Achim E. Gau

Accumulation of Pathogenesis-Related Proteins in the Apoplast of a Susceptible Cultivar of Apple (Malus domestica cv. Elstar) after Infection by Venturia inaequalis and Constitutive Expression of PR Genes in the Resistant Cultivar Remo

Leaves of apple (Malus domestica cv. Elstar) were infected with a cloned isolate of the apple scab Venturia inaequalis. The intercellular washing fluid (IWF) of these plants was collected and the variation in the composition of proteins in the IWF was analysed by SDS-PAGE and two-dimensional gel electrophoresis during and after the infection with V. inaequalis, the causal agent of apple scab. The subsequent analysis of induced proteins by electron spray ionization quadrupole time of flight mass spectroscopy revealed the presence of β-1,3-glucanase, chitinase, thaumatin-like protein and a cysteine-like protease in M. domestica leaves infected by V. inaequalis. These results were confirmed by immunoblotting with antibodies against some of these proteins. Moreover, a non-specific lipid transfer protein was identified in uninfected leaves: the amount declined to a non-detectable level within the first week after infection by V. inaequalis. The analysis of the IWF of M. domestica cv. Remo, bearing resistances to apple scab, powdery mildew and fire blight, showed a protein pattern comparable to that of the IWF from V. inaequalis infected leaves from cultivar Elstar indicating the constitutive production at least of some of the pathogenesis-related proteins in the resistant cultivar.

Presence of a flavoprotein in O2-evolving Photosystem II preparations from the cyanobacterium Anacystis nidulans

Abstract A highly active O2-evolving Photosystem II complex which was greatly depleted of phycobiliproteins was isolated from the cyanobacterium Anacystis nidulans. This complex contained the flavoprotein with l -amino acid oxidase activity which we have previously shown to be present in thylakoid preparations of this cyanobacterium (Pistorius, E.K. and Voss, H. (1982) Eur. J. Biochem. 126, 203–209). One of the most prominent polypeptides in this O2-evolving Photosystem II complex had a molecular weight of 49 kDa. This polypeptide co-chromatographed on SDS-polyacrylamide gels with the purified l -amino acid oxidase which consists of two subunits of 49 kDa. The antagonistic effect of CaCl2 on the two examined reactions could also be demonstrated with this O2-evolving Photosystem II complex: CaCl2 stimulated photosynthetic O2 evolution, but inhibited the l -amino acid oxidase activity. Both reactions were inhibited by o-phenanthroline. These results further support a functional relationship between the flavoprotein with l -amino acid oxidase activity and Photosystem II activities in A. nidulans. However, we only found 1 mol FAD per 350–650 mol chlorophyll, although 1 gatom Mn per 5–10 mol chlorophyll was present. When we assume a photosynthetic unit of about 40 chlorophylls, then in most preparations the FAD values were more than a factor of 10 too low. Results which we obtained with the purified l -amino acid oxidase showed that the FAD values were in most enzyme samples lower than the theoretically expected value of 2 mol FAD per mol enzyme. Moreover, in some cases the absorption spectrum of the enzyme showed substantial deviations from the spectrum of oxidized FAD. These experiments indicated that the flavin in the enzyme could partly exist in a form which was different from ‘authentic oxidized FAD’. We do not yet know the chemical nature of this ‘modified flavin’.

L-Amino Acid Oxidases with Specificity for Basic L-Amino Acids in Cyanobacteria

Abstract The two closely related fresh water cyanobacteria Synechococcus elongatus PCC 6301 and Synechococcus elongatus PCC 7942 have previously been shown to constitutively express a FAD-containing l-amino acid oxidase with high specificity for basic L-amino acids (L-arginine being the best substrate). In this paper we show that such an enzyme is also present in the fresh water cyanobacterium Synechococcus cedrorum PCC 6908. In addition, an improved evaluation of the nucleotide/amino acid sequence of the l-amino acid oxidase of Synechococcus elongatus PCC 6301 (encoded by the aoxA gene) with respect to the FAD-binding site and a translocation pathway signal sequence will be given. Moreover, the genome sequences of 24 cyanobacteria will be evaluated for the occurrence of an aoxA-similar gene. In the evaluated cyanobacteria 15 genes encoding an l-amino acid oxidase-similar protein will be found.

Up-regulation of pathogenesis-related proteins in the apoplast of Malus domestica after application of a non-pathogenic bacterium.

The intercellular washing fluid (IWF) of Malus domestica cv. Holsteiner Cox before and after application of the non-pathogenic bacterium Pseudomonas fluorescens Bk3 to the leaves was investigated in a comparative manner. SDS-PAGE in combination with ESI Q-ToF mass spectrometry, and homology search in relevant data bases revealed the highly up-regulated expression of several pathogenesis-related plant proteins in the apoplast of the leaves treated with P. fluorescens. These proteins were β-1,3-glucanase, chitinase, thaumatin-like protein, ribonuclease-like protein, and a hevein-like protein. Moreover, a 9 kDa non-specific lipid transfer protein was significantly reduced after the application of P. fluorescens. The possible relevance of a pre-treatment of apple cultivars with the non-pathogenic bacterium P. fluorescens Bk3, as an alternative method to the treatment with fungicides, for increasing the resistance of susceptible apple cultivars against an infection with the fungus Venturia inaequalis is discussed.

Isolation and Partial Characterization of a Manganese Requiring ʟ-Arginine Metabolizing Enzyme Being Present in Photosystem II Complexes of Spinach and Tobacco

Abstract A low ʟ-arginine metabolizing enzyme (L-AME) activity leading to ornithine, urea and additional products not identified so far could be detected in photosystem II (PS II) membranes of spinach and of the chlorophyll deficient tobacco mutant Su/su. The detectable L-AME activity was very low in untreated PS II membranes, but increased significantly (about 10 fold) when the extrinsic peptides (psbO, P and Q gene products) were removed - suggesting that the L-AME is exposed at the lumen side of PS II. It was possible to isolate the detergent-solubilized protein from CaCl2-washed PS II membranes of spinach by a combination of anion and cation exchange columns. On the basis of SDS PAGE the protein was homogenous and had an apparent molecular mass of 7 kDa. N-terminal sequencing of the polypeptide gave a contiguous sequence of 20 amino acids showing no homologies to PS II polypeptides as yet sequenced. After chromatography of the L-AME on an anion exchange column at pH 9.5 (last purification step) a completely inactive enzyme was obtained. Maximal reactivation was achieved by dialyzing the protein against Hepes-NaOH buffer in the pH range of 6.5 to 7.5 containing 100 mᴍ chloride or sulfate (being the most effective anions). The L-AME activity was totally dependent on manganese added to the reaction mixture. Moreover, there were indications of a second cation binding site being more sequestered and requiring bound Ca2+ or Mn2+ for activity (Sr2+ was less effective and Mg2+ was ineffective). There are indications that the protein contains a redox active group - possibly an aminoacid- derived quinonoid (based on a redox cycling assay with glycine and nitroblue tetrazolium). The capability of this PS II associated protein to bind the cofactors of water oxidation and having a redox active group (preliminary results) suggests that this protein might be functional in photosynthetic water oxidation. This is further supported by the fact that the isolated L-AME has a low catalase activity

Immunological Identification of Polypeptides in Photosystem II Complexes from the Cyanobacterium Anacystis nidulans

The minimal O2 evolving photosystem II (PS II) complex of higher plants consists of at least 8 polypeptides: the reaction center peptides D-1 and D-2, the two apoproteins of cytochrome b559 (9 and 4.3 kDa), a recently identified 4.8 kDa peptide, two chlorophyll a binding peptides of 47 and 43 kDa as the immediate antenna proteins and the extrinsic Mn stabilizing peptide (1,2,3). Recently it has been shown that a minimal complex which was isolated from O2 evolving spinach PS II complexes and which only consisted of D-1, D-2, the two cytochrome b559 binding peptides and a 4.8 kDa peptide, can still catalyze photochemical charge separation (3,4). In a hypothetical model, Barber (5) suggested that all additional cofactors required for water oxidation might also be associated with this complex and that only the extrinsic 33 kDa peptide is required in addition for stabilization of Mn at the reaction center. This model has not yet been experimentally proven, but would imply that a separate mangano enzyme does not exist (1,2).

Further investigations about the flavin in the L-amino acid oxidase and a possible flavin in photosystem II complexes from the cyanobacterium Anacystis nidulans

The absorption spectrum of the previously purified ʟ-amino acid oxidase from the cyanobacterium Anacystis nidulans has shown considerable variation with each preparation and the spectrum in several preparations was quite different from the absorption spectrum of other simple flavoproteins (E. K. Pistorius and A. E. Gau, Biochim. Biophys. Acta 849, 203, 1986). Here we show that the spectral complexity and variability of the ʟ-amino acid oxidase can be largely explained by the presence of a modified flavin derivative of yet unknown structure besides oxidized FAD and FAD semiquinone. After removal from the enzyme this modified chromophore has absorption maxima at 260, 396 and in the 600 nm region. This derivative of FAD seems to be formed in variable amounts during the purification of the enzyme. On the other hand, extraction of Anacystis photosystem II complexes which contain the flavoprotein, almost exclusively yields modified flavin derivatives and practically no authentic oxidized FAD. The spectrum of the chromophores which have been extracted from photosystem II complexes at different purification stages, is either similar (although not identical) to the spectrum of the chromophore extracted from the isolated ʟ-amino acid oxidase or similar to the spectrum of reduced flavin. All extracted chromophores show a fluorescence emission in the 420 to 560 nm region when excited with light of 390 nm. These results indicate that the flavin present in the ʟ-amino acid oxidase protein as well as in photosystem II complexes from A. nidulans rapidly undergoes modification reactions of yet unknown nature to yield several closely related FAD derivatives. This might possibly be the reason why so far no flavin has been detected in photosystem II. The presence of such modified flavin derivatives in photosystem II complexes of A. nidulans as shown here is an additional support of our hypothesis that an unusual flavin is functional on the donor side of photosystem II.