Ultraviolet-B radiation causes tendril coiling in Pisum sativum.

Low dose UV-B radiation (UV-B(BE,300) = 0.1 W m(-2)), but neither UV-A radiation, ozone and NaCl stress, nor wounding, caused tendril coiling in Pisum sativum. This coiling occurred with both attached and detached tendrils and can be used as a specific UV-B stress marker in pea.

Molecular markers for UV-B stress in plants: alteration of the expression of four classes of genes in Pisum sativum and the formation of high molecular mass RNA adducts.

Sixteen ultraviolet-B radiation-regulated pea genes were identified. Functionally, the corresponding proteins were divided into four groups. (i) Chloroplast-localized proteins. Genes for these proteins were down-regulated, underlining the deleterious effects of UV-B on this organelle. A novel down-regulated photosystem I light-harvesting chlorophyll a/b-binding protein gene (PsLhcA4), was cloned and sequenced. (ii) Protein turnover enzymes. Levels of mature mRNAs for the PU1 and PsUBC4 genes, encoding proteins of the ubiquitin protein degradation pathway, were up- and down-regulated, respectively, implying alteration of plant cell protein content by changes in both gene expression and protein degradation. (iii) Proteins involved in intracellular signalling. Expression of genes for small GTPases, rab and rho homologues, were altered. (iv) Phenylpropanoid or flavonoid biosynthesis. Expression of three genes encoding enzymes in these pathways were up-regulated and one of them, the novel PsC450R1, was cloned and sequenced. Moreover, unexpected high molecular mass psbA RNA adducts were found to appear after UV-B exposure. In addition, a large increase in corresponding high molecular mass adducts were also found for PsLhcA4, and PsUBC4 mRNA and 23S rRNA. These RNA species do not contain protein and probably appear due to cross-linking of two or more RNA molecules, or are the result of UV-B-induced failure of transcription termination.

Cloning, expression, and molecular characterization of a small pea gene family regulated by low levels of ultraviolet B radiation and other stresses.

A pea (Pisum sativum) DNA fragment (termed MB3) was isolated by differential display of cDNAs obtained from total leaf RNA of ultraviolet B (UV-B) radiation-treated plants. Longer cDNAs were cloned by rapid amplification of cDNA ends in the 3' to 5' direction. Three different, but very similar, cDNAs were cloned, sadA, sadB, and sadC, the major difference between them being a 36-bp deletion in the coding region of sadB. Southern blotting confirmed the occurrence of at least three genes in the pea genome. Database comparisons of the SAD protein sequences revealed high identity (46%) and similarity (77%) with a putative tomato (Lycopersicon esculentum) short-chain alcohol dehydrogenase. Very low levels of UV-B radiation (the biologically effective radiation normalized to 300 nm = 0.08 W m(-2)) was shown to up-regulate expression, a dose considerably lower than that needed to induce expression of the well-known UV-B defensive chalcone synthase and phenylalanine ammonia lyase genes. RNase protection assay revealed that primarily sadA and sadC mRNA accumulation was enhanced by UV-B. In addition to UV-B irradiation, ozone fumigation, wounding, aluminum stress, and salt stress induced increased transcript levels of the sad genes in pea.

The mRNA-binding ribosomal protein S26 as a molecular marker in plants: molecular cloning, sequencing and differential gene expression during environmental stress.

One condition for using a gene as a transcriptional marker for environmental stress is its specific and differential expression. In order to be used as such a marker, the ribosomal protein S26 cDNA from pea (Pisum sativum L.) was cloned and fully sequenced. The gene (PsRPS26) was shown to be differentially regulated by ozone and UV-B radiation in opposite ways. Ozone gave rise to increased mRNA levels, whereas UV-B led to a decrease in S26 transcript abundance. Thus, the expression of PsRPS26 can be used as a molecular marker to differentiate between these two environmental stresses.

Occurrence, overexpression and partial purification of the protein (majastridin) corresponding to the URF6 gene of the Rhodobacter blasticus atp operon.

Antibodies were produced against two antigenic peptides of a protein, which was named majastridin, corresponding to the URF6 gene of the Rhodobacter blasticus atp operon [Tybulewicz, V. L. J., Falk, G. & Walker, J. E. (1984) J. Mol. Biol. 179, 185-214]. A protein band of the expected size is labelled by immunoblotting in Western blots containing the cytosolic fractions from Rb. blasticus and Paracoccus denitrificans but not from Escherichia coli or Rhodospirillum rubrum. Although the protein is present during the entire life cycle of a Rb. blasticus culture, it is most abundant early during the stationary phase. Plasmid constructs of the URF6 gene for overexpression in E. coli were made. These constructs were designed to obtain proteins both with and without His-tagging. In both cases, a protein product was visible in induced cells. The His-tagged protein was purified to 85% on a Ni column and, further, to at least 95% by anion-exchange chromatography. By N-terminal sequencing of the His-tagged protein, its identity was confirmed.

A 3-hydroxy-3-methylglutaryl-CoA lyase gene in the photosynthetic bacterium Rhodospirillum rubrum.

A 1.2 kb long DNA segment from Rhodospirillum rubrum has been sequenced (EMBL/GenBank accession number: U41280). This DNA segment includes the first sequenced gene for a putative 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, termed hmgL, from a photosynthetic organism. The sequenced segment also contains a ribosome-binding site and two clusters of possible-35 and -10 promotor sequences preceding the hmgL gene. Translation of the gene would yield a 303 amino-acid-long protein with a calculated molecular weight of 31.1 kDa. This protein shows 55-60% identity and approx. 75% similarity, including conservative substitutions, with the three eukaryotic and the single prokaryotic HMG-CoA lyases which previously have been sequenced. The R. rubrum enzyme showed stronger homology to the chicken HMG-CoA lyase than to the other bacterial protein. Significant sequence similarity was also found with homocitrate synthases from nitrogen-fixing prokaryotes. In contrast to the other sequenced prokaryotic HMG-CoA lyase (from Pseudomonas mevalonii), the R. rubrum hmgL does not seem to appear in an operon together with a HMG-CoA reductase. The hmgL gene was transcribed in photosynthetically grown cells as judged by amplification of cDNAs synthesised from DNA-free total RNA. In addition, HMG-CoA lyase activity was found in R. rubrum cells grown anaerobically in the light with leucine as the carbon source.