Biological function of ;pathogenesis-related' proteins: four PR proteins of tobacco have 1,3-beta-glucanase activity

Three of the ten acidic `pathogenesis-related' (PR) proteins known to accumulate in Nicotiana tabacum cv Samsun NN reacting hypersensitively to tobacco mosaic virus, namely −O, −N and −2, have been shown to have 1,3-β-glucanase (EC 3.2.1.39) activity. By using sera raised against each protein purified to homogeneity close serological relationships have been demonstrated between the three proteins. The same specific sera cross-reacted with a basic protein which is also a 1,3-β-glucanase induced by virus infection and which can be considered as a new basic pathogenesis-related protein of tobacco. Protein PR-O and the basic 1,3-β-glucanase display about the same specific enzymatic activity, i.e. 50-fold and 250-fold higher than specific activities of proteins PR-N and -2 respectively.     

Sunflower (Helianthus annuus L.) Pathogenesis-Related Proteins (Induction by Aspirin (Acetylsalicylic Acid) and Characterization)

Sunflower leaf discs floated on a solution containing aspirin (acetylsalicylic acid) produced a set of new proteins extractable at pH 5.2 and excreted into the intercellular space. More than 80% of the proteins found in the intercellular fluids of induced leaf discs have been identified as pathogenesis-related (PR) proteins by their immunological relationship with tobacco PR proteins. Members of the four major classes of PR proteins have been characterized. Sunflower PR proteins of type 1 (PR1) and of type 3 (PR3) were found to have acidic isoelectric points, whereas the induced PR protein of type 2 (PR2) had a basic isoelectric point. Members of the type 5 PR proteins (PR5), known in tobacco as thaumatin-like proteins, showed a more complex pattern. Multiple sunflower PR5 isomers of similar molecular weight but of different isoelectric points were excreted from the cells in response to the aspirin treatment. PR2 and PR3 proteins were found at very low basal levels in untreated leaves, whereas PR1 and PR5 proteins could not be detected at all in the same extracts. Glucanase and chitinase activities were always associated with PR2 and PR3 proteins in partially purified sunflower extracts. All of these data indicate that, in response to aspirin treatment, sunflower plants produce a complete set of PR proteins characterized by an apparently exclusively extracellular localization.     

Plant chitinase/lysozyme isoforms show distinct substrate specificity and cleavage site preference towards lipochitooligosaccharide Nod signals

The ability of 16 chitinases from seven different plant species to hydrolyze a collection of several structurally related lipochitooligosaccharides (LCOs) of Rhizobium was analyzed. It was found that the enzymes differed to a large extent in their activity on different LCOs. Differences were attributed to (i) the relative activity on different LCOs as substrate (e.g. sulfated versus non-sulfated LCOs); (ii) the relative cleavage site preference on a given LCO molecule (hydrolysis of either the second, third or fourth glycosidic bond from the non-reducing end of the molecule); and (iii) the stereochemistry of the reaction (retention or inversion of the anomeric configuration). A graphic representation of the different substrate specificities resulted in a ‘fingerprint’ that is characteristic for a given enzyme or a family of related enzymes. By comparing the LCO-fingerprint of unknown enzymes with those obtained for already characterized proteins, it is possible to identify new glycosyl hydrolases. The high diversity of substrate specificity found among plant chitinases may reflect variations in the natural substrates of the enzymes, such as substitutions on the chitin moiety of fungal cell walls or, in plants, the presence of putative endogenous substrates related to LCOs.     

Substrate specificities of tobacco chitinases

Ten tobacco chitinases (1,4-N-acetyl-β-D-glucosaminide glycanhydrolase, EC 3.2.1.14) were purified from tobacco leaves hypersensitively reacting to tobacco mosaic virus. The 10 enzymes, which belong to five distinct structural classes of plant chitinases, were incubated with several potential substrates such as chitin, a β-1,4 N-acetyl-D-glucosamine (GlcNAc) polymer, chitosan (partially deacetylated chitin), chitin oligomers of variable length and bacterial cell wall. Tobacco chitinases are all endo-type enzymes that liberate oligomers from chitin and are capable of processing the chito-oligomers further at differential rates. Chitin reaction products were separated and quantified by HPLC and differential kinetics of oligomer accumulation and degradation were observed with the distinct classes of chitinases. Depending on the substrate to be hydrolysed, each isoform displayed a different spectrum of activity. For example, class I isoforms were the most active on chitin and (GlcNAc)_4–6 whereas class III basic isoforms were the most efficient in inducing bacterial lysis. Class V and class VI chitinases were shown to more readily hydrolyse chitin oligomers than the chitin polymer itself. Together, these data indicate that the 10 tobacco chitinases represent complementary enzymes which may have synergistic effects on their substrates. This paper discusses their implication in plant defense by attacking pathogen's structural components and in plant development by maturing signal molecules.     

Purification and properties of the three o-diphenol-O-methyltransferases of tobacco leaves

Three o-diphenol-O-methyltransferases (OMTs I, II and III) which catalyse the monomethylation of various o-diphenols using S-adenosyl-L-methionine as methyl donor were isolated and purified about 210-, 70-, and 70-fold, respectively, from leaves of Nicotiana tabacum cv Samsun NN. They had slightly different MWs (93 000, 90 000 and 100000 for OMTs 1, 11 and Ill respectively) and slightly different pls (5.21, 4.80 and 4.74). The activities of all three enzymes were very stable when stored at 0° but they had different sensitivities to ultrafiltration and to heat treatment (45°). In none of the enzymes was there any change in reaction rate when Mg²⁺ ions or EDTA were added. The three enzymes exhibited very high and similar affinities towards the substrate S-adenosylmethionine and the reaction product S-adenosylhomocysteine, but they differed markedly in specificities towards the various o-diphenolic substrates. Relative methylation efficiencies were estimated from the calculation of the V/K_m ratios that led to the following decreasing order of best substrates: 5-hydroxyferulic acid > caffeic acid > homo-catechol > esculetin > protocatechuic aldehyde > catechol > hydrocaffeic acid > chlorogenic acid, for OMT I, and: homocatechol > catechol > protocatechuic aldehyde > esculetin ≈ cafreic acid > 5-hydroxyferulic acid, for both OMTsIIandIII. Most of the o-diphenols assayed were methylated exclusively in the meta position, but all three tobacco OMTs showed both para and meta-directing activities with protocatechuic acid, protocatechuic aldehyde and escultin. Since K_m values towards the two position of methylation were always found to be identical, we conclude that each enzyme bears only one catalytic site.     

Isolation and characterization of six pathogenesis-related (PR) proteins of Samsun NN tobacco

The purification to homogeneity of pathogenesis-related (PR) proteins R and S from Nicotiana tabacum cv. Samsun NN leaves has been achieved by using a combination of conventional and high-performance chromatographic supports. The same procedure allowed the purification and the characterization of four other proteins which displayed some properties characteristic of tobacco PR proteins and were shown to accumulate in tobacco leaves in response to virus infection. They can be, therefore, considered as new tobacco PR proteins which we designate as PR-s₁,-s₂,-r₁ and-r₂. The relative electrophoretic mobilities (Rf) under non-denaturing conditions were estimated to 0.30 for PR-r₁ and-r₂, 0.25 for Pr-R, 0.20 for PR-s₁ and-s₂ and 0.15 for PR-S. On SDS gels PR proteins R and S possessed the same apparent molecular weight (M _r 24000) as did PR-proteins s₁ and r₁ (M _r 14500) and PR-s₂ and-r₂ (M _r 13000). However, proteins s₁, s₂, r₁ and r₂ had identical electrophoretic mobilities on SDS gels when the loading sample buffer contained no reducing agent. Polyclonal antisera were raised against PR proteins R and S and used in immunoblotting experiments. Proteins R and S were shown to be serologically closely related. No cross-reaction was detected with any of the four new tobacco PR proteins r₁, r₂, s₁ and s₂ or with the previously described PR proteins, i.e. PR-1a,-1b,-1c,-2,-N,-O,-P and-Q.     

Enzymes of the phenylpropanoid pathway and the necrotic reaction of hypersensitive tobacco to tobacco mosaic virus

Pronounced increases in activity of phenylalanine ammonia-lyase (PAL), cinnamic acid-4-hydroxylase (CAH) and caffeic acid-O-methyltransferase (OMT)