Genetic control of purothionins in wheat: problems of the aneuploid analysis when searching for regulatory genes

The study of the genetic control of purothionins in wheat endosperm illustrates some of the problems and pitfalls faced in aneuploid analysis of regulatory effects. Biochemical and genetic evidence is presented indicating that the possible regulatory effect of genes located in group 5 chromosomes on the expression of the purothionin structural genes located in group 1 chromosomes is not actually operating "in vivo"

Thionins: plant peptides that modify membrane permeability in cultured mammalian cells

Thionins, which are high-sulphur polypeptides present in the endosperm of wheat and related species, have been found to prevent growth and to inhibit macromolecular synthesis in cultured mammalian cells. Baby hamster kidney (BHK) cells were markedly more sensitive to thionins than the other cell lines tested (monkey CV1, mouse L, human HeLa). A thionin concentration of 5 μ/ml (1 μM) completely blocked translation in BHK cells. It was later found that omission of both calcium and magnesium ions from the medium strongly enhanced the inhibitory effects of thionins (BHK cells, 80% inhibition, 0.5 μ/ml). Several lines of evidence indicate that thionins might act at the membrane level. Indeed, both the 86Rb+ content and the nucleotide pool of BHK cells were drastically decreased at thionin concentrations that inhibited translation. In addition, thionin concentrations that did not affect macromolecular synthesis in these cells, allowed inhibition of translation by antibiotics, such as hygromycin Bthat are not able to cross the cell plasma membrane by themselves. Our results suggest that the inhibition of protein, RNA and DNA synthesis in BHK cells might be a consequence of membrane leakiness induced by thionin treatment. In this respect, particularly striking was the parallelism found between 86Rb+ leakage and inhibition of protein synthesis by treatment with different genetic variants of thionins (α1 purothionin, α2 purothionin, β purothionin from wheat; hordothionin from barley), as well as with the viscotoxins, which are homologous polypeptides from the European mistletoe

Cloning and nucleotide sequence of a cDNA encoding the precursor of the barley toxin a-hordothionin

A cDNA library, prepared from developing barley endosperm, was screened for thionin recombinants. Clone pTH1 was that with the largest insert out of three identified. The longest reading frame in the 610-base-pair insert codes for a protein of 127 amino acids that includes an internal sequence of 45 amino acids, which is identical to that obtained for the α-hordothionin by direct protein sequencing. The deduced thionin sequence is preceded by a leader sequence of 18 residues and followed by a sequence that corresponds to an acidic protein of 64 amino acids. This structure supports previous evidence indicating that thionin is synthesized as a much larger precursor, which undergoes two processing steps: the cotranslational cleavage of a leader sequence and the post-translational one of a larger peptide. The size of the mRNA was estimated to be about 950 bases by Northern analysis. Thionin concentration in mature endosperm of barley cv. Bomi was about twice that of its high-lysine mutant Risç 1508. The same difference was observed in thionin mRNA in the corresponding developing endosperms, indicating that gene expression is partially blocked in the mutant at a pretranslational leve

Synthesis and processing of thionin precursors in developing endosperm from barley (Hordeum vulgare)

Thionin is a lysine-rich polypeptide (mol. wt. 5000) which is synthesized in developing barley endosperm from - 8 days to - 30 days after anthesis. Two thionin precursors (THPl and THP2) have been identified using monospecific antibodies (A-TH) prepared against the mature protein. THPl, which is the only polypeptide recognized in vitro by A-TH, is encoded by a 7.5S mRNA obtained from membrane-bound polysomes, and its alkylated derivative has an apparent mol. wt. of 17 800. THP2, which is selected together with mature thionin by A-TH among labelled proteins in vivo, differs from THPl in apparent mol. wt. (17 400 alkylated) and in electrophoretic mobility at pH 3.2. Both THPl and THP2 are competed out of the antigen-antibody complex by purified thionin. The conversión of THP2 into thionin, which has been demonstrated in a pulse-chase experiment in vivo, is a post-translational process. As it has not been possible to detect THPl in vivo it is assumed that it is converted cotranslationally into THP2. Final deposition of thionin as an extrínsic membrane protein, possibly associated with the endoplasmic reticulum, has been tentatively established on the basis of subcellular fractionation experíments

Inhibition of eukaryotic cell-free protein synthesis by thionins from wheat endosperm

Thionins are polypeptide toxins of about 5000 molecular weight, present in the endosperms of many Gramineae, which modify membrane permeability and inhibit macromolecular synthesis in cultured mammalian cells. Evidence is presented that they inhibit in vitro protein synthesis at micromolar concentrations in cell-free systems derived from wheat germ or from rabbit reticulocytes. Inhibition seems to occur by direct binding of mRNA by the toxin, as judged by the ability of thionins to mediate retention of RNA in nitrocellulose filters and by the dependence of inhibitory concentrations on the amount of exogenous RNA added to the wheat-germ translation system. Commercial preparations of wheat-germ have been found to include some endosperm contamination (up to 15%), which may result in at least partially inhibitory concentrations of the toxin in the cell-free extracts

The CM-proteins from cereal endosperm: Immunochemical relationships

The CM-proteins, which are salt-soluble proteins that can be extracted with chloroform: methanol (2: 1, v/v), seem to be present in the endosperm of all the cereal species investigated. Antibodies raised against a mixture of the barley CM-proteins (A-H) cross-reacted with wheat and rye proteins in Ouchterlony tests and a detailed study was carried out for purified wheat (CM1, CM2. CM3. CM3') and barley (CMa, CMb, CMc, CMd) CM-proteins. [35Sl-Cysteine-labelled endosperm proteins from wheat and barley were investigated by immuno-precipitation, electrophoresis and fluorography using the antibodies (A-H) and also those to a mixture of wheat CM-proteins and to CMd. There was complete antigenic identity for all the wheat proteins and CMd, some of the other proteins showed partial antigenic identity. Previously proposed genetic and biochemical relationships among these proteins were confirmed in the present study

CM-proteins and thionins in cereals: characterization and cloning of cDNA

The study of cereal albumins and globulins has lagged somewhat behind that of the prolamins, which nave been considered as typical reserve proteins. However, these protein fractions merit closer attention for a variety of reasons. The main individual albumins and globulins are at least as abundant as many prolamin components, and it can be speculated that in a tissue, such as the cereal endosperm, which is completely consumed during germination, al 1 abundant proteins may play a reserve role. They have also a higher proportion of essential amino acids, as compared with the prolamins, and thus may be releyant in connec^ tion with the genetic alteration of overall grain composition. Finally, a high proportion of the main components of these protein fractions have inhibitory and even toxic properties, which may be related to the protection of this tissue during development and germination, and might influence the nutritional valué of the cereal products. We report here the characterization in barley of cDNA clones encoding two major groups of proteins: the CM-proteins, a family that inciudes inhibitors of trypsin and a-amylase, and the thionins, a group of high-lysine toxic polypeptides

Chromosomal location and expression of genes encoding low molecular weight proteins in wheat and related species

A review of recent work on the chromosomal location and expression of genes encoding moderately abundant, presumably non-storage endosperm proteins from wheat, barley and rye is presented. Related aspects, such as regulatory genetic effects, in vivo and in vitro synthesis, types of processing, deposition sites, and molecular cloning are also discussed. The relevance of these studies in connection with basic endosperm biology, genetic manipulation of quality and agronomic traits, and the evolution of these important crops is briefly emphasized

Characterization and analysis of thionin genes

The general designation of thionins has been proposed for a family of homologous proteins that have been isolated from different tissues in a wide range of plant taxa and have been variously named purothionins, viscotoxins, crambins, etc. (see Garcia-Olmedo et al., 1989). The possible involvement of thionins in plant defense was first suggested, on the basis of their in vitro toxicity to plant pathogens, by Fernandez de Caleya et al., (1972). Those observations had been prompted by earlier reports concerning the antimicrobial properties of these polypeptides (Stuart and Harris, 1942; Balls and Harris, 1944). Work on the thionins, which has been actively pursued over the past half-century, has been recently reviewed in detail (Garcia-Olmedo et al., 1989). For this reason, earlier work will only be partially summarized in the present chapter, which will focus on recent developments concerning thionin genes and their potential role in plant defense mechanisms

The thionins: a protein family that includes purothionins, viscotoxins and crambins

Almost half a century ago, a crystalline protein material was obtained from lipid extracts of wheat endosperm and designated "purothionin" (Trvpwo, wheat; 9 e i w v , sulphur) on account of its high sulphur content (Balls et al., 1942a,b). This material, which was thought to be the oxidized form of a powerful oxidation-reduction system, was found to have baetericidal and fungicidal properties (Stuart and Harris, 1942), to inhibit fermentation of wheat mashes (Balls and Harris, 1944), and to be toxic to laboratory animáis (Coulson et al., 1942)