Intracellular thionins of barley. A second group of leaf thionins closely related to but distinct from cell wall-bound thionins

Leaf thionins of barley have been identified as a novel class of cell wall proteins, toxic to plant pathogenic fungi, and possibly involved in the defense mechanism of plants (Bohlmann, H., Clausen, S., Behnke, S., Giese, H., Hiller, C., Reimann-Philipp, U., Schrader, G., Barkholt, V., and Apel, K., (1988) EMBO J. 7, 1559-1565). In the present work a second subfraction of thionins has been detected within the leaf cell, mainly in the vacuole. Thionins of both groups are closely related to each other. They are toxic to phytopathogenic fungi as well as to plant protoplasts, they share similar amino acid sequences, and their synthesis in etiolated seedlings of barley is down-regulated by light. Despite these similarities each of the two subfractions of thionins could be clearly distinguished by its subcellular distribution. In ultrathin sections of embedded etiolated leaf material, cell wall thionins could be immunogold labeled specifically by an antiserum raised against a fusion protein of Escherichia coli beta-galactosidase and the 15,000 Mr precursor polypeptide of thionins. This antiserum did not react with intracellular thionins. Inversely, intracellular thionins were recognized specifically by an anti-serum raised against soluble leaf thionins. The possible function of intracellular thionins as part of a defense mechanism has been discussed.     

Leaf thionins, a novel class of putative defence factors

Leaf thionins of barley have been identified as a novel class of highly abundant polypeptides with antifungal activity, which are present in walls and vacuoles of barley leaf cells. Similar thionins occur not only in monocotyledonous but also in various dicotyledonous plants. The leaf thionins of barley are encoded by a complex multigene family, which consists of at least 50–100 members per haploid genome. The toxicity of these thionins for plant pathogenic fungi and the fact that their synthesis can also be triggered by pathogens strongly suggest that leaf thionins are involved in the mechanism of plant defence against microbiol infection.     

The effect of chemical stress on the polypeptide composition of the intercellular fluid of barley leaves

The effect of chemical stress on the polypeptide composition of the intercellular fluid of barley (Hordeum vulgare L.) and tomato (Lycopersicon esculentum Mill.) leaves has been studied. In some dicotyledonous plant species, including tomato, exposure to chemical stress leads to the denovo synthesis of intercellular proteins known as pathogenesis-related proteins which have been implicated to be part of a defence mechanism. In barley, however, no such changes in the polypeptide composition of the intercellular fluid could be detected. On the other hand, similar stress conditions induce in barley a strong accumulation of mRNA encoding leaf-specific thionins. These barley thionins represent a novel class of cell-wall proteins toxic to phytopathogenic fungi and are possibly involved in the defence mechanism. These proteins could not be detected in tomato plants. In contrast to the pathogenesis-related proteins of dicotyledonous plants, the leaf-specific thionins of barley are not present in the intercellular fluid of leaves. These results indicate that barley may have evolved a different mechanism to cope with the presence of stress.Abbreviations PAGE polyacrylamide gel electrophoresis - PR pathogenesis-related - SDS sodium dodecyl sulfate     

Leishmania donovani: Thionins, plant antimicrobial peptides with leishmanicidal activity

The leishmanicidal activity of plant antibiotic peptides (PAPs) from the principal families, such wheat thionins, a barley lipid transfer protein and potato defensins and snakins were tested in vitro against Leishmania donovani. Only thionins and defensins were active against this human pathogen at a low micromolar range of concentrations. Thionins resulted as the most active peptides tested until now. They collapsed ionic and pH gradients across the parasite plasma membrane together with a rapid depletion of intracellular ATP without affecting mitochondrial potential. Hence the lethal effect of thionins was mostly associated to permeabilization of the plasma membrane leading to an immediate death of the parasite. The present work is the first evidence for leishmanicidal activity in plant peptides. Future prospects for their development as new antiparasite agents on human diseases are considered.     

Subcellular localization of type I thionins in the endosperms of wheat and barley

Summary Thionins are cysteine-rich polypeptides of about 5,000 Da. Localization at the subcellular level of type I endosperm thionins has been carried out by immunogold labeling, using an antibody that recognizes type I thionin variants. In developing wheat and barley caryopses, sectioned at different times between 13 and 24 days after flowering, this type of thionins was only detected around protein bodies from cells of the starchy endosperm, using light microscopy. Electron microscopy revealed that these proteins were located in electron-dense spheroids in the periphery of protein bodies, at the earlier stages, whereas later the label appeared also as a thin layer around these organelles.Abbreviations DAF days after flowering - RER rough endoplasmic reticulum     

The cytotoxic plant protein, beta-purothionin, forms ion channels in lipid membranes

Thionins are small cysteine-containing, amphipathic plant proteins found in seeds and vegetative tissues of a number of plant genera. Many of them have been shown to be toxic to microorganisms such as fungi, yeast, and bacteria and also to mammalian cells. It has been suggested that thionins are present in seeds to protect them, and the germinating seedling, from attack by phytopathogenic microorganisms, but the mechanism by which they kill cells remains unclear. Using electrophysiological measurements, we have shown that beta-purothionin from wheat flour can form cation-selective ion channels in artificial lipid bilayer membranes and in the plasmalemma of rat hippocampal neurons. We suggest that the generalized toxicity of thionins is due to their ability to generate ion channels in cell membranes, resulting in the dissipation of ion concentration gradients essential for the maintenance of cellular homeostasis.     

Thionins: properties,possible biological roles and mechanisms of action

Thionins are low-molecular-weight proteins (M _r ca. 5000) occurring in seeds, stems, roots and leaves of a number of plant species. The different members of this family of plant proteins show both sequence and structural homology, and are toxic to bacteria, fungi, yeasts and various naked cells in vitro. Toxicity requires an electrostatic interaction of the positively charged thionin with the negatively charged phospholipids making up the membrane, followed by either pore formation or a specific interaction with a certain lipid domain. This domain might be composed of phosphoinositides, which mediate transduction of environmental signals in eukaryotes. Their in vitro toxicity to plant pathogenic bacteria and fungi could reflect a direct role in plant defence, although, in view of the many divergent activities displayed by thionins both in vitro and in vivo, a biological role other than inhibition of microbial growth is equally plausible.     

Isolation and characterization of cDNAs encoding viscotoxins of mistletoe (Viscum album).

Viscotoxins have been isolated from leaf homogenates of European mistletoe (Viscum album L.) and purified to apparent homogeneity. Antisera raised against these polypeptides were used to screen a cDNA expression library in lambda gt11. Two positive clones have been isolated, one encoding a full-length preprotein of viscotoxin A3 and the other encoding the precursor of viscotoxin B. Besides the viscotoxin domain the precursor contained a signal sequence and an acidic polypeptide domain. Similar higher molecular mass precursor proteins have been described for thionins of leaves and seeds of barley. Even though the acidic part of the viscotoxin precursor is much shorter than the corresponding domain of the precursors of the leaf and seed thionins of barley, both the negative charge and the number and the relative position of cysteine residues have been conserved within the acidic domain. This result is consistent with our proposal that the acidic domain of the thionin precursor may play an important role in keeping the thionin inactive within the plant cell.     

Cultivar-related differences in the distribution of cell-wall-bound thionins in compatible and incompatible interactions between barley and powdery mildew

Leaf-specific thionins of barley (Hordeum vulgare L.) have been identified as a novel class of cell-wall proteins toxic to plant-pathogenic fungi and possibly involved in the defence mechanism of plants. The distribution of these polypeptides has been studied in the host-pathogen system of barley and Erisyphe graminis DC.f.sp. hordei Marchal (powdery mildew). Immunogold-labelling of thionins in several barley cultivars indicates that resistance or susceptibility may be attributed to the presence or absence of thionins at the penetration site in walls and papillae of epidermal leaf cells.All of the leaf-specific thionin genes are confined to the distal end of the short arm of chromosome 6 of barley. None of the genes for cultivarspecific resistance to powdery mildew which have previously been mapped on barley chromosomes are found close to this locus.     

Antimicrobial Peptides from Plants

Peptides with antimicrobial properties are present in most if not all plant species. All plant antimicrobial peptides isolated so far contain even numbers of cysteines (4, 6, or 8), which are all pairwise connected by disulfide bridges, thus providing high stability to the peptides. Based on homologies at the primary structure level, plant antimicrobial peptides can be classified into distinct families including thionins, plant defensins, lipid transfer proteins, and he vein- and knottin-type antimicrobial peptides. Detailed three-dimensional structure information has been obtained for one or more members of these peptide families. All antimicrobial peptides studied thus far appear to exert their antimicrobial effect at the level of the plasma membrane of the target microorganism, but the different peptide types are likely to act via different mechanisms. Antimicrobial peptides can occur in all plant organs. In unstressed organs, antimicrobial peptides are usually most abundant in the outer cell layer lining the organ, which is consistent with a role for the antimicrobial peptides in constitutive host defense against microbial invaders attacking from the outside. Thionins are predominantly located intracellularly but are also found in the extracellular space, whereas most plant defensins and lipid transfer proteins are deposited exclusively in the extracellular space. In a number of plant species, a strong induction of genes expressing either thionins, plant defensins, or lipid transfer proteins has been observed on infection of the leaves by microbial pathogens. Hence, antimicrobial peptides can also take part in the inducible defense response of plants. Constitutive expression in transgenic plants of heterologous antimicrobial peptide genes has been achieved, which in some cases has led to enhanced resistance to particular microbial plant pathogens.     

硫堇蛋白及细胞防御素在植物抗病性育种中的研究

硫堇蛋白及细胞防御素是一类广泛存在于植物细胞中并对细菌、真菌等病原微生物具有抑制或杀灭作用的小分子量多肽抗生素。二者在分子量、空间结构及某些化学性质具有相似性,近年来在植物抗病性育种中得以应用。对植物硫堇蛋白及细胞防御素的分类、结构、作用机制以及在植物抗性育种的应用实例进行综述。     

Leaf-specific thionins of barley-a novel class of cell wall proteins toxic to plant-pathogenic fungi and possibly involved in the defence mechanism of plants

A novel class of highly abundant polypeptides with antifungal activity has been detected in cell walls of barley leaves. Similar polypeptides known as thionins occur not only in monocotyledonous but also in various dictoyledonous plants. The leaf-specific thionins of barley are encoded by a complex multigene family, which consists of at least 50-100 members per haploid genome. All of these genes are confined to chromosome 6. The toxicity of these thionins for plant pathogenic fungi and the fact that their synthesis can also be triggered by pathogens strongly suggest that thionins are a naturally occurring, inducible plant protein possibly involved in the mechanism of plant defence against microbial infections.     

The effect of light on the biosynthesis of leaf-specific thionins in barley, Hordeum vulgare

In barley seedlings grown in the dark large amounts of thionin-specific mRNAs are present, the concentration of which rapidly declines once the seedling is exposed to light. This rapid light effect is mediated by a complex interaction of possibly two photoreceptors, phytochrome and a blue-light-absorbing photoreceptor. Parallel to the decline in mRNA content, the de novo synthesis of leaf-specific thionins ceases rapidly upon illumination of etiolated seedlings. However, thionins which have accumulated before the onset of illumination remain stable within the seedling at high concentrations. In younger leaves of mature, nonstressed barley plants grown under a 16-h-light/8-h-dark cycle thionins are still present, although at much lower concentrations. In these plants, synthesis and accumulation of thionins occur predominantly in the meristematic zone at the leaf basis, which is shielded from light through the sheath of the preceding leaf. In mature light-adapted barley plants, mRNA encoding leaf-specific thionins may reaccumulate if these plants are exposed to pathogens or other stresses. Thus, the inhibitory effect of light on the biosynthesis of thionins may be overruled by stress- and pathogen-induced signals.     

Structural characterization of hellethionins from Helleborus purpurascens

Thionins are relatively small-sized multiple-cystine peptides that are probably involved in the plant defense against pathogens. As such, these peptides constitute promising candidates for engineered plant resistance in the agricultural industry. More recently, thionins have been proposed as potential immunotoxins in tumor therapy. In the search for pharmacologically active natural products, a new family of thionins was recently discovered in the roots of Helleborus purpurascens that accordingly were termed hellethionins. The structural characterization by NMR of one representative member of this family, i.e., of hellethionin D, clearly reveals that thionins from different sources share a highly conserved overall fold. In fact, the well-defined 3D structure of hellethionin D is very similar to those reported so far for viscotoxins, purothionins, or crambin, although distinct differences could be detected in the C-terminal portion, especially for loop 36-39. These differences may derive from the unusual distribution of charged residues in the C-terminal half of the peptide sequence compared to other thionins and from the uncommon occurrence of four contiguous threonine residues in loop 36-39. As expected, reduction of the disulfide bonds in hellethionin D leads to complete unfolding, but upon oxidative refolding by air oxygen in the presence of glutathione the correct isomer is recovered in high yields, confirming the very robust fold of this class of bioactive cystine peptides.     

Synergistic Enhancement of the Antifungal Activity of Wheat and Barley Thionins by Radish and Oilseed Rape 2S Albumins and by Barley Trypsin Inhibitors

Although thionins and 2S albumins are generally considered as storage proteins, both classes of seed proteins are known to inhibit the growth of pathogenic fungi. We have now found that the wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) thionin concentration required for 50% inhibition of fungal growth is lowered 2- to 73-fold when combined with 2S albumins (at sub- or noninhibitory concentrations) from radish (Raphanus sativus L.) or oilseed rape (Brassica napus L.). Furthermore, the thionin antifungal activity is synergistically enhanced (2- to 33-fold) by either the small subunit or the large subunit of the radish 2S albumins. Three other 2S albumin-like proteins, the barley trypsin inhibitor and two barley Bowman-Birk-type trypsin inhibitor isoforms, also act synergistically with the thionins (2- to 55-fold). The synergistic activity of thionins combined with 2S albumins is restricted to filamentous fungi and to some Gram-positive bacteria, whereas Gram-negative bacteria, yeast, cultured human cells, and erythrocytes do not show an increased sensitivity to thionin/albumin combinations (relative to the sensitivity to the thionins alone). Scanning electron microscopy and measurement of K+ leakage from fungal hyphae revealed that 2S albumins have the same mode of action as thionins, namely the permeabilization of the hyphal plasmalemma. Moreover, 2S albumins and thionins act synergistically in their ability to permeabilize fungal membranes.     

Immunocytochemical Localization of Cell Wall-Bound Thionins and Hydroxyproline-Rich Glycoproteins in Fusarium culmorum-Infected Wheat Spikes

Abstract In the present study, a rabbit polyclonal antiserum against cell wall‐bound thionins from barley leaf and a mouse monoclonal antibody against hydroxyproline‐rich glycoproteins (HRGP) from maize were used to investigate the subcellular localization of thionins and HRGP or extensins in Fusarium culmorum‐infected wheat spikes by means of the immunogold labelling technique. The proteins were localized in cell walls of different tissues including the lemma, ovary and rachis, while the cytoplasm and organelles in these tissues showed almost no labelling. However, accumulation of thionins and HRGP in infected wheat spikes of resistant wheat cultivars differed distinctly from those of susceptible cultivars. Compared with the healthy tissues, labelling densities for the two types of proteins in cell walls of the infected lemma, ovary and rachis increased only slightly in the susceptible cultivar Agent, while in cell walls of infected tissues of the resistant cultivar Arina labelling densities of thionins and HRGP increased markedly. These findings indicated that accumulation of thionins and HRGP in cell walls of infected resistant wheat spikes may be involved in defence responses to infection and in spreading of F. culmorum.     

Intracellular Localization of Jasmonate-Induced Proteins in Barley Leaves

The plant growth substance jasmonic acid and its methyl ester (JA-Me) induce a set of proteins (jasmonate-induced proteins, JIPs) when applied to leaf segments of barley (Hordeum vulgare L. cv. Salome). Most of these JIPs could be localized within different cell compartments by using a combination of biochemical and histochemical methods. Isolation and purification of various cell organelles of barley mesophyll cells, the separation of their proteins by one-dimensional polyacrylamide gel electrophoresis and the identification of the major abundant JIPs by Western blot analysis, as well as the immuno-gold labelling of JIPs in ultrathin sections were performed to localize JIPs intracellularly. JIP-23 was found to be in vacuoles, peroxisomes, and in the granular parts of the nucleus as well as within the cytoplasm; JIP-37 was detected in vacuoles and in the nucleoplasm; JIP-66 is a cytosolic protein. Some less abundant JIPs were also localized within different cell compartments: JIP-100 was found within the stromal fraction of chloroplasts; JIP-70 is present in the peroxisome and the nucleus; JIP-50 and JIP-6 accumulate in vacuoles. The location of JIP-66 and JIP-6 confirms their possible physiological role deduced from molecular analysis of their cDNA.