John E. Nielsen

Characterization of a new antifungal chitin-binding peptide from sugar beet leaves

The intercellular washing fluid (IWF) from leaves of sugar beet (Beta vulgaris L.) contains a number of proteins exhibiting in vitro antifungal activity against the devastating leaf pathogen Cercospora beticola (Sacc.). Among these, a potent antifungal peptide, designated IWF4, was identified. The 30-amino-acid residue sequence of IWF4 is rich in cysteines (6) and glycines (7) and has a highly basic isoelectric point. IWF4 shows homology to the chitin-binding (hevein) domain of chitin-binding proteins, e.g. class I and IV chitinases. Accordingly, IWF4 has a strong affinity to chitin. Notably, it binds chitin more strongly than the chitin-binding chitinases. A full-length IWF4 cDNA clone was obtained that codes for a preproprotein of 76 amino acids containing an N-terminal putative signal peptide of 21 residues, followed by the mature IWF4 peptide of 30 residues, and an acidic C-terminal extension of 25 residues. IWF4 mRNA is expressed in the aerial parts of the plant only, with a constitutive expression in young and mature leaves and in young flowers. No induced expression of IWF4 protein or mRNA was detected during infection with C. beticola or after treatment with 2,6-dichloroisonicotinic acid, a well-known inducer of resistance in plants.

Pectin methyl esterase from orange fruit: characterization and localization by in-situ hybridization and immunohistochemistry

Abstract. Pectin methyl esterase (PME) from orange (Citrus sinensis L.) fruit peels has been purified by ammonium sulphate precipitation, and ion-exchange and gel-filtration chromatography. Characterization of the enzyme revealed a 36-kDa protein with an isoelectric point >9, a pH optimum at 7 and temperature optimum at 50 °C. The substrate specificity and kinetic experiments showed that the affinity of PME for pectin was highly dependent on the degree of esterification (DE) of the pectin, with Km values of 0.7 mg ml-1 for pectin with a DE of 70% and 17 mg ml-1 for pectin with a DE of 25%. The sequences of the NH2-terminal end of digested peptides from the mature protein were obtained. A DNA fragment of 501 bp was cloned by polymerase chain reaction amplification using degenerate primers and was further used for screening of a cDNA library. Two cDNA clones were isolated encoding PMEs of 584 amino acids and 362 amino acids, respectively, including a putative signal peptide. The deduced amino acid sequence showed full identity to the sequenced peptides. Polyclonal antibodies raised against orange peel PME were used for immunohistochemistry. The main localization of PMEs was in the outer cell layers of the juice vesicles, in the outer cell layers of the lamellae between the segments and in the inner cell layers of the albedo in the peel. In-situ hybridization showed that the mRNA is very abundant in the fruit and was found in the same cell layers as the native enzyme. A very intensive staining for PME mRNA was also seen in the core and in the flavedo close to the oil glands.

Detection, expression and specific elimination of endogenous β-glucuronidase activity in transgenic and non-transgenic plants

Abstract With the aim of making a system using the GUS gene as a selection gene, the effects of two toxin glucoronides were tested on transgenic tobacco cells ( Nicotiana tabacum L.) containing a β-glucuronidase (GUS) gene from Escherichia coli and on non-transgenic cells. No significant difference in toxicity was observed between transgenic and non-transgenic cells. We found that this most probably was due to the activity of an endogeneous GUS enzyme, which could be detected in all plant species tested, e.g. tobacco, sugar beet ( Beta vulgaris L.), oilseed rape ( Brassica napus L.), pea ( Pisum sativum L.), wheat ( Triticum sativum L.) and rhubarb ( Rheum rhaponticum L.) This indicates that GUS may be ubiquitous to plants contrary to earlier assmptions. The endogenous enzyme is active at pH 4–5 and the activity is eliminated without reducing the introduced GUS activity when pH is elevated. In addition the endogenous GuS can be selectively inhibited at high temperatures. Modifications according to these findings can be employed in standard GUS assays to avoid misinterpretations when the expression of tissue specific promoters is tested.

New antifungal proteins from sugar beet (Beta vulgaris L.) showing homology to non-specific lipid transfer proteins

Two novel, nearly identical antifungal proteins, IWF1 and IWF2, were isolated from the intercellular washing fluid (IWF) of sugar beet leaves. The proteins were purified to homogeneity and their amino acid sequences were determined. They are basic, monomeric proteins of 91 amino acid residues, 89 of which are identical. Both proteins show strongin vitro antifungal activity againstCercospora beticola, the casual agent of leaf spot disease in sugar beet. Based on primary sequence homology, including the presence of 8 conserved cysteine residues, IWF1 and IWF2 are related to the family of plant non-specific lipid transfer proteins (nsLTPs). Antibodies were raised against IWF2 after conjugation to diphtheria toxoid. The amino acid sequence data was used to generate a polymerase chain reaction (PCR) clone, employed for the isolation of a cDNA clone encoding a closely related isoform IWFA, which differs from IWF1 by two amino acid substitutions only. The induction and subcellular localization of these proteins were studied by western and northern blotting analyses after treatment with 2,6-dichloroisonicotinic acid (INA), a compound capable of inducing resistance againstC. beticola, and after fungal infection. The following observations were made: (1) the proteins were present in leaves of non-INA-treated and uninfected control plants, (2) they were only slightly induced by INA treatment and during infection withC. beticola, and (3) they were present both intra- and extracellularly. However, their strong antifungal potentials together with immunohistological investigations, the proteins accumulating in contact with the fungus and in autolysing cells, suggested a role of these proteins in plant defence. Finally, immunohistology revealed a remarkable expression pattern of the IWF1 and IWF2 proteins, or serologically related proteins, in sugar beet styles, in that single or a few scattered papillae and a few cells in the lower transmitting tissue strongly and specifically reacted with the antibody.

Distribution of pectin methyl esterase and acetylesterase in the genus Citrus visualized by tissue prints and chromatography

The distribution of pectin methyl esterase (PME) and acetylesterase (AE) in orange, lime, lemon, grapefruit and clementine fruits has been investigated. Isoforms from PME and AE have been separated by cation exchange chromatography. PME isoforms separated in two main groups for all citrus extracts. For AE isoforms, only one major AE isoform was seen in orange, whereas several isoforms were separated in extracts from grapefruit, lime and lemon. The overall localization of AE and PME in the different citrus fruits was investigated by tissue print immunolocalization. In orange, grapefruit and clementine, PME was immunolocalized in the juice vesicles, whereas the PME localization in the juice vesicles was significantly weaker in lemon and lime. The distribution of AE in the juice vesicles differed from the distribution of PME in the sense that PME was more restricted to the periphery of the juice vesicles, whereas AE was also found in the more central parts of the juice vesicles. The immunological detection indicated that PME is involved in the ripening process, but this is probably not the only function for AE. The distribution of AE in paraffin sections of developing lime fruits showed that AE was present in almost all parts of the fruit deposited in the cell wall and intracellularly.

Isolation and expression of two cDNA clones encoding UDP-galactose epimerase expressed in developing seeds of the endospermous legume guar

Abstract UDP-galactose 4′-epimerase (UDPG epimerase) catalyses the reversible conversion of UDP- d -glucose to UDP- d -galactose. This compound is a precursor for the biosynthesis of various galactosides and cell wall polymers, including galactomannan which is the main storage polysaccharide in endospermous legumes. Using functional complementation of a UDPG epimerase deficient Escherichia coli mutant (PL-2) by a cDNA expression library from immature guar ( Cyamopsis tetragonoloba ) seeds, galactose metabolising colonies with UDPG epimerase activities comparable to wild type level were obtained. Two cDNA clones (GEPI42 and GEPI48) encoding two different UDPG epimerases were isolated. Re-transformation of PL-2 by plasmid DNA, isolated from either of the two clones, resulted in numerous galactose-metabolising colonies, all with high UDPG epimerase activities. GEPI42 and GEPI48 encoded proteins with 354 and 350 amino acid residues, respectively, corresponding to deduced molecular weights of 39 286 and 38 373 Dalton, respectively. The amino acid sequence identity was 66.9%. Southern analysis of genomic guar DNA confirmed the origin and distinctness of the two UDPG epimerase genes. Analysis by immunohistochemistry showed the presence of significant levels of UDPG epimerase antigen in the endosperm of immature seeds with rapid galactomannan biosynthesis. In the endosperm of seeds close to maturity where galactomannan deposition has ceased, no antigen was detected. These data indicate that one or both of the two cloned UDPG epimerase genes are expressed in guar endosperm at a developmental stage where galactomannan biosynthesis occurs, suggesting that one or both may be involved in this process.

Immunohistological localization of a basic class IV chitinase in Beta vulgaris leaves after infection with Cercospora beticola

Abstract The subcellular distribution of a basic class IV chitinase (Ch4) in Cercospora beticola -infected sugar beets has been studied by immunohistological analysis. Ch4 was primarily deposited in extracellular globuli proximal to the necrosis. High levels of Ch4 were observed in the vicinity of the necrotic lesions, low levels were found at distant sites from the necrosis. This plant-pathogen interaction thus gives rise to a local induction of Ch4, whereas no apparent systemic induction of Ch4 was found. After infection with Cercospora , a tolerant sugar beet cultivar accumulated Ch4 faster than a susceptible one, and the protein was spatially distributed over a comparatively larger area in the tolerant variety. In the tolerant beet cultivar symptoms appear a few days later, and the number of necroses are reduced and smaler than in the sensitive cultivar. In uninfected sugar beet leaves, Ch4 is present in low amounts in epidermis, stomata and xylem cells. A specific autofluorescence and a high endogenous β-glucuronidase activity in Cercospora were used to demonstrate that this pathogen did not penetrate the tissue beyond the necrosis.

Isolation, characterization and immuno localization of orange fruit acetyl esterase

Abstract Acetyl esterase (AE) has been purified to homogeneity from orange peels. The purification steps included cation exchange chromatography and gel filtration. The enzyme has affinity for triacetin and sugar beet pectin with K M of 39 mM and K M of 26 mg/ml, respectively. AE has a MW of 42 kD and is a monomer. The isoelectric point is at pH > 9. Immuno localization using polyclonal antibodies raised against AE showed that AE was widely distributed in orange fruit but with more intensive immunological detection in the outer part of the peels e.g. albedo and flavedo and in the segments (juice vesicles). The results indicate that AE is located at the site where the major fraction of pectin is deposited.

α-1,4-Glucan lyase-producing endophyte of Gracilariopsis sp. (Rhodophyta) from China

Purification of α-1,4-glucan lyase from red algae and fungi has previously been reported. The α-glucan lyase converts α-glucans to 1,5- anhydro-d-fructose. In this study, an endophyte was detected between the algal cells of Gracilariopsis sp., but not penetrating the cell walls. Histological staining was consistent with the endophyte being fungal and immunohistochemistry revealed that it possessed an α-1,4-glucan lyase differing from that of the host. The endophyte α-glucan lyase was recognized by antisera against both fungal and red algal α-glucan lyases, whereas the α-glucan lyase found in the algal tissue was recognized only by the antisera against algal α-glucan lyase. The lyases occur independently in the host and in the endophyte, and were sometimes detected simultaneously. The endophyte was only observed in Gracilariopsis sp. collected in China; no endophyte could be detected in Gracilariopsis sp. from California or in Gracilaria chilensis.