Bjarne M. Stummann

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.

Localization and nucleotide sequence of the gene for the membrane polypeptide D2 from pea chloroplast DNA

SummaryThe gene for the membrane polypeptide D2 has been mapped on the pea (Pisum sativum) chloroplast genome. The nucleotide sequence of the gene and its flanking regions is presented. The only large open reading frame in the sequence codes for a protein of MW 39.5 kD. A potential ribosome binding site is located 6 nucleotides upstream from the initiation codon and there are two sets of putative promotor sequences in the 5′ flanking region. The polypeptide has a high content of hydrophobic amino acids, predominatly grouped in clusters of 20 or more residues. The 3′ end of the D2 gene is overlapped by 50 nucleotides of a second open reading frame (UORF I) which is at least 369 nucleotides long. Based on current data we suggest the D2 polypeptide to be a constituent of photosystem II (PSII).

Characterization of an ethylene receptor family with differential expression in rose (Rosa hybrida L.) flowers

Abstract To analyze differences in flower longevity and ethylene sensitivity, we isolated Rosa hybrida gene fragments with sequence similarity to the Arabidopsis thaliana ethylene receptor gene-family. A rose gene (RhETR1) highly similar to AtERS1 had been previously sequenced. Here, we report the isolation of three additional partial rose genes (RhETR2–4) belonging to different sub-groups of ethylene receptor genes. RhETR2 clusters with AtETR1, RhETR4 with AtERS1 and RhETR1, whereas RhETR3 shows high sequence similarity to AtETR2 and AtERS2. Expression analysis of RhETR2 and RhETR3 revealed that they are differentially expressed. RhETR2 is expressed at a constitutive level throughout flower development whereas RhETR3 expression increases in senescing flowers of the cultivar Bronze which has a short floral life while it remains at low levels in the long-lasting flowers of the cultivar Vanilla. Expression of both genes was increased by ABA and ethylene treatment, but transcript abundance differed between rose cultivars with different postharvest performance. These results indicate that differences in flower life among rose cultivars could be due to differences in receptor levels.

Sequence of two genes in pea chloroplast DNA coding for 84 and 82 kD polypeptides of the photosystem I complex

SummaryThe genes encoding the two P700 chlorophyll a-apoproteins of the photosystem I complex were localized on the pea (Pisum sativum) chloroplast genome. The nucleotide sequence of the genes and the flanking regions has been determined. The genes are separated by 25 bp and are probably cotranscribed. The 5′ terminal gene (psaA1) codes for a 761-residue protein (MW 84.1 kD) and the 3′ terminal gene (psaA2) for a 734-residue protein (MW 82.4 kD). Both proteins are highly hydrophobic and contain eleven putative membrane-spanning domains. The homology to the corresponding polypeptides from maize are 89% and 95% for psaA1 and psaA2, respectively. A putative promoter has been identified for the psaA1 gene, and potential ribosome binding sites are present before both genes.

A cDNA clone encoding a 10.8 kDa photosystem I polypeptide of barley

A cDNA clone encoding the barley photosystem I polypeptide which migrates with an apparent molecular mass of 16 kDa on SDS‐polyacrylamide gels has been isolated. The 634 bp sequence of this clone has been determined and contains one large open reading frame coding for a 15 457 Da precursor polypeptide. The molecular mass of the mature polypeptide is 10 821 Da. The amino acid sequence of the transit peptide indicates that the polypeptide is routed towards the stroma side of the thylakoid membrane. The hydropathy plot of the polypeptide shows no membrane‐spanning regions.

Involvement of ABA in postharvest life of miniature potted roses

Exogenous application of ABA (abscisic acid) to intact miniature potted rose plants (Rosa hybrida L.) resulted in deterioration of postharvest quality of two cultivars. Spraying with ABA increased leaf drop and accelerated flower senescence in ‘Vanilla’ and ‘Bronze’, while bud drop was only induced in ‘Bronze’. Application of ABA to detached rose flowers accelerated their senescence, indicating that the observed senescence promoting effect was not a secondary response resulting from ABA-induced leaf senescence and abscission. ABA-treatment increased ethylene production in ‘Bronze’ flowers, while no ethylene production was measured in flowers of ‘Vanilla’, or in the leaves of both cultivars. Pre-treatment with 1-MCP (1-methylcyclopropene) delayed ABA promoted flower senescence in ‘Bronze’, suggesting that the effect of ABA is at least partly mediated by ethylene. The senescence promoting effect of ABA on leaf drop and flower life of ‘Vanilla’ flowers was not counteracted by 1-MCP pre-treatment. The cultivar ‘Vanilla’ had a low ABA level at all flower stages, while ABA content of the ‘Bronze’ petals was high in buds, lower in open flowers, and increased during flower senescence. An increased ABA content after ethylene treatment in ‘Vanilla’ suggests that ethylene, natural or exogenous, can increase ABA levels of flowers.

STRUCTURE AND EXPRESSION OF A NITRITE REDUCTASE GENE FROM BEAN (PHASEOLUS VULGARIS) AND PROMOTER ANALYSIS IN TRANSGENIC TOBACCO

A structural gene encoding nitrite reductase (NiR) in bean (Phaseolus vulgaris) has been cloned and sequenced. The NiR gene is present as a single copy encoding a protein of 582 amino acids. The bean NiR protein is synthezised as a precursor with an amino-terminal transit peptide (TP) consisting of 18 amino acid residues. The bean NiR transit peptide shows similarity to the TPs of other known plant NiRs.The NiR gene is expressed in trifoliate leaves and in roots of 20-day old bean plants where transcript accumulation is nitrate-inducible. Gene expression occurs in a circadian rhythm and induced by light in leaves of dark-adapted plants.A particular 100 bp sequence is present in the promoter and in the first intron of the NiR gene. Several copies of this 100 bp sequence are present in the bean genome. Comparisons between the promoter of the bean NiR gene and of two bean nitrate reductase genes (NR1 and NR2) show a limited number of conserved motifs, although the genes are presumed to be co-regulated. Comparisons are also made between the bean NiR promoter and the spinach NiR promoter.Transformation of tobacco plants with the bean NiR promoter fused to the GUS reporter gene (β-glucuronidase) shows that the bean NiR promoter is nitrate-regulated and that the presence of the 100 bp sequence influences the level of GUS activity. NiR-coding sequences are not required for nitrate regulation but have a quantitative effect on the measured GUS activity.

Isolation and characterization of four somatic embryogenesis receptor-like kinase (RhSERK) genes from miniature potted rose (Rosa hybrida cv. Linda)

The isolation and expression analysis of four partial gene sequences from rose (Rosa hybrida cv. Linda) belonging to the receptor-like kinase gene superfamily are reported. These genes have been designated RhSERK1 to RhSERK4 (Accession No. EF631967 to EF631970) as they exhibit high sequence identities with genes from the somatic embryogenesis receptor-like kinase (SERK) family in other plant species. The RhSERK genes are differentially expressed in non-embryogenic callus, embryogenic callus, mature somatic embryos and a range of tissues from intact plants, indicating a broad role in plant growth and development. However, the expressions of RhSERK3 and RhSERK4 were approximately fivefold higher in embryogenic callus than in non-embryogenic callus, and they are even higher when compared to tissues from intact plants. In addition, RhSERK4 expression was approximately eightfold higher in somatic embryos than in embryogenic callus. These results suggest that the expression pattern of RhSERK3 and RhSERK4 may be used as a marker of somatic embryogenesis.

Structure of a 3.2 kb region of pea chloroplast DNA containing the gene for the 44 kD photosystem II polypeptide

SummaryThe gene for the 44 kD chlorophyll a-binding photosystem II polypeptide has been localized on the pea (Pisum sativum) chloroplast genome. The nucleotide sequence of the gene and its flanking regions has been analyzed. The gene codes for a polypeptide of 473 amino acid residues and is possibly cotranscribed with the gene for the D2 photosystem II polypeptide with which it has 50 bp in common. The amino acid sequences of the 44 kD polypeptides from pea, spinach and maize are approximately 95% homologous. Within the 1 kb fragment 3′ to the 44 kD gene a 93 bp tRNA-Ser (UGA) gene and an open reading frame of 62 codons (ORF 62) were identified. Both show high homology to corresponding genes 3′ to the 44 kD genes from spinach, maize and barley. The 44 kD gene and ORF 62 are encoded in the same strand, and have putative promoter sequences, ribosome binding sites and transcription termination signals.

Comparison of postharvest properties of closely related miniature rose cultivars (Rosa hybrida L.)

Abstract The miniature rose cultivar ‘Vanilla Kordana’ is a useful genetic resource for improving display life and ethylene resistance of miniature potted roses. To investigate the significance of ethylene production and sensitivity as determinants of display life two mutants (‘Goldy, Safari’) selected on external traits and one offspring (‘Champagner’) of ‘Vanilla’ were analysed. All cultivars exhibited a shorter postharvest life and higher ethylene sensitivity than ‘Vanilla’. The cultivar ‘Amber’, which was crossed with ‘Vanilla’ to produce ‘Champagner’, had the shortest display life of the investigated cultivars and was highly ethylene sensitive. In response to exogenous ethylene, ‘Vanilla’ and the related cultivars exhibited differences in display life, chlorophyll loss, leaf and bud drop. The two mutants had a clearly higher ethylene production than ‘Vanilla’, suggesting that this is the main reason for their shorter flower life. ‘Vanilla’ and the mutants did not show autocatalytic stimulation of ethylene production. The ethylene production in ‘Champagner’, which results from crossing of ‘Vanilla’ and ‘Amber’, was lower than in both parents and in the mutants. Ethylene exposure induced increased ethylene production in leaves and partly in flowers of the cultivars ‘Amber’ and ‘Champagner’. This autocatalytic ethylene production may be a major reason for the short display life of these two cultivars. The results indicate that not only initial ethylene production but also degree of autocatalytic stimulation is a major factor determining display life.