms which synthesise chlorophyll or bacteriochlorophyll. Soluble protein preparations containing >10 mg protein/ml, obtained by gentle lysis of barley plastids and Rhodobacter sphaeroplasts, inserted Mg2+ into deuteroporphyrin IX in the presence of ATP at rates of 40 and 8 pmoles/mg protein per min, respectively. With barley extracts optimal activity was observed with 40 mM Mg2+. The activity was inhibited by micromolar concentrations of chloramphenicol. Mutations in each of three genetic loci, Xantha-f, -g and -h, in barley destroyed the activity. However, Mg-chelatase activity was reconstituted in vitro by combining pairwise the plastid stroma protein preparations from non-leaky xantha-f, -g and -h mutants. This establishes that, as in Rhodobacter, three proteins are required for the insertion of magnesium into protoporphyrin IX in barley. These three proteins, Xantha-F, -G and -H, are referred to as Mg-chelatase subunits and they appear to exist separate from each other in vivo. Active preparations from barley and Rhodobacter yielded pellet and supernatant fractions upon centrifugation for 90 min at 272 000 × g. The pellet and the supernatant were inactive when assayed separately, but when they were combined activity was restored. Differential distribution of the Mg-chelatase subunits in the fractions was established by in vitro complementation assays using stroma protein from the xantha-f, -g, and -h mutants. Xantha-G protein was confined to the pellet fraction, while Xantha-H was confined to the supernatant. Reconstitution assays using purified recombinant BchH, BchI and partially purified BchD revealed that the pellet fraction from Rhodobacter contained the BchD subunit. The pellet fractions from both barley and Rhodobacter contained ribosomes and had an A260:A280 ratio of 1.8. On sucrose density gradients both Xantha-G and BchD subunits migrated with the plastid and bacterial ribosomal RNA, respectively.

Chlorina mutants of barley (Hordeum vulgare L.)

We have examined 31 newchlorina mutants of barley using in vivo absorption spectroscopy, 77 K fluorescence emission spectroscopy, room temperature fluorescence induction kinetics, HPLC separation of pigments and SDS-PAGE. Based on these properties they can be placed into 4 groups. The first group consists of 10 mutants which are allelic to the chlorophyllb-lesschlorina-f2 and comprises five strongly and three slightly leaky mutants at this locus. The decrease in chlorophyllb content was correlated with a corresponding decrease in the amount of chlorophylla/b-proteins in the thylakoids. One mutant (chlorina 106) was found which had a very low chlorophyllb content and a deficiency in Chla/b-P2, but was not allelic tochlorina-f2. The second group ofchlorina, mutants has unusual fluorescence properties, with a high Fm/Fo ratio. Gaussian deconvolution of the 77 K fluorescence emission spectra revealed an increase in the amplitude of components emitting at 694 and 718 nm. The possibility is discussed that these properties result from the absence of the PSI connecting antenna LHCI-680. The third group has a Fs/Fo ratio <1, and other properties consistent with a partial deficiency in the chlorophylla-proteins of PSII reaction centres. The fourth group consists ofchlorina mutants which are very similar to wild-type in the properties examined, differing mainly in having a lower chlorophyll content.

Biochemical mutant in barley renders chemical stabilization of beer superfluous

The recessive mutantant-13 isolated from Foma barley after a mutagen treatment with ethyl methanesulfonate is shown to be blocked in the formation of anthocyanins, catechins and proanthocyanidins (=anthocyanogens). The mutant has been propagated, malted on a pilot scale and the malt used for pilot brews of beer. Foma barley has been malted and brewed for comparison. Malt, wort and beer made from the mutant was free of the polyphenols that are found, when Foma is used as raw material. The beer brewed from the proanthocyanidin-and catechin-free mutant had satisfactory haze stability. In colour and flavourant-13 beer was indistinguishable from Foma beer, but in haze stability the former was drastically superior to the latter.

Ribulose-1,5-bisphosphate carboxylase as a nuclear and chloroplast marker

SummaryThe data on the primary structure of ribulose-1,5-bisphosphate carboxylase/oxygenase are reviewed. Examples of their use as markers and in the elucidation of the evolution, adaptation and function of this key enzyme are given.

Genetic engineering in the adaptation of plants to evolving human needs

Genetic engineering is a technique for isolating individual genes from plants and for multiplying cloning these genes in bacteria and yeast. This makes it possible to determine the structure and the function of individual genes. It is hoped that procedures can be worked out to incorporate into the genomes of crop plants useful or even improved genes and thereby aid in the continuous adaptation of agricultural crops to human needs. Before describing the present state of gene technology as related to plant breeding, I would like to briefly emphasize that genetic engineering is a technique which is added to our arsenal of existing breeding techniques and will be complementing but not replacing the conventional breeding procedures. Our major crop plants, rice, wheat, maize, barley, soybeans but also tobacco, sugar beets and rape have been bred by man some more than 4000 years ago; some a few hundred years ago. At first, plant breeding was exercised by selecting bigger grain, non-shattering ears and diesease resistant plants from the variations produced in nature by crosses and mutation. Records of artificial fertilization of the date palm in Assyria date back to centuries before the time of Christ, but we have no record whether the genomes from Triticum monococcum, Triticum searsii and Triticure tauschii were combined into the 42 chromosomic bread wheat by artificial pollination. Perhaps artificial pollination also accounts for the amphidiploid tobacco, which may have been produced in South America in pre-Colombian times by hand pollination of Nicotiana sylvestris with pollen from Nicotiana tomentosiformis and accidental chromosome doubling in the hybrid. To this day plant breeding is mostly carried out empirically by crossing promising lines with each other and by selecting plants with improved yield, disease resistance and better climatic adaptation. Especially since the end of the last century our knowledge of genetics and biochemistry has improved steadily and thereby given us increased opportunities to apply this knowledge to plant breeding in a rational manner 29. We can cite a few examples to illustrate this. Hybrid vigor was discovered by Joseph Gottlieb K61reuter in Germany in 1760 and was also recommended by him as a means of increasing yield. In 1908, after Carlsberg Laboratory alumnus Wilhelm Johannsen had clarified the theoretical basis for the distinction of pure genetic lines versus populations and coined the word gene for Mendels units, George H. Shull in the United States produced by inbreeding pure lines of maize and established that single and double hybrids of these lines exhibit superiority in yield as a consequence of gene heterozygosity. Research at universities and breeding work at seed producing industries has led to the first commercial hybrid corn varieties in the early 1930s. In 1935 an average of 1.5 tons of maize was harvested per hectare and less than 1% of the area in the United States of America was planted with the new hybrid varieties. In 1968, 5.1 tons per hectare were harvested and 90% of the area was planted with hybrid maize. This enormous increase in yield is due to the combined results of plant breeding and improved agricultural practice. Further increases have been achieved over the last 15 years. While the success of maize breeding examplifies the importance of suitable new combinations of genes, the triploid monogerm sugar beet introduced in the 1960s illustrates the use of a morphological mutation and polyploidy to adapt crop plants to modern society 18. Originally, seeds of the sugar beet were produced in clusters on the flowering stem and when sown each cluster gave rise to several seedlings, which had to be singled by hand. In the monogerm lines, only 1 seedling is produced from each cluster and the labor effort to cultivate 1 hectare could be reduced from 290 man-hours to 13. In his thesis from the Carlsberg Laboratory, 10jvind Winge proposed in

Expression of an Erwinia pectate lyase in three species of Aspergillus

Abstract Transgenic filamentous fungi of the species Aspergillus niger, A. nidulans and A. awamori expressing and secreting Erwinia carotovora subsp. atroseptica pectate lyase 3 (PL3) were generated. Correct processing of the pre-enzyme was achieved using the A. niger pectin lyase A (PEL A) signal peptide. With the prepro-peptide of A. niger polygalacturonase II, secreted enzymes still possessed the 6- aa pro-sequence, indicating the importance of the conformation of the precursor protein for correct cleavage of the signal sequence. PL3 expression was markedly increased in media optimized for limited protease activity, and reached 0.4, 0.8 and 2.0 mg/l for expression in A. niger, A. awamori and A. nidulans, respectively. Glycans attached to the PL3 enzymes exhibited species-specific differences, and an increase of molecular mass coincided with reduced specific activities of the enzymes.

Chloroplast and Nucleus: Concerted Interplay Between Genomes of Different Cell Organelles. The Emil Heitz Lecture

Emil Heitz (1928) discovered that the chromo-centres of the interphase nucleus constitute heterochromatic portions of chromosomes as contrasted to their euchromatic parts. His is the introduction of the terms heterochromatin and euchromatin as well as the recognition of different categories of heterochromatin. In spite of the large effort by cytologists and biochemists to elucidate the features of this chromatin differentiation a notion of Heitz in 1955 remains essentially true today. No definitive answer has been obtained as to the nature of the structural difference between eu- and heterochromatin nor is the genetic and functional significance of heterochromatin clear. Unfortunately, up to this day heterochromatin is frequently used as a scape-goat.