The defensive role of nonspecific lipid-transfer proteins in plants.

Plant nonspecific lipid-transfer proteins stimulate the transfer of a broad range of lipids between membranes in vitro. In view of their ability to inhibit bacterial and fungal pathogens, their distribution at high concentrations over exposed surfaces and in the vascular system, and the response of Ltp-gene expression to infection with pathogens, they are now thought to be active plant-defense proteins.

Genetic control of sterol esterification in developing wheat endosperm.

1. The action of gene Pln, previously characterized by the sterol ester patterns of mature whole wheat kernels, has been found to be restricted to the endosperm and not to affect the embryo, the pericarp or the seed coat. 2. The dominant allele Pln, which determines a sterol ester pattern with palmitate as the main ester, is also responsible for a low level of free sterol at maturity. A high level of free sterol is associated with the recessive allelet pln, which determines an ester pattern with linoleate as the main ester. 3. Divergence between the two phenotypes starts at about 21 days after anthesis, when cell proliferation has been completed, the aleurone layer has differentiated, and only cell enlargement is taking place. A marked increased in esterification, mainly by palmitate, which is controlled by the dominant allele, is concomitant with a sharp decrease in free sterol. 4. The increased net esterification is non-specific with respect to 4-demethyl sterols, because it affects the four main ones, namely sitosterol, stigmasterol, campesterol and cholesterol.

The barley alpha-thionin promoter is rich in negative regulatory motifs and directs tissue-specific expression of a reporter gene in tobacco.

The promoter of the barley alpha-thionin gene (1.6 kb) fused to the beta-glucuronidase (GUS) gene directs temporally-controlled, tissue-specific expression in the endosperm of transgenic tobacco. The nucleotide sequence of this promoter shows negative regulatory motifs which have been functionally analyzed in other genes.

New alpha-amylase and trypsin inhibitors among the CM-proteins of barley (Hordeum vulgare).

Barley CM-proteins are a group of at least five salt-soluble components (CMa-e) that can be selectively extracted from endosperm with chloroform/methanol mixtures. N-terminal sequences of proteins CMa, CMb and CMc have been determined and found to be homologous to those previously determined for CMd and CMe, an observation which confirms that their structural genes are members of a dispersed multi-gene family. The purified CM-proteins were tested against trypsin and against alpha-amylases from saliva, pancreas, Aspergillus oryzae, Tenebrio molitor and barley. Besides CMe, which was known to be a trypsin inhibitor, CMc also showed antitrypsin activity, whereas CMa was specifically active against the alpha-amylase from T. molitor and no inhibitory activity was found for proteins CMb and CMd. The evolutionary implications of these findings are discussed.

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.

Control of galactosyl diglycerides in wheat endosperm by group 5 chromosomes.

Lower levels of monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) have been found in tetraploid wheats as compared with those in hexaploid wheats. The same difference has been found between hexaploid cultivars and tetraploid lines derived from them by D genome extraction. A lower level of MGDG and DGDG is also present in Triticum carthlicum (AABB) as compared with Aegilops squarrosa (DD) or with the synthetic T. spelta (AABBDD) obtained from them. Analysis of the appropriate nullitetrasomic and ditelosomic lines indicates that a gene or genes located in the short arm of chromosome 5D are responsible for the observed difference and that group 5 chromosomes can be ranked as to their influence on the MGDG and DGDG levels in the order 5B > 5D > 5A and 5D > 5B > 5A, respectively. These results further support our previous identification of DGDG as the lipid factor responsible for petroleum ether solubility of lipopurothionins. Since DGDG contributes to baking quality by improving the retention of fermentation gases, the present observations imply that the difference in bread-making quality between the two types of wheat is not due only to proteins contributed by the D genome.

Differential Allelic Expression at a Locus Encoding an Endosperm Protein in Tetraploid Wheat (TRITICUM TURGIDUM).

Two hydrophobic endosperm proteins, designated CM3 and CM3', have been purified from appropriate cultivars of tetraploid wheat (T. turgidum) and characterized. They are inherited as though encoded by alleles at a single locus, designated Cm3a and Cm3b, respectively. The net amount of protein molecules has been measured for each of the alleles at one, two and three doses. The amount of CM3' is 50%-65% of that found for CM3. For both, there is a linear gene dosage response. These effects were observed not only in the parental material and the reciprocal F(1) generations, but also in the segregating F(2) generation, indicating that the quantitative difference depends on differences in the structural gene or is controlled by regulatory or modifier gene(s) linked to it.

Gene expression in alloploids: genetic control of lipopurothionins in wheat.

Lipopurothionins are complexes of basic polypeptides and polar lipids found in petroleum ether extracts of wheat endosperm. Location of the structural genes for the protein moiety and of genes probably controlling the lipid moiety has been achieved by analysis of compensated nulli-tetrasomic and ditelosomic lines of Triticum aestivum L. cv. Chinese Spring, as well as of other genetic stocks. There are two electrophoretic variants of the apoprotein designated alpha and beta purothionins. Structural genes for alpha purothionins are located in the long arm of chromosomes 1B and 1D, and for the beta variant in the long arm of 1A. These genes have been tentatively designated Pur-A1, Pur-B1, and Pur-D1. The aminoacid composition of purified alpha and beta purothionins from Triticum aestivum (genomes AABBDD) and T. durum (AABB), and of beta purothionin from T. monococcum (AA) is also consistent with this conclusion and suggests that the alpha purothionin encoded by gene Pur-B1 probably differs from that encoded by gene Pur-D1 in at least three positions of the aminoacid sequence. A gene (or genes) located in the short arm of chromosome 5D markedly affects the level of lipopurothionin but does not affect apoprotein synthesis. It is concluded that they control the lipid moiety which is required for solubility in petroleum ether.

Snakin-1, a peptide from potato that is active against plant pathogens.

A new type of antimicrobial peptide, snakin-1 (SN1), has been isolated from potato tubers and found to be active, at concentrations < 10 microM, against bacterial and fungal pathogens from potato and other plant species. The action of SN1 and potato defensin PTH1 was synergistic against the bacterium Clavibacter michiganensis subsp. sepedonicus and additive against the fungus Botrytis cinerea. Snakin-1 causes aggregation of both gram-positive and gram-negative bacteria. The peptide has 63 amino acid residues (M(r) 6,922), 12 of which are cysteines, and is unrelated to any previously isolated protein, although it is homologous to amino acid sequences deduced from cloned cDNAs that encode gibberellin-inducible mRNAs and has some sequence motifs in common with kistrin and other hemotoxic snake venoms. A degenerate oligonucleotide probe based on the internal sequence CCEECKC has been used to clone an SN1 cDNA. With the cDNA used as probe, one copy of the StSN1 gene per haploid genome has been estimated and expression of the gene has been detected in tubers, stems, axillary buds, and young floral buds. Expression levels in petals and carpels from fully developed flowers were much higher than in sepals and stamens. The expression pattern of gene StSN1 suggests that protein SN1 may be a component of constitutive defense barriers, especially those of storage and reproductive plant organs.

Relative effects on virulence of mutations in the sap, pel, and hrp loci of Erwinia chrysanthemi.

We constructed strains of Erwinia chrysanthemi EC16 with multiple mutations involving three virulence systems in this bacterium, namely pel (coding for the major pectate lyases pelABCE), hrp (hypersensitive response and pathogenicity), and sap (sensitivity to antimicrobial peptides). The relative effects on virulence of those mutations have been analyzed on potato tubers and chicory leaves. In potato tubers, the sap mutation (BT105) had a greater effect in the reduction of the virulence than the pel (CUCPB5006) and hrp (CUCPB5039) mutations. This reduction was similar to that observed in the pel-hrp double mutant (CUCPB5037). The analysis of the strains affected in Pel-Sap (BT106), Hrp-Sap (BT107), and Pel-Hrp-Sap (BT108) suggested that the effects of these mutations are additive. In chicory leaves, the mutation in the sap locus appeared to have a greater effect than in potato tubers. The competitive indices of strains BT105, UM1005 (Pel-), CUCPB5039, and CUCPB5037 have been estimated in vivo and in vitro. These results indicate that the mutation in the hrp locus can be complemented in vivo by coinfection, whereas the mutations in pel and sap cannot.

Evidence against a direct antimicrobial role of H2O2 in the infection of plants by Erwinia chrysanthemi.

We have investigated the role of bacterial resistance to oxidative stress in pathogenesis. The oxyR gene from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It is closely related to that found in Escherichia coli (88% overall amino acid identity). An E. chrysanthemi oxyR mutant strain was constructed by marker exchange. After induction with a sublethal dose of H2O2, this mutant was more sensitive to H2O2 and showed reduced levels of catalase and glutathione reductase activities, compared with the wild type. The oxyR mutant was unable to form individual colonies on agar plates unless catalase was added exogenously. However, it retained full virulence in potato tubers and tobacco leaves. These results suggest that the host-produced H2O2 has no direct antimicrobial effect on the interaction of E. chrysanthemi with the two plant species.

Linked sucrose synthase genes in group-7 chromosomes in hexaploid wheat (Triticum aestivum L.).

A cDNA library from developing wheat endosperm was screened for sucrose-synthase clones using a maize cDNA probe corresponding to the Sh1 locus under non-stringent conditions. Five positive clones were isolated and initially classified into two types on the basis of their relative ability to hybridize with the probe and of their partial restriction maps. Determination of the nucleotide sequences indicated homology between the two types of wheat clones, with type 1 showing higher homology to the maize Sh1 locus than to type-2 sequences. The inserts cloned in plasmids pST8 (type 1) and pST3 (type 2) were used as probes to determine the chromosomal locations of the two types of genes. DNAs from compensated nulli-tetrasomic and ditelosomic lines of wheat cultivar Chinese Spring were cleaved with EcoRI and analysed in Southern blots. DNA segments of the two types were thus identified in the short arms of chromosomes 7A, 7D, and, possibly, 7B. The two types of linked loci have been designated Ss1 and Ss2, respectively.

Differential expression of two types of sucrose synthase-encoding genes in wheat in response to anaerobiosis, cold shock and light.

The expression of two types of sucrose synthase-encoding genes, Ss1 and Ss2, in hexaploid wheat (Triticum aestivum, L.), has been investigated using type-specific probes, corresponding to the 250-270 bp C-terminal portions of the respective cDNA clones. Both types of genes are highly expressed in developing endosperm, where the expression of the Ss2 type slightly precedes in time that of the Ss1 type. Expression of Ss genes is lower in etiolated leaves and in roots than in endosperm. In the first two tissues, the Ss1 mRNA is much more abundant than the Ss2 mRNA, and the Ss1 mRNA level sharply increases in response to anaerobiosis and to cold shock (6 degrees C), while the level of Ss2 mRNA is not significantly affected. Upon illumination of etiolated leaves, the Ss1 level mRNA decreases significantly and the Ss2 mRNA level increases.

Nucleotide sequence and endosperm-specific expression of the structural gene for the toxin alpha-hordothionin in barley (Hordeum vulgare L.).

A barley genomic library, obtained by cloning in the vector lambda EMBL-4, was screened with a cDNA probe encoding the alpha-hordothionin toxin. A positive clone, designated lambda TH1, was selected for further characterization. The coding and flanking regions of the alpha-hordothionin gene (Hth-1) were sequenced. Hth-1 has two introns of 420 and 91 nucleotides (nt), respectively. The promoter region has the following main features: one TATA box; three CATC boxes; an enhancer-like sequence, starting at nt position -282 from the first ATG codon, which is homologous to sequences appearing at similar positions in other endosperm genes; two versions of an 18-nt sequence that is more highly repeated in structural domains of several prolamin genes; two extensive regions close to the first ATG codon that are homologous to a sequence located much further upstream in the B-hordein promoter. The transcription start point was determined at nt positions -46 to -47, both by the S1 nuclease-protection and by the primer-extension assays. A maximum of 2-4 copies of the Hth-1 gene per haploid genome was determined by Southern-blot hybridization. Expression of the Hth-1 gene was detected during the cell proliferation stage of endosperm development (maximum at 13-16 days after pollinization) and was not detected in either etiolated or green coleoptiles.

A fatty-acid-binding protein from wheat kernels.

A protein of about 7 kDa (W-FABP) has been isolated from mature wheat kernels by H2O extraction and gel filtration of the extract, followed by two steps of high-performance liquid chromatography. The N-terminal amino acid-sequence has been determined up to the 28th residue and found to be identical (except for positions 4 and 5) to that deduced from a barley cDNA (EMBL X15257), which had been improperly classified as a non-specific lipid transfer protein (LTP2). Similarly with LTPs, W-FABP does bind fatty acids, but in contrast, it is not significantly homologous to LTPs, it is not recognized by LTP antibodies, it has a more acidic isoelectric point (pH < 6.8 vs. pH > 9.6), and it does not show antibiotic properties.

Lipid transfer proteins (nsLTPs) from barley and maize leaves are potent inhibitors of bacterial and fungal plant pathogens.

Four homogeneous proteins (Cw18, Cw20, Cw21, Cw22) were isolated from etiolated barley leaves by extraction of the insoluble pellet from a Tris-HCl (pH 7.5) homogenate with 1.5 M LiCl and fractionation by reverse-phase high-performance liquid chromatography. All 4 proteins inhibited growth of the pathogen Clavibacter michiganensis subsp. sepedonicus (EC50s = 1-3 x 10(-7) M) and had closely related N-terminal amino acid sequences. The complete amino acid sequences of proteins Cw18 and Cw21 were determined and found to be homologous to previously described, non-specific lipid transfer proteins from plants (32-62% identical positions). The proteins also inhibited growth of the bacterial pathogen Pseudomonas solanacearum (EC50s = 3-6 x 10(-7) M) and the fungus Fusarium solani (EC50s = 3-20 x 10(-6) M). A homologous protein from maize leaves (Cw41) was purified in a similar manner and also found to have inhibitory properties. A synergistic effect against the fungus was observed when protein Cw21 was combined with thionins. A defense role for non-specific lipid transfer proteins from plants is proposed.

Purification and antipathogenic activity of lipid transfer proteins (LTPs) from the leaves of Arabidopsis and spinach.

Two homogeneous proteins active in vitro against the bacterial pathogen Clavibacter michiganensis subsp. sepedonicus were obtained from a crude cell-wall preparation from the leaves of Columbia wild-type Arabidopsis. The N-terminal amino acid sequences of these proteins allowed their identification as lipid transfer proteins (LTP-a1, LTP-a2); the LTP1-a1 sequence was identical to that deduced from a previously described cDNA (EMBL M80566) and LTP-a2 was quite divergent (44% identical positions). These proteins were not detected in the cytoplasmic fraction by Western-blot analysis. Proteins LTP-s1 and LTP-s2 were similarly obtained from spinach leaves; LTP-s1 was 91% identical to a previously purified spinach LTP (Swiss Prot P10976), and LTP-s2 was moderately divergent (71% identical positions). About 1/3 of the total LTPs were detected in the cytoplasmic fraction from spinach by Western-blot analysis. Concentrations of these proteins causing 50% inhibition (EC-50) were in the 0.1-1 microM range for the bacterial pathogens C. michiganensis and Pseudomonas solanacearum and close to 10 microM for the fungal pathogen Fusarium solani.

Effects of thionins on beta-glucuronidase in vitro and in plant protoplasts.

Thionins cause the irreversible inactivation of beta-glucuronidase (GUS) in vitro in a dose- and time-dependent manner. The enzyme is also sensitive to externally added thionins when expressed in the cytoplasmic compartment of tobacco protoplasts transformed with the Gus gene under the 35S promoter of the cauliflower mosaic virus. In protoplasts transformed with the Gus gene fused to a signal peptide, where GUS is translocated into the lumen of the endoplasmic reticulum, the activity is significantly increased both by externally-added and by transiently-expressed thionin, suggesting that it interferes with GUS secretion.

Novel defensin subfamily from spinach (Spinacia oleracea).

Antimicrobial peptides (So-D1-7) were isolated from a crude cell wall preparation from spinach leaves (Spinacia oleracea cv. Matador) and, judged from their amino acid sequences, six of them (So-D2-7) represented a novel structural subfamily of plant defensins (group IV). Group-IV defensins were also functionally distinct from those of groups I-III. They were active at concentrations < 20 microM against Gram-positive (Clavibacter michiganensis) and Gram-negative (Ralstonia solanacearum) bacterial pathogens, as well as against fungi, such as Fusarium culmorum, F. solani, Bipolaris maydis, and Colletotrichum lagenarium. Fungal inhibition occurred without hyphal branching. Group-IV defensins were preferentially distributed in the epidermal cell layer of leaves and in the subepidermal region of stems.

Selective disulphide linkage of plant thionins with other proteins.

Thionins are shown to form disulphide linkages with other proteins. The reaction with bacterial enzymes beta-glucuronidase and neomycin phosphotransferase II could be prevented and reversed with dithiothreitol and blocked with N-ethylmaleimide. Other cysteine-rich low-molecular-weight toxic peptides from plants (LTP-3 from barley and P19 from potato) did not react as the thionins. Certain cysteine-containing proteins, such bovine serum albumin, ovalbumin and cytochrome c, reacted with thionins, while others, including carbonic anhydrase, soybean trypsin inhibitor, bovine-lung trypsin inhibitor and phosphorylase B did not. Selectivity of the reaction with a periplasmic component of the phytopathogenic bacterium Pseudomonas solanacearum was also shown.