Occurrence and properties of Petunia peroxidase a

T. Hendriks

Research output: Thesisinternal PhD, WU


<p>Peroxidases are probably the most extensively studied enzymes in higher plants. Various isoenzymes occur as soluble proteins in the apoplast and in the vacuole, or are bound to membranes and cell walls. Their occurrence is often organ-specific and developmentally controlled, and there is circumstantial evidence that they function in growth, differentiation and defence. In Chapter I biochemical, physiological and genetic aspects of higher-plant peroxidases are reviewed, particularly in relation to the peroxidase system in petunia ( <u>Stimoryne</u> Rafin., formerly <u>Petunia</u> Juss.). It is pointed out that only by studying genetically characterized systems a full understanding of the biochemical and physiological characteristics of the various peroxidase isoenzymes can be obtained.<p>In petunia eight different peroxidase isoenzymes have been genetically characterized and the occurrence of two more is inferred (Chapters 1 and 2). Of these , the most prominent one in leaves and stems is peroxidase a (PRXa), which migrates upon gel electrophoresis as a fast-moving, anionic set of three (sometimes four) molecular forms (mozymes). PRXa also occurs in petals, but is absent from roots. Leaves also contain the intermediatemoving anionic isoenzyme PRXb and the fast-moving cationic peroxidase bands of PRXc. PRXa and PRXc were shown to be the major peroxidases present in extracellular extracts obtained by the vacuum infiltration method, whereas PRXb was the most active peroxidase in isolated mesophyll protoplasts. The three isoenzymes differed in their affinity for Concanavalin A (Con A)-Sepharose; whereas PRXb bound firmly, and was released only in the presence of mannose or glucose, PRXa and PRXc were only partly retained. Since all peroxidases are glycoproteins, these results suggest that the nature of the oligosaccharide chains could be important in determining the cellular location of the peroxidases (Chapter 3).<p>The localization of PRXa in the apoplast was exploited for its purification by preferentially extracting the enzyme from leaves by the vacuum infiltration method (Chapter 4). The electrophoretic variant (allozyme) PRXa1, the product of the allele <u>prxA1</u> , present in the petunia cv. Roter Vogel, was purified over 1300-fold by two sequential acetone precipitations, gel filtration and chromatofocusing. The purified enzyme had a RZ (Reinheits Zahl; A <sub><font size="-1">404nm</font></sub> /A <sub><font size="-1">280nm</font></sub> ) of 3.6, an apparent molecular weight of about 37 kD and an isoelectric point of 3.8. The three molecular forms had slightly different molecular weights and were separated by affinity chromatography on a Con A-Sepharose column. The absorption spectrum of PRXa showed maxima at 404 (Soret band), 496 and 636 nm. Further spectral analy sis revealed that freshly isolated PRXa contains a bound hydrogen donor which is lost upon storage. The pH optimum for the reaction with hydrogen peroxide and guaiacol was 5.0 and the specific activity of the enzyme 61 mkat/g protein. The rate constants for its reaction with hydrogen peroxide and guaiacol were 2.6 x 10 <sup><font size="-2">6</font></SUP>and 1.9 x 10 <sup><font size="-2">6</font></SUP>M <sup><font size="-2">-1</font></SUP>s <sup><font size="-2">-1</font></SUP>, respectively. The reaction with guaiacol was inhibited by various aromatic compounds, notably by trihydroxylated and <u>ortho</u> - and <u>para</u> -dihydroxylated phenolic derivatives. Cinnamic-acid derivatives were more inhibitory than phenol or benzoic-acid derivatives. Coniferyl alcohol was completely inhibitory and was polymerized to presumed lignin-like material. Together with its localization in the apoplast, this substrate preference suggests that PRXa functions in the polymerization of lignin in the cell wall (Chapter 4).<p>A specific antiserum raised in a rabbit against the purified PRXa was used to investigate as to how far peroxidases in other Solanaceous species are antigenically related to the petunia enzyme. Polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE) followed by immunoblotting revealed the presence of cross-reacting proteins in all species tested. To determine whether these proteins represented peroxidases, thorn-apple ( <u>Datura stramonium</u> L.), tobacco ( <u>Nicotiana tabacum</u> L.), sweet pepper ( <u>Capsicum annuum</u> L.), potato ( <u>Solanum tuberosum</u> L.) and tomato ( <u>Lycopersicon esculentum</u> Mill.) were selected for further analysis. Immunoblots after native PAGE revealed that the antiserum specifically recognized the fastmoving anionic peroxidases that are localized in the apoplast. Despite their antigenic relatedness, these peroxidases differed with respect to heat stability and apparent molecular weight, possibly as a result of differences in glycosylation. Apparently, the <u>Solanaceae</u> contain orthologous genes encoding peroxidase isoenzymes homologous to petunia PRXa. The antiserum did not react with peroxidases from horseradish, turnip, African marigold, maize and oats (Chapter 5).<p>Since PRXa is encoded by a single gene, the occurrence of three molecular forms must result from post-transcriptional or post-translational modifications. In Chapter 6 the nature of this heterogeneity was further analysed. Upon incubation with α-mannosidase the larger PRXa1.1 and PRXa1.2 were converted into immunoreactive products resembling the smaller PRXa1.3, suggesting that the three forms of PRXa1 differ in the number of terminal mannose residues. Some α-mannosidese activity was found to be present in the apoplast of leaves and may be responsible for the conversion of PRXa1.1 into PRXa1-2 and PRXa1.3 during leaf development. Lectin-affinity blotting suggested that PRXa contains small, complex-type carbohydrate chains; gas-chromatographic analysis indicated the presence of xylose, arabinose, fucose, mannose, glucose and galactose (Chapter 6).<p>In Chapter 7 the distribution of the soluble peroxidase activity in tissues from petals, leaves and stems was investigated. In petals from fully expanded flowers, the upper epidermis contained nearly 20 % of the peroxidase activity, but no activity was present in protoplasts isolated from this tissue. The peroxidase activity in the upper epidermis was extra-cellular and consisted of PRXa. In protoplasts from the remaining tissue (mesophyll and lower epidermis) PM was the only peroxidase present; it was responsible for about 45 % of the total activity in petals. In leaves, the lower epidermis contained 14 - 40 % <em></em> of the peroxidase activity, depending on their stage of development. Up to half of this activity was localized extracellularly. In stems, the epidermis contained nearly 80 <em>0</em> of the peroxidase activity and 30 % <em></em> of this activity was localized extracellularly. All extracellular extracts contained merely PRXa, suggesting that PRXa is localized mainly in the epidermis. By pressing transversely cut leaves and stems on membrane filters and using these dip-blots to localize the enzyme <u>in</u><u>situ</u> , it was shown that both total peroxidase activity and immunoreactive PRXa are localized mainly in the epidermis, in leaves particularly at the site of the midrib and veins. The epidermal localization of PRXa is suggestive of a role in modulating growth rate or in defence, either by the deposition of lignin, suberin or cutin, or by increased cross-linking of various cell-wall polymers, or both (Chapter 7).<p>In order to analyse the developmentally-controlled regulation of PRXa synthesis, it was attempted to obtain a cDNA probe for PRXa mRNA. Using a Poly(A) <sup><font size="-2">+</font></SUP>-RNA fraction isolated from leaves, a possible precursor of PRXa was detected upon immunoprecipitation of <u>in</u><u>vitro</u> translation products, and an extended product of about 110 nucleotides was detected after reverse transcription primed with a mixed oligonucleotide probe based on a conserved amino-acid sequence in other plant peroxidases. The incorporation of <sup><font size="-2">35</font></SUP>S-methionine and <sup><font size="-2">32</font></SUP>P-CTP, respectively, was very low, suggesting that peroxidase mRNA was present at a low concentration only. A cDNA library in the bacteriophage λgt11 was constructed and screened with the antiserum or the oligonucleotide probe, but no positive clones were detected. Similarly, no peroxidase-specific clones were found upon screening a corrola- specific cDNA library with the antiserum (Chapter 8).<p>In Chapter 9 possible functions of PRXa are discussed. Based on its extracellular localization, its organ and tissue specificity, and its catalytic properties, a function in the rigidification of cell walls in response to internal and external signals seems likely. Petunia PRXa strongly resembles a supposedly lignin-forming peroxidase in tobacco, and seems to be different from a suberin- forming peroxidase induced in potato tubers and tomato fruits upon wounding. Accordingly, in contrast to the total peroxidase activity, PM activity does not increase in floating leaf discs, indicating that it is not induced upon wounding. The identification of genetically defined peroxidase isoenzymes in members of the <u>Solanaceae</u> as being involved in specific physiological processes will provide insight into the function and the regulation of the peroxidase system in this plant family.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Bruinsma, J., Promotor, External person
  • van Loon, L.C., Promotor, External person
Award date10 Mar 1989
Place of PublicationS.l.
Publication statusPublished - 1989


  • ornamental plants
  • solanaceae
  • peroxidases
  • catalase
  • petunia


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