Thionin genes specifically expressed in barley leaves

Complementary-DNA (cDNA) clones encoding thionin were identified as one of the most frequent types of clones in a cDNA library constructed from total polyadenylated RNA from young barley leaf cells. One full-length clone codes for a precursor protein that starts with a signal peptide (28 amino acids) followed by the mature thionin (46 amino acids) and terminated by a long acidic extension (63 amino acids). The amino-acid sequence of the leaf thionin is 52% homologous to thionins from barley endosperm and in the C-terminal extension the homology decreases to 41%. In contrast, the leaf thionin is 72% homologous to viscotoxin from mistletoe leaves. Leaf thionin is coded by a multigene family with an estimated nine to eleven genes and analysis of the cDNA clones showed that at least two extremely homologous genes are expressed. Northern hybridization experiments indicate that the leaf thionin genes are not expressed in endosperm and roots. In leaves, the expression of the thionin genes is strongly repressed by light.Abbreviations cDNA complementary DNA - poly(A)RNA polyadenylated RNA     

cDNA-derived identification of novel thionin precursors in Viscum album that contain highly divergent thionin domains but conserved signal and acidic polypeptide domains

The existence of new thionin variants in Viscum album has been deduced from cDNA sequences. Unlike the viscotoxins and related thionins previously found in different members of the Viscaceae, these novel thionins contain eight rather than six cysteine residues. In this respect they resemble thionins described previously from various cereals and from Pyrularia pubera, which also contain eight cysteine residues at identical positions. All of the new thionins of V. album are encoded as higher-molecular-weight precursors consisting of a signal peptide, a thionin domain and an acidic polypeptide domain. While the deduced amino acid sequences of the thionin domains of different precursor molecules are highly divergent, the two other domains are conserved among all of the variants and are distinct from the corresponding domains of thionin precursors of other plant species.     

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.     

Organ-specific expression of highly divergent thionin variants that are distinct from the seed-specific crambin in the crucifer Crambe abyssinica

Most thionins of higher plants are toxic to various bacteria, fungi, and animal and plant cells. The only known exception is the seed-specific thionin, crambin, of the crucifer Crambe abyssinica. Crambin has no net charge, is very hydrophobic and exhibits no toxicity. In the present work, the organization of the crambin precursor polypeptide was deduced from cDNA sequences. The precursor shows a domain structure similar to that of the preproprotein of other thionins, which contains a signal peptide, a thionin domain and a C-terminal amino acid extension. Unlike the thionin precursors studied thus far, both the thionin domain and the C-terminal amino acid extension of the crambin precursor have no net charge and are hydrophobic, thus facilitating their interaction, by analogy to that proposed for the corresponding domains of other thionin precursors that have positive and negative charges. The existence of a large number of novel and highly variable thionin variants in Crambe abyssinica has been deduced from cDNA sequences that were amplified by the polymerase chain reaction (PCR) from RNA of seeds, leaves and cotyledons. While the deduced amino acid sequences of the thionin domains of most of these thionin precursor molecules are highly divergent, the two other domains are conserved. Most of the predicted thionin variants are positively charged. The presence of positively charged residues in the thionin domains consistently correlates with the presence of a negatively charged residue in the C-terminal amino acid extension of the various thionin precursors. The different thionin variants are encoded by distinct sets of genes and are expressed in an organ-specific manner.     

Organ-specific expression of highly divergent thionin variants that are distinct from the seed-specific crambin in the crucifer Crambe abyssinica

Most thionins of higher plants are toxic to various bacteria, fungi, and animal and plant cells. The only known exception is the seed-specific thionin, crambin, of the crucifer Crambe abyssinica. Crambin has no net charge, is very hydrophobic and exhibits no toxicity. In the present work, the organization of the crambin precursor polypeptide was deduced from cDNA sequences. The precursor shows a domain structure similar to that of the preproprotein of other thionins, which contains a signal peptide, a thionin domain and a C-terminal amino acid extension. Unlike the thionin precursors studied thus far, both the thionin domain and the C-terminal amino acid extension of the crambin precursor have no net charge and are hydrophobic, thus facilitating their interaction, by analogy to that proposed for the corresponding domains of other thionin precursors that have positive and negative charges. The existence of a large number of novel and highly variable thionin variants in Crambe abyssinica has been deduced from cDNA sequences that were amplified by the polymerase chain reaction (PCR) from RNA of seeds, leaves and cotyledons. While the deduced amino acid sequences of the thionin domains of most of these thionin precursor molecules are highly divergent, the two other domains are conserved. Most of the predicted thionin variants are positively charged. The presence of positively charged residues in the thionin domains consistently correlates with the presence of a negatively charged residue in the C-terminal amino acid extension of the various thionin precursors. The different thionin variants are encoded by distinct sets of genes and are expressed in an organ-specific manner.     

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.     

Extreme divergence of a novel wheat thionin generated by a mutational burst specifically affecting the mature protein domain of the precursor.

A new type of neutral thionin (type V), specifically expressed in developing wheat endosperm, has been found to be encoded by a set of single-copy genes located in the long arms of chromosomes 1A, 1B and 1D, within less than 10,000 base-pairs of those corresponding to the highly basic type-I thionins. Divergence between types I and V has occurred through a process of accelerated evolution that has affected the amino acid sequence of the mature thionin but not the precursor domains corresponding to the N-terminal signal peptide and the long C-terminal acidic peptide. This process involved a deletion and a non-synonymous nucleotide substitution rate equal to the synonymous rate in the thionin sequence.     

Isolation and characterization of cDNAs coding for leaf-specific thionins closely related to the endosperm-specific hordothionin of barley (Hordeum vulgare L.)

Summary In etiolated barley seedlings a highly abundant mRNA encoding a 15 000 M_r polypeptide is present whose concentration rapidly declines upon illumination. The amino acid sequence of the 15 000 M_r polypeptide has been deduced from cDNA sequences and the polypeptide has been identified as a high-molecular-weight thionin precursor. Closely related thionins, most of them highly toxic, have been described previously in several higher plants. In cereals the occurrence of these thionins has been thought to be confined to the seeds. Our present data demonstrate that, in additon to endosperm-specific hordothionin mRNA, a closely related but distinct second group of thionin mRNAs is present in the barley leaf during early seedling development. Since the appearance of the bordothionin mRNA is not controlled by light, two different gene families for thionins exist whose expression seems to be under a different mode of developmental control.Dedicated to Prof. W. Halbsguth on the occasion of his 75th birthday     

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.     

The A- and B-chains of carboxypeptidase I from germinated barley originate from a single precursor polypeptide

Carboxypeptidase I from germinated barley (Hordeum vulgare) grain consists of two peptide chains linked by disulfides; the A- and B-chains contain 266 and 148 amino acid residues, respectively (Sorensen, S. B., Breddam, K., and Svendsen, I. (1986) Carlsberg Res. Commun. 51, 475-485). A cDNA library prepared from mRNA isolated from scutella of 2-day germinated barley has now been screened with a mixed oligonucleotide encoding a peptide fragment of the A-chain. Nucleotide sequence analysis of a 1443-nucleotide pair cDNA clone revealed that both chains of the enzyme are translated from a single mRNA. The coding region of the A-chain is located at the 5'-end of the cDNA and is separated from the B-chain coding region by a 165-nucleotide pair linking region. The B-chain coding region is followed by a stop codon, a 187-nucleotide pair 3'-untranslated sequence, and a short polyadenylic acid tail. The results indicate that the A- and B-chains of barley carboxypeptidase I arise by endoproteolytic excision of a 55-residue linker peptide from a single precursor polypeptide chain. The putative linker peptide is rich in proline, lysine, and arginine residues, has an apparent pI of 11.9, and appears to be excised by cleavage of peptide bonds on the COOH-terminal side of serine residues.     

Isolation and Characterization of a Thionin Proprotein-processing Enzyme from Barley

Thionins are plant-specific antimicrobial peptides that have been isolated from the endosperm and leaves of cereals, from the leaves of mistletoes, and from several other plant species. They are generally basic peptides with three or four disulfide bridges and a molecular mass of ∼5 kDa. Thionins are produced as preproproteins consisting of a signal peptide, the thionin domain, and an acidic domain. Previously, only mature thionin peptides have been isolated from plants, and in addition to removal of the signal peptide, at least one cleavage processing step between the thionin and the acidic domain is necessary to release the mature thionin. In this work, we identified a thionin proprotein-processing enzyme (TPPE) from barley. Purification of the enzyme was guided by an assay that used a quenched fluorogenic peptide comprising the amino acid sequence between the thionin and the acidic domain of barley leaf-specific thionin. The barley TPPE was identified as a serine protease ({"type":"entrez-protein","attrs":{"text":"BAJ93208","term_id":"326526063","term_text":"BAJ93208"}}BAJ93208) and expressed in Escherichia coli as a strep tag-labeled protein. The barley BTH6 thionin proprotein was produced in E. coli using the vector pETtrx1a and used as a substrate. We isolated and sequenced the BTH6 thionin from barley to confirm the N and C terminus of the peptide in planta. Using an in vitro enzymatic assay, the recombinant TPPE was able to process the quenched fluorogenic peptide and to cleave the acidic domain at least at six sites releasing the mature thionin from the proprotein. Moreover, it was found that the intrinsic three-dimensional structure of the BTH6 thionin domain prevents cleavage of the mature BTH6 thionin by the TPPE.     

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     

Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli

Thionins are antimicrobial plant peptides produced as preproproteins consisting of a signal peptide, the thionin domain, and a so-called acidic domain. Only thionin itself has been isolated from plants. To study the processing of the precursor, it has to be produced in a heterologous system. Since both domains contain several cysteines and, due to the known antimicrobial activity of the thionin, we tested the expression of all four Arabidopsis proproteins as fusion proteins. Periplasmic expression as fusion with maltose binding protein was not successful but cytoplasmic expression as His-tagged TRX fusion proteins with a TEV recognition sequence resulted in proteins of correct size. Use of the SHuffle strain C3030 further improved the expression. Fusion proteins inhibited growth of Escherichia coli. They could be cleaved by TEV protease, releasing authentic proproteins without any additional amino acid at the N-terminus.     

Cloning and nucleotide sequence of a cDNA encoding the precursor of the barley toxin alpha-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 alpha-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 Ris? 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 level.     

A new class of N-hydroxycinnamoyltransferases. Purification,cloning, and expression of a barley agmatine coumaroyltransferase (EC 2.3.1.64)

Agmatine coumaroyltransferase (ACT), which catalyzes the first step in the biosynthesis of antifungal hydroxycinnamoylagmatine derivatives, was purified to apparent homogeneity from 3-day-old etiolated barley (Hordeum vulgare L.) seedlings. The enzyme was highly specific for agmatine as acyl acceptor and had the highest specificity for p-coumaroyl-CoA among various acyl donors with a specific activity of 29.7 nanokatal x mg(-1) protein. Barley ACT was found to be a single polypeptide chain of 48 kDa with a pI of 5.20 as determined by isoelectric focusing. The 15 N-terminal amino acid residues were identified by micro-sequencing of the native protein and were used to clone a full-length barley ACT cDNA that predicted a protein of 439 amino acid residues. The sequence was devoid of N-terminal signal peptide, suggesting a cytosolic localization of barley ACT. Recombinant ACT produced and affinity-purified from Escherichia coli had a specific activity of 189 nanokatal x mg(-1) protein, thus confirming the identity of the purified native protein. A partial cDNA sequence for ACT was obtained from wheat that predicted a protein of 353 amino acid residues and had 95% sequence identity to barley ACT. Two motifs in the amino acid sequence reveal that barley ACT represents a new class of N-hydroxycinnamoyltransferases belonging to the transferase superfamily. The barley ACT is unique in producing the precursor of hordatine, a proven antifungal factor that may be directed toward Blumeria graminis.     

Abnormal germling development by brown rust and powdery mildew on cer barley mutants

The barley leaf rust fungus forms appressoria over host leaf stomata and penetrates via the stomatal pore. High levels of avoidance to leaf rust fungi have been described in some wild accessions of Hordeum species where a prominent wax layer on the stomata inhibits triggering of fungal appressorium differentiation. Leaf rust avoidance has not yet been found in H. vulgare. Since cuticular leaf waxes are implicated in the avoidance trait, we screened 27 eceriferum (cer) mutant lines of H. vulgare for avoidance to barley leaf rust. These mutations affect leaf waxes. Reduction in numbers of germ tubes forming appressoria over stomata was found in some lines, but the greatest reduction (ca 30%) was less than previously found in wild barley spp. or in an accession of H. chilense used here as a check. In one line (cer-zh654), avoidance was due to a combination of factors. Firstly, fewer germ tubes oriented towards stomata and so failed to contact them. Secondly, some germ tubes that encountered stomata did not form appressoria but over-grew them. In this line, therefore, the fungus tended to fail both to locate and to respond to stomata. The appressoria of barley powdery mildew form on leaf epidermal cells that they penetrate directly. On certain cer lines, a proportion of germlings of the barley powdery mildew fungus developed abnormally, suggesting that germlings failed to recognise and/or respond to the leaf surface waxes on these mutants.     

Organization and expression of two tandemly oriented genes encoding ribulosebisphosphate carboxylase/oxygenase activase in barley

We have isolated and structurally characterized genomic DNA and cDNA sequences encoding ribulose-1,5-bisphosphate carboxylase/oxygenase (Rbu-P2 carboxylase) activase from barley (Hordeum vulgare L.). Three Rbu-P2 carboxylase activase (Rca) polypeptides are encoded in the barley genome by two closely linked, tandemly oriented nuclear genes (RcaA and RcaB); cDNAs encoding each of the three Rbu-P2 carboxylase activase polypeptides were isolated from cDNA libraries of barley leaf mRNA. RcaA produces two mRNAs, which encode polypeptides of 42 and 46 kDa, by an alternative splicing mechanism identical to that previously reported for spinach and Arabidopsis Rca genes (Werneke, J.M., Chatfield, J.M., and Ogren, W. L. (1989) Plant Cell 1, 815-825). RcaB is transcribed to produce a single mRNA, which encodes a mature peptide of 42 kDa. Genomic Southern blots indicate that RcaA and RcaB represent the entire Rbu-P2 carboxylase activase gene family in barley. The genes share 80% nucleotide sequence identity, and the 42-kDa polypeptides encoded by RcaA and RcaB share 87% amino acid sequence identity. Coding regions of the two barley Rca genes are separated by 1 kilobase pair of flanking DNA. DNA sequence motifs similar to those thought to control light-regulated gene expression in other nuclear-encoded plastid polypeptide genes are found at the 5' end of both barley Rca genes. Probes specific to three mRNAs were used to determine the relative contribution each species makes to the total Rca mRNA pool.