Organ-specific expression of the stress-related anionic peroxidases in cucumber flowers

The study revealed a marked qualitative and quantitative differences in the pattern of expression of three stress-related cucumber (Cucumis sativus L., cv. Laura) isoperoxidases. Activity staining, as well as the protein gel blot (Western blotting) confirmed that the proteins studied are differentially expressed in both male and female sepals, in male but not in female pedicels and in, pistil. By using antibodies specific to three stress-related peroxidases, one serologically related pistil-specific anionic peroxidase was detected. This specific band had never been observed in other flower organs. Differential appearance of the stress-related peroxidase isoenzymes in both male and female flowers demonstrates that these proteins are developmentally regulated, showing an organ-specific expression.     

Homology of Plant Peroxidases: AN IMMUNOCHEMICAL APPROACH

Antisera specific for the basic peroxidase from horseradish (Amoracea rusticana) were used to examine homology among horseradish peroxidase isoenzymes and among basic peroxidases from root plants. The antisera cross-reacted with all tested isoperoxidases when measured by both agar diffusion and quantitative precipitin reactions. Precipitin analyses provided quantitative measurements of homology among these plant peroxidases. The basic radish (Raphanus sativus L. cv. Cherry Belle) peroxidase had a high degree of homology (73 to 81%) with the basic peroxidase from horseradish. Turnip (Brassica rapa L. cv. Purple White Top Globe) and carrot (Daucus carota L. cv. Danvers) basic peroxidases showed less cross-reaction (49 to 54% and 41 to 46%, respectively). However, the cross-reactions of antisera with basic peroxidases from different plants were greater than were those observed with acidic horseradish isoenzymes (30 to 35%). These experiments suggest that basic peroxidase isoenzymes are strongly conserved during evolution and may indicate that the basic peroxidases catalyze reactions involved in specialized cellular functions. Anticatalytic assays were poor indicators of homology. Even though homology among isoperoxidases was detected by other immunological methods, antibodies inhibited only the catalytic activity of the basic peroxidase from radish.     

Structural determinants of plant peroxidase function

The classical plant peroxidases are a well-studied group of heme-containing enzymes for which many different functions have been proposed. In the majority of plant species investigated they occur as distinctive isoenzymes which can be constitutive or induced in response to external factors such as wounding, stress and attack by pathogens. More than 70 peroxidase isoenzymes are predicted to occur in Arabidopsis thaliana alone, according to recent analysis of the complete peroxidase gene family of this model plant. Understanding this enzymatic diversity and its functional significance is a major focus of structural and mechanistic studies of plant peroxidases. The three-dimensional structures of plant peroxidases from Arabidopsis, barley, horseradish, peanut and soybean have now been determined by X-ray crystallography together with the structures of several catalytic intermediates and substrate complexes that are relevant to enzyme function. On this basis, specific roles for particular amino acid residues and structural motifs or regions have been proposed or in some cases, confirmed. Some of these have been investigated experimentally using site-directed mutagenesis and other techniques. An overview of recent developments will be presented that reflects our current understanding of structure and function in this important group of enzymes.     

Hormonal regulation and distribution of peroxidase isoenzymes in the Cucurbitaceae

Ethylene enhanced the levels of peroxidases in the roots, stems, leaves, and cotyledons of 2-week-old cucumber Cucumis sativus cv Poinsett 76 seedlings. Antibodies to the isoelectric point (pl) 9 and pl 4 isoenzymes were used in a radial immuno-diffusion assay to demonstrate that ethylene induced similar peroxidases in other cultivars of C. sativus, other species of Cucumis and other genera of Cucurbitaceae. Examination of ethylene-induced peroxidases, using isoelectric focusing gels, demonstrated the presence of a series of other peroxidases, mostly slightly acidic, whose isoelectric focusing pH was approximately 6. These pl 6 peroxidases were partially purified on a cation exchange column. Ouchterlony double diffusion gels indicated that these proteins cross-reacted with antibodies to both the pl 9 and pl 4 peroxidase. The data presented here suggest that the induction of peroxidase isoenzymes during ethylene-induced senescence is a common response in this family of plants. In addition, antibody and isoelectric focusing studies indicate that both acidic and basic peroxidase are highly conserved in members of this family.     

Interrelationship between lignin deposition and the activities of peroxidase isoenzymes in differentiating tracheary elements of Zinnia

In a culture system in which single cells isolated from the mesophyll of Zinnia elegans L. differentiate to tracheary elements (TEs), two inhibitors of phenylalanine ammonia-lyase (EC 4.3.1.5), L-α-aminooxy-β-phenylpropionic acid (AOPP) at 10 μM inhibited lignification without reducing the number of TEs formed. These inhibitors caused intracellular changes in peroxidase (EC 1.11.1.7) activities. The inhibitors increased the activity of peroxidases bound to the cell walls and especially the activity of peroxidase bound ionically to the cell walls. In contrast, the activity of extracellular peroxidase decreased. There were five isoenzymes, P1-P5, in the ionically bound peroxidase of cultured Zinnia cells. Among the isoenzymes, P4 and P5 appeared to be specific for TE differentation. Treatment with AOPP and AIP resulted in increases in the activities of P2, P4 and P5 isoenzymes, with the most prominent increase in P5 activity. The addition of lignin precursors, including coniferyl alcohol, to the AOPP-treated cells restored lignification, and suppressed the alteration of peroxidase isoenzyme patterns caused by AOPP. The relationship between the wall-bound peroxidases and lignification during TE differentiation is discussed in the light of these results.     

Study of Antioxidant Enzymes Activity during Organogenesis and In Vitro Propagation of Asiatic Hybrid Lily

A protocol for indirect differentiation of shoots / roots from leaf callus of Asiatic hybrid lily was developed through in vitro methods. The involvement of antioxidant enzymes, like, SOD, POX and CAT, and their isoenzymes during organogenesis in the morphogenetic callus was stud ied.The activity of these enzymes was increased during early development and differentiation of callus. SOD activity increased significantly as compared to POX and CAT during root formation, while it decreased in shoot formation and the decrease was significant in POX and CAT enzymes. The results indicate that the organogenesis is a very complicated biological process involving up and down regulation of a number of antioxidant enzymes, which seem to play an important role during organogenesis of Lilium callus.     

Indole-3-acetic acid content and glutamine synthetase activity in the pericarp,and peroxidase activity and isoenzymes in the meso- and exocarp of growing peach fruits

The indole-3-acetic acid (IAA) content in peach pericarp (Prunus persica L. Batsch cv. Merry) was highest at early stage I of development (～200 ng/g fresh wt), decreased to the lowest level during stage II, and rose again at stage III to 60–70 ng/g fresh wt. High activity of glutamine synthetase was found in the pericarp during stage I. The soluble peroxidase activity was highest in the meso- and exocarp at stage II, and isoenzymatic changes in this fraction corresponded to the transition from cationic isoenzymes, predominant at stage I, to anionic isoenzymes at stage III. The ionically bound peroxidase activity in these tissues was highest at stage I. The three developmental stages showed marked differences in auxin content and enzyme activities; for peroxidases these changes reflect a developmental expression pattern for the isoenzymes.     

Transformation of Industrial Dyes by Manganese Peroxidases from Bjerkandera adusta and Pleurotus eryngii in a Manganese-Independent Reaction

We investigated the transformation of six industrial azo and phthalocyanine dyes by ligninolytic peroxidases from Bjerkandera adusta and other white rot fungi. The dyes were not oxidized or were oxidized very little by Phanerochaete chrysosporium manganese peroxidase (MnP) or by a chemically generated Mn³⁺-lactate complex. Lignin peroxidase (LiP) from B. adusta also showed low activity with most of the dyes, but the specific activities increased 8- to 100-fold when veratryl alcohol was included in the reaction mixture, reaching levels of 3.9 to 9.6 U/mg. The B. adusta and Pleurotus eryngii MnP isoenzymes are unusual because of their ability to oxidize aromatic compounds like 2,6-dimethoxyphenol and veratryl alcohol in the absence of Mn²⁺. These MnP isoenzymes also decolorized the azo dyes and the phthalocyanine complexes in an Mn²⁺-independent manner. The reactions with the dyes were characterized by apparent K_m values ranging from 4 to 16 μM and specific activities ranging from 3.2 to 10.9 U/mg. Dye oxidation by these peroxidases was not increased by adding veratryl alcohol as it was in LiP reactions. Moreover, the reaction was inhibited by the presence of Mn²⁺, which in the case of Reactive Black 5, an azo dye which is not oxidized by the Mn³⁺-lactate complex, was found to act as a noncompetitive inhibitor of dye oxidation by B. adusta MnP1.     

Basic peroxidases in isolated vacuoles of nicotiana tabacum L.

Vacuoles of tobacco mesophyll and of suspension-cultured cells were isolated in order to study the localization of peroxidase isoenzymes. Only basic peroxidases were detectable by electrophoretic separation of the vacuolar sap. Some of the basic peroxidases have formerly been described as an ionically bound cell-wall fraction. This fraction, however, was found to be an artifact produced by incomplete cell breakage. Reinvestigation of isolated cell walls confirmed that mainly acidic peroxidases are localized in the cell walls where they move freely or are bound. As a consequence of former and present results we think it probable that all of the peroxidase isoenzymes are secretory proteins because they have to be transported from the sites of synthesis in the cytoplasm to the sites of function, the extracytoplasmic spaces, cell wall (acidic peroxidases), and vacuole (basic peroxidases).Abbreviation ER endoplasmic reticulum - PAGE polyacrylamide gel electrophoresis     

Predominant expression of a peroxidase gene in staminate flowers of Mercurialis annua

A direct correlation between the feminizing process of male plants of Mercurialis annua by benzylaminopurine and the disappearance of male-specific peroxidases was established. In order to understand the process of hormonal regulation of the sexual differentiation and peroxidase activity, two peroxidase cDNA clones were selected from a male flower cDNA library with degenerated oligonucleotide probes encoding a peroxidase-specific site. These clones contained three peroxidase-specific sequences and an Eco RI restriction site. The two Eco RI fragments of one cDNA clone were subcloned and used as probes to analyse the expression of corresponding gene(s). The northern blot analysis showed hybridization on a single mRNA band with a predominant expression in male flowers. The feminizing treatment with a cytokinin induced the progressive disappearance of the hybridization signal in correlation with the disappearance of male specific isoperoxidases.     

A pollinators' eye view of a shelter mimicry system

Background and Aims

‘Human-red’ flowers are traditionally considered to be rather unpopular with bees, yet some allogamous species in the section Oncocyclus (genus Iris, Iridaceae) have evolved specialized interactions with their pollinators, a narrow taxonomic range of male solitary bees. The dark-red, tubular flowers of these irises are nectarless but provide protective shelters (i.e. a non-nutritive form of reward) primarily to male solitary bees (Apidae, Eucerini) that pollinate the flowers while looking for a shelter. An earlier study on orchids suggested that species pollinated predominantly by male solitary bees produce significantly larger amounts and larger numbers of different n-alkenes (unsaturated cuticular hydrocarbons). Whether or not this also applies to the Oncocyclus irises and whether pollinators are attracted by specific colours or scents of these flowers is unknown.

Methods

Using Iris atropurpurea, recording of pollinator preferences for shelters with different spatial parameters was combined with analyses of floral colours (by spectrophotometry) and scents (by gas chromatography–mass spectrometry) to test the hypotheses that (a) pollinators significantly prefer floral tunnels facing the rising sun (floral heat-reward hypothesis), and that (b) flowers pollinated predominantly by male solitary bees produce significantly larger amounts and larger numbers of unsaturated cuticular hydrocarbons (n-alkenes) in their floral scent (preadaptation to sexual-deception hypothesis).

Key Results

Male bees do not significantly prefer shelters facing the rising sun or with the presence of high absolute/relative amounts and numbers of n-alkenes in the floral scent.

Conclusions

The results suggest that the flowers of I. atropurpurea probably evolved by pollinator-mediated selection acting primarily on floral colours to mimic large achromatic (‘bee-black’) protective shelters used preferentially by male solitary bees, and that pollinator visits are presumably not the result of an odour-based sexual stimulation or motivated by an increased morning floral heat reward in tunnels facing the rising sun.     

Gene expression in Mercurialis annun flowers: In vitro translation and sex genotype specificity. Male-specific cDNA cloning and hormonal dependence of a corresponding specific RNA

Summary Sequences specifically expressed in flowers of each sex type of Mercurialis annua have been demonstrated by a comparative analysis of the translation products of their poly(A)⁺ RNA populations (wheat germ system, two-dimensional electrophoresis). This method confirms previous results of hybridization kinetics: the staminate flowers of the normal fertile male (wild type) and restored fertile male strain (identical morphology) and those of the sterile male genotype contain specific poly(A)⁺ RNAs and sequences shared by sets of two of these males, as well as numerous common sequences. The pistillate flowers of the constructed female strain 19_–5 (carrying male sterility determinants) also contains specific poly(A)⁺ RNA compared to identical flowers of a normal female genotype. In vitro translation, however, showed a specificity (not revealed by hybridization kinetics) in the normal female genotype compared to a normal male genotype and to the female strain 19_–2 (female strain cDNAs hybridized 100% to poly(A)⁺ RNAs of male and female 19_–5 genotypes). Cloning certain specific sequences (cDNAs) of the fertile male wild type and using one cDNA as probe also confirms the previously described male specificity. Moreover, the hormonal dependence of RNA corresponding to a specific male probe is demonstrated: its kinetics of disappearance are a function of the action of the feminizing hormone (cytokinins). These results are in agreement with our hypothesis of sexual organogenesis: sexual hormones controlled by regulator genes of the sexual genotype induce, in definite cell lineages of bipotential meristems, the expression of genes specific for male or female expression.     

Antigenic relationships between petunia peroxidase a and specific peroxidase isoenzymes in other Solanaceae

Summary A highly specific rabbit antiserum raised against peroxidase (PRXa) from petunia (Petunia hybrida) was used to investigate the antigenic relatedness of peroxidases in the Solanaceae. After SDS-PAGE of crude leaf extracts from a large number of species of this family, immunoblotting revealed that cross-reacting protein bands were present in all species tested. In order to determine whether these protein bands represent peroxidases, the peroxidase isoenzymes in thorn apple (Datura stramonium L.), tobacco (Nicotiana tabacum L.), sweet pepper (Capsicum annuum L.), potato (Solanum tuberosum L.), and tomato (Lycopersicon esculentum Mill.) were further analyzed. Immunoblots obtained after native PAGE revealed that the antiserum only recognized fast-moving peroxidase isoenzymes that are localized in the apoplast. Despite their serological relatedness, these peroxidases differed with respect to heat stability and apparent molecular weight. Differences in avidity for the petunia PRXa antiserum were suggested by immunoprecipitation with antibodies bound to protein A-Sepharose. The antiserum did not react with peroxidases from horseradish (Armoracea rusticana Gaertn., Mey and Scherb), turnip (Brassica napus L.), African marigold (Tagetes cresta L.), maize (Zea mays L.), and oats (Avena sativa L.). Apparently, the Solanaceae contain orthologous genes encoding the fast-moving anionic peroxidases homologous to petunia PRXa.     

Phylogeny, topology, structure and functions of membrane-bound class III peroxidases in vascular plants

Peroxidases are key player in the detoxification of reactive oxygen species during cellular metabolism and oxidative stress. Membrane-bound isoenzymes have been described for peroxidase superfamilies in plants and animals. Recent studies demonstrated a location of peroxidases of the secretory pathway (class III peroxidases) at the tonoplast and the plasma membrane. Proteomic approaches using highly enriched plasma membrane preparations suggest organisation of these peroxidases in microdomains, a developmentally regulation and an induction of isoenzymes by oxidative stress. Phylogenetic relations, topology, putative structures, and physiological function of membrane-bound class III peroxidases will be discussed.     

Activity of antioxidant enzymes and secondary metabolites during in vitro regeneration of Sterculia urens

The changes in the activities of antioxidant enzymes and amounts of proteins, phenols, and flavonoids in regenerating and non-regenerating calli during organogenesis of Sterculia urens were monitored. Maximum growth of calli and the most efficient regeneration of shoots occurred on Murashige and Skoog (MS) medium supplemented with 0.5 mg dm^?3 benzylaminopurine (BAP) + 2 or 4 mg dm^?3 naphtalene acetic acid (NAA). Peroxidase (POD), catalase, and superoxide dismutase activities increased in the regenerating calli but decreased in the non-regenerating calli. Six POD isoenzymes were detected. Protein content decreased in the non-regenerating calli and increased significantly during regeneration of shoots from callus. Total phenols and flavonoids increased in the non regenerating calli. SDS-PAGE analysis revealed a role of many proteins in organogenesis.     

Role of phytochrome B in organ formation processes in Cucumis sativus L.

The role of phytochrome B in the organogenesis process in the apical and axillary shoot meristems during early ontogenesis stages in cucumber Cucumis sativus L. at photoperiods (day/night) 10/14, 16/8 h, and continuous light in comparison with wild type plants and phytochrome B-deficient mutant (lh-mutant) was investigated. In mutant phytochrome B, deficiency caused inhibition of initiation of leaves both in the main shoot and lateral shoots and increased the number of flower buds (IV stage of organogenesis). With continuous light, the number of lateral shoots and flowers during stage IV of organogenesis in wild-type plants increased twofold in comparison with the mutant. Short-term temperature drops did not induce floral ontogenesis in mutants but increased the number of off-shoots in both experimental variants during a long photoperiod and continuous lighting. We propose that phytochrome B, by increasing the compactness of chromatin, may facilitate coordination of ontogenesis processes with changing environmental conditions.     

Purification and characterization of peroxidases from the dye-decolorizing fungus Bjerkandera adusta

A peroxidase oxidizing Mn²⁺ (MnP) is described for the first time in Bjerkandera adusta, a fungus efficiently degrading xenobiotic compounds. The MnP appeared as two isoenzymes, which were purified to homogeneity together with two lignin peroxidases (LiP). Their N-terminal sequences were identical, but the MnP isoenzymes showed more basic isoelectric points and differences in amino acid composition and catalytic properties. The B. adusta LiP is similar to LiP from Phanerochaete chrysosporium. However, the interest of the MnP described here is related to its ability to catalyze Mn²⁺-mediated as well as Mn²⁺-independent reactions on aromatic compounds, which may be of use for applications in biotechnology and environmental technology.     

Chemical components of male and female flowers of Schisandra chinensis

Schisandra chinensis (Turcz.) Baill. is one of the important traditional medicinal plants in East Asia. It is a dioecious plant with aromatic flowers. The female and male flowers of S. chinensis possess slightly different fragrance characteristics. The overall scent of S. chinensis flowers is quite similar to that of Syringa dilatata (Korean lilac) flowers. Hence, this study aimed to understand the aromatic profile of the hexane extract from female and male flowers of S. chinensis and to compare their profile with the hexane extract of Korean lilac flowers. The chemical composition of hexane extract was determined by gas chromatography and mass spectrometry (GC-MS) analysis. In total, 67 different components were detected in the hexane extract of female (48) and male flowers (51) of S. chinensis; 32 of which were common to both female and male flowers. In regards to gender difference, 16 components were found only in female flowers, and 19 components were found only in male flowers. The results revealed that the most abundant components in the hexane extract of both female and male flowers were lilac alcohol C (9.53 and 7.00%), lilac alcohol A (6.55 and 5.71%), n-hexadecanoic acid (6.21 and 6.96%), linoleic acid (5.14 and 7.61%), β-elemene (5.12 and 1.99), and lilac aldehyde D (4.13 and 4.97%). The data suggest that the major compounds in the hexane extract of S. chinensis flowers were generally similar, but they varied quantitatively according to gender. The presence of 10 components in both S. chinensis and Korean lilac flowers may be responsible for their similar fragrance characteristics. It could be concluded that the different fragrance characteristics of these flowers may be due to the presence of several gender-specific aromatic compounds in minor percentages.