Tissue-specific localization of aspartic proteinase in developing and germinating barley grains

Resting seeds of several plant species, including barley grains, have been reported to contain aspartic proteinase (EC 3.4.23) activity. Here, the expression of the Hordeum vulgare L. aspartic proteinase (HvAP) was studied in developing and germinating grains by activity measurements as well as by immunocytochemical and in-situ hybridization techniques. Southern blotting suggests the presence of one to two HvAP-encoding genes in the barley genome, while Northern analysis reveals a single 2.1-kb mRNA in grains and vegetative tissues. Western blotting with antibodies to HvAP shows the same subunit structure in different grain parts. In developing grains, HvAP is produced in the embryo, aleurone layer, testa and pericarp, but in the starchy endosperm HvAP is present only in the crushed and depleted area adjacent to the scutellum. During seed maturation, HvAP-encoding mRNA remains in the aleurone layer and in the embryo, but the enzyme disappears from the aleurone cells. The enzyme, however, remains in the degenerating tissues of the testa and pericarp as well as in resting embryo and scutellum. During the first three days of germination, the enzyme reappears in the aleurone layer cells but is not secreted into the starchy endosperm. The HvAP is also expressed in the flowers, stem, leaves, and roots of barley. The wide localization of HvAP in diverse tissues suggests that it may have several functions appropriate to the needs of different tissues.Abbreviations DAA days after anthesis - DTT dithiothreitol - HvAP Hordeum vulgare aspartic proteinase Both authors have contributed equally to this workWe thank Mart Saarma, Pia Runeberg-Roos, Alan Schulman and Yrjö Helariutta for helpful discussions during the study, Tiina Arna and Sari Makkonen for their help in proteinase activity experiments as well as Jaana Korhonen (Department of Pathology, University of Helsinki), Salla Marttila and Ilkka Porali (Department of Biology, University of Jyväskylä, Jyväskylä, Finland) for their advice on microscopical techniques. We also thank Liisa Pyhälä and Leena Liesirova for the production of the antibodies to HvAP at the National Public Health Institute, Helsinki. This study was supported by grants from the Ministry of Agriculture and Forestry and the Academy of Finland.     

A corolla-and carpel-abundant,non-specific lipid transfer protein gene is expressed in the epidermis and parenchyma of Gerbera hybrida var. Regina (Compositae)

We are examining the floral organ differentiation in Compositae by isolating and characterizing corolla abundant genes. Differential screening of a cDNa library made from the ray floret corolla of Gerbera hybrida var. Regina revealed an abundant cDNA clone which is expressed in the corolla but not in leaves. This cDNA (gltp1) codes for a polypeptide similar to non-specific lipid transfer proteins of the plants. The gltp1 gene is expressed only in the corolla and carpels and is developmentally regulated during corolla development. The gltp1 mRNA accumulates both in epidermal cell layers and in the mesophyll of the corolla. In the stylar part of the carpel, the gltp1 mRNA can be detected in the epidermal and in parenchymal cells but not in the transmitting tissue. Analogous patterns of gltp1 expression in the corolla and carpel may indicate that similar genetic programmes operate during the development of these two tissues.     

Two polyketide synthases are necessary for 4‐hydroxy‐5‐methylcoumarin biosynthesis in Gerbera hybrida

Gerbera (Gerbera hybrida) is an economically important ornamental species and a model plant of the Asteraceae family for flower development and secondary metabolism. Gerberin and parasorboside, two bitter tasting glucosidic lactones, are produced in high amounts in nearly all gerbera tissues. Gerbera and its close relatives also produce a rare coumarin, 4‐hydroxy‐5‐methylcoumarin (HMC). Unlike most coumarins, 5‐methylcoumarins have been suggested to be derived through the acetate‐malonate pathway. All of these polyketide‐derived glucosylated molecules are considered to have a role in defense against herbivores and phytopathogens in gerbera. Gerbera expresses three genes encoding 2‐pyrone synthases (G2PS1–3). The enzymes are chalcone synthase‐like polyketide synthases with altered starter substrate specificity. We have shown previously that G2PS1 is responsible for the synthesis of 4‐hydroxy‐6‐methyl‐2‐pyrone (triacetolactone), a putative precursor of gerberin and parasorboside. Here we show that polyketide synthases G2PS2 and G2PS3 are necessary for the biosynthesis of HMC in gerbera, and that a reductase enzyme is likely required to complete the pathway to HMC. G2PS2 is expressed in the leaf blade and inflorescences of gerbera, while G2PS3 is strictly root specific. Heterologous expression of G2PS2 or G2PS3 in tobacco leads to the formation of 4,7‐dihydroxy‐5‐methylcoumarin, apparently an unreduced precursor of HMC, while ectopic expression in gerbera leads to HMC formation in tissues where nontransgenic tissue does not express the genes and does not accumulate the compound. Using protein modelling and site‐directed mutagenesis we identified the residues I203 and T344 in G2PS2 and G2PS3 to be critical for pentaketide synthase activity.     

Structural characterization and biological implications of di-zinc binding in the ferroxidase center of Streptococcus pyogenes Dpr

Dps proteins contain a ferroxidase site that binds and oxidizes iron, thereby preventing hydroxyl radical formation by Fenton reaction. Although the involvement of a di-iron ferroxidase site has been suggested, X-ray crystal structures of various Dps members have shown either one or two iron cations with various occupancies despite the high structural conservation of the site. Similarly, structural studies with zinc, a redox-stable replacement for iron, have shown the binding of either one or two zinc ions. Here, the crystal structure of Streptococcus pyogenes Dpr in complex with zinc reveals the binding of two zinc cations in the ferroxidase center and an additional zinc-binding site at the surface of the protein. The results suggest a structural basis for the protection of Streptococcus pyogenes in zinc stress conditions and provide a clear evidence for a di-zinc and di-iron ferroxidase site in Streptococcus pyogenes Dpr protein.     

Large scale interaction analysis predicts that the <Emphasis Type="Italic">Gerbera hybrida</Emphasis> floral E function is provided both by general and specialized proteins

Background  

The ornamental plant Gerbera hybrida bears complex inflorescences with morphologically distinct floral morphs that are specific to the sunflower family Asteraceae. We have previously characterized several MADS box genes that regulate floral development in Gerbera. To study further their behavior in higher order complex formation according to the quartet model, we performed yeast two- and three-hybrid analysis with fourteen Gerbera MADS domain proteins to analyze their protein-protein interaction potential.     

Probiotic bacteria as potential detoxification tools: assessing their heavy metal binding isotherms

Dietary exposure to heavy metals may have detrimental effects on human and animal health, even at low concentrations. Specific probiotic bacteria may have properties that enable them to bind toxins from food and water. We assessed the interaction of probiotic bacteria with cadmium and lead in vitro as an initial screening step to identify strains for heavy metal decontamination in food and intestinal models. Binding isotherms for cadmium and lead were characterized for Lactobacillus rhamnosus LC-705, Propionibacterium freudenreichii subsp. shermanii JS and a mix of them used by the food industry. Differences among the strains and their combinations in binding performance at a range of concentrations between 0.1 and 100 mg.L-1 were evaluated with the Langmuir model for biosorption. The effects of pH, contact time, and viability on the binding capacities were also investigated. All strains and their combinations were found to bind cadmium and lead efficiently at low concentration ranges commonly observed in foods. However, the two strains and their combinations differed significantly in their maximum binding capacities and affinities represented by the Langmuir constants Qmax and b, respectively. The binding seemed to occur instantaneously and in a pH-dependent manner, which can be perfectly described by a segmented linear-plateau model.