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MICROSCOPIC VISUALIZATION AND ELECTROPHORETIC SEPARATION OF APHID PEROXIDASE
Department of Biochemistry and Molecular Biology, University of Natural Sciences and Humanities in Siedlce, Poland
Localization of the enzyme, peroxidase (POD)
in the gelling saliva (the solid stylet sheath) and in the midgut of aphid, Sitobion
avenae F. and Rhopalosiphum padi L. is recognized in this paper, by
the sensitive staining procedure with 3,3', 5,5' – tetramethylbenzidine and H2O2,
(TMBZ-H2O2). POD activity is not identified in soluble
fraction of salivary protein diffused around of the stylet sheath and released
in the watery saliva, and that is extremely easly visualized with the Coomassie
Brillant Blue R-250 reagent both in the agarose gel and on the sucrose syrup
layer punctured by aphids. Moreover, localization, quantification and molecular
weight of aphid POD isoenzymes after their separation by sodium dodecyl sulfate
– polyacrylamide gel electrophoresis (SDS-PAGE) and staining by TMBZ-H2O2/Coomassie
Blue are shown. Generally, for salivary glands, midgut and whole aphid organism
two POD bands/isoenzymes are established. Real significance of aphid POD for
formation of stylet sheath, elimimination of H2O2 generated
in aphid tissues as result of red-ox metabolism and detoxication/toxication of
phenolic allelochemicals is specified. TMBZ-H2O2 appears
as excellent/suitable technique for the microscopical visulization of POD reaction
in various aphid tissues and agarose or polyacrylamide gels.
Localization of the enzyme, peroxidase (POD) in the gelling saliva (the solid stylet sheath) and in the midgut of aphid, Sitobion avenae F. and Rhopalosiphum padi L. is recognized in this paper, by the sensitive staining procedure with 3,3', 5,5' – tetramethylbenzidine and H2O2, (TMBZ-H2O2). POD activity is not identified in soluble fraction of salivary protein diffused around of the stylet sheath and released in the watery saliva, and that is extremely easly visualized with the Coomassie Brillant Blue R-250 reagent both in the agarose gel and on the sucrose syrup layer punctured by aphids. Moreover, localization, quantification and molecular weight of aphid POD isoenzymes after their separation by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE) and staining by TMBZ-H2O2/Coomassie Blue are shown. Generally, for salivary glands, midgut and whole aphid organism two POD bands/isoenzymes are established. Real significance of aphid POD for formation of stylet sheath, elimimination of H2O2 generated in aphid tissues as result of red-ox metabolism and detoxication/toxication of phenolic allelochemicals is specified. TMBZ-H2O2 appears as excellent/suitable technique for the microscopical visulization of POD reaction in various aphid tissues and agarose or polyacrylamide gels.
Key words: peroxidase (POD), POD activity, protein, aphid, salivary secretions, stylet sheath, watery saliva, midgut, isoenzyme, TMBZ-H2O2 technique, SDS–PAGE, Coomassie Blue stain.
The enzyme peroxidase (donor: hydrogen peroxide oxidoreductase, EC 18.104.22.168) is well known to biochemists as POD and also as 'horseradish peroxidase' and catalyses the following reaction: donor + hydrogen peroxide (H2O2) → oxidized donor + 2H2O. POD is the specific enzyme for the hydrogen acceptor, and in vivo only H2O2 is required for this bio-catalysis. In contrast, POD is not specific for the hydrogen donor and catalyses the dehydrogenation/oxidation of a large number of chemicals, in particular phenols and polyphenols, and in addition also aromatic amines. Due to two facts, firstly that the oxidation of phenols and polyphenols catalysed by POD leads to production of toxic quinones and secondly that en route toxic H2O2 is eliminated, POD in biochemical toxicology is considered/classified both as toxifying and detoxifying enzyme. Although the real POD reaction consists of the transfer of hydrogen from a donor to H2O2, there are examples of POD acting like oxidase and monoxygenase. On the other hand, apart from the true POD, peroxidative reactions with certain substrates i.e. phenols can be catalysed by the enzyme catalase [2,6,21,22,24].
POD in bacteria, higher plants and mammals is the longest and the best studied. Consequently, an enormous research is available on POD of these organisms, in various aspects i.e. subcellular and tissue occurrence, chemical and molecular structure, biochemical functions and implications for biological systems. Moreover, the range of application of POD assay is greatly increasing not only in the basic biochemical research and for clinical diagnosis, but also for environmental and industrial biotechnologies .
The scientific interest in POD of Aphididae started since 1970 by its discovery in the salivary secretion of Macrosiphum rosae . Subsequently, the salivary and midgut POD has been reported in a variety of aphid species e.g. Acyrthosiphon pisum, Aphis fabae, A. gossipi, Myzus persicae, M. rosae, S. avenae, R. padi [8,13,14,15,17,20,26,28,29,30]. Yet, in a number of studies POD activity was indefinite and inconsistent e.g. such results obtained Miles and Harrewijn for the watery saliva of A. fabae and M. persicae .
In fact, POD activity occurrence is demonstrated in Aphididae, but its tissue location, molecular structure and isoenzyme polymorphism and as well biochemical and toxicological function towards phytochemicals with 'anti-aphid' activity, remain still speculative.
There is much to be improved about methodologies for detection of realatively low level of POD activity in tissues. The application of the sodium dedocyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the tool well know to biochemists in the field of enzymology research and the use of sensitive staining i.e. 3,3', 5,5' – tetramethylbenzidine – H2O2 procedure (TMBZ-H2O2), common in cyto-/histochemistry for microscopical visualization of enzymes, are required in further studies on the POD of Aphididae [1,2,4,7,19,22,23]. In this paper the POD activity of Sitobion avenae F. and Rhopalosiphum padi L. is re-examined by TMBZ-H2O2 and SDS-PAGE; in particular: (1) location/light microscopic visualization in the salivary secretions secreted into the agarose gel and on sucrose syrup and also in dissected salivary glands and midgut; (2) electrophoretic profiles and molecular weights of isoenzymes after separating of protein samples of salivary glands, alimentary canals and whole aphids, unstarved and starved, reared on susceptible and resistance wheat varieties. Moreover, detection/occurrence of protein in the salivary secretions of the aphids by the classical stain with Coomassie Brillant Blue R-250 is showed.
MATERIAL AND METHODS
Aphids. The adults of R. padi and S. avenae, both unstarved and starved for 24 hrs, reared on susceptible and resistant winter wheat cultivars at L 18: D 8 photoperiod and 21ºC were used in the experiments.
Chemicals. Coomassie Brillant Blue R-250 was obtained from Bio-Rad (Mississauga, ON, USA). All other reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
Detection of POD activity and protein in salivary secretions. The adults set in plastic ring were allowed to probe/puncture through stretched Parafilm M® membrane agarose (1.25% w/v) – sucrose (30% w/v) for 15 hrs at 21°;C . For POD activity the gel immersed in freshly prepared TMBZ (6.3 mM) – H2O2 (30mM) incubation medium, pH 6.5, in the dark for 1 to 2 hrs [2,23]. The blue staining demonstrated POD reaction and was examined by light microscopy. For POD analysis in the watery fraction of saliva the modified method by Miles  was used, and aphids were allowed to puncture a very thin layer of 80% w/v sucrose syrup containing the TMBZ-H2O2, for eventual demonstration of blue stained spots (Fig. 1).
For microscopical visualization of protein both in gel and sucrose syrup probed/punctured by aphids the Coomasie Blue stain (0.1% Comasie Brillant Blue R-250; 20% methanol; 10% acetic acid) was used [4,23]. Ten replications were done for all procedures.
|Fig. 1. Diagram illustrating how to separate the salivary secretions of aphid by the 'artificial' diets; A – when aphid stylet 'enters' into thick layer i.e. the agarose gel, generally the gelling saliva is secreted; B – when aphid inserts the stylets into very thin layer e.g. syrup of sucrose (80% w/v) that is easly pierced and then only the watery saliva is injected .|
Assay of POD activity in salivary glands and midgut. Tissues, freshly dissected in isotonic saline, rinsed in distilled water and incubated by the TMBZ-H2O2 technique for 1 hr; POD – positive area of tissues stained blue [2,4,23].
Control for POD. For control experiments the TMBZ/H2O2 in the reaction mixture was absent or the inhibitor, 10 mM sodium cyanide was added to the incubation medium [2,19,22].
Electrophoresis experiments. Around one thousand of pairs of salivaery glands, midguts and whole aphids was grinded in Na-phosphate buffered 15% (w/v) sucrose, pH 7.0 and centrifuged at 3000 x g for 15 min. For the electrophoretic studies 35 µl of extracts were used. The electrophoresis was performed on slab gels of 100 x 60 mm. The separating slab gel contained 12.5% (w/v) acrylamide, pH 8.8 and the stacking gel contained 3.0% (w/v) acrylamide, pH 6,8 and bromophenol blue was the tracking dye. Initial current was 9 mA/gel, but when proteins completely entered the gel, the current was increased to 16mA/gel. When the electrophoretic separation was over, the gels were incubated for the POD activity in freshly prepared the TMBZ (6.3 mM) solution in acetate buffered 30% methanol, pH 6.5 for 1 to 2 hrs at room temperature in the dark. Next H2O2 was added to final concentration of 30 mM. The staining was visible within 3 min and increased in intensity over the next 30 min. The gels were placed once or twice in acetate – buffered 30% isopropanol solution, pH 6.5 for clearing of gel background and enhancing the staining intensity. Visualization of the protein fractions was performed with the Coomasie Blue stain [1,4,7,8,23]. Molecular weights of POD isoenzymes were estimated using the standard/marker proteins with mol.wt. from 10 to 200 kiloDaltons (kDa).
How protein is distributed in aphid salivary secretions. Fig. 2A demonstrates protein in the gelling saliva; two different protein fractions appear to be easly identified namely, the stable fraction, focused/closed within of the stylet sheaths and the soluble fraction diffused around of the stylet sheaths. Protein is also easly visualized with the Coomassie Blue stain in the watery saliva (Fig. 2B).
|Fig. 2. Light micrographs of a salivary secretions, salivary glands and alimentary canals of S. avenae incubated for microsopic visualization of protein (with the Coomassie Blue stain), and POD activity (with the TMBZ – H2O2 medium)|
|A – the intense protein staining within/'inside' and around of the stylet sheaths
B – the blue-violet coloured 'spots' of protein presence in the watery saliva, visible on surface of the sucrose syrup layer pierced by aphids
C – the POD reaction is distinctly visible only within the salivary sheath
D – this figure illustrates the POD activity inhibition (lack of staining) in consequence of 10 mM NaCN added into the incubation medium
E – the POD reaction is visible in the salivary glands and midgut, freashly prepared from unstarved aphid
F – visibility of the POD reaction in the midgut tissues of aphid starved for 24 hrs, to eliminate the possible effect of the ingested plant POD.
The same results were obtained for R. padi.
Localization of POD activity in aphid. The POD activity was easly identified inside of the stylet sheath with the sensitive TMBZ-H2O2 technique. On the other hand, no activity of this enzyme was visualized for the soluble protein fraction of the gelling saliva, diffusing around of the stylet sheath (Fig. 2C); the negative results was abtained also for the watery saliva. The blue-stained 'areas' visible distinctly on Fig. 2E indicate the POD action in vivo; in particular, in the midgut and also in the salivary gland cells. While, Fig. 1F shows/illustrates that the activity/reaction of POD is not zero in consequence of lack of feeding/starvation for 24hrs.
Electrophoretic separation of proteins in gels. The PAGE gels stained for protein with the Coomasie Brillant Blue R-250 reagent demonstrated for all tested samples a variety of bands/fractions with molecular weights ranged from about 10 kDa up to 200 kDa. Several bands of protein corresponded to the TMBZ – H2O2 staining patterns (Fig. 3).
|Fig. 3. Light micrographs of a salivary secretions, salivary glands and alimentary canals of S. avenae incubated for microsopic visualization of protein (with the Coomassie Blue stain), and POD activity (with the TMBZ-H2O2 medium)|
POD activity in SDS-PAGE gels. In developed gels containing proteins of salivary glands two bands showed positive reaction with the TMBZ-H2O2 medium, used as indicator of the POD activity. For alimentary canal and whole aphid samples two or three bands of the POD activity were visible; namely, distinctly defined two bands and one weakly stained minor band (Fig. 3).
Samples of whole aphids reared/fed on winter wheat varieties, resistant and susceptible displayed two bands of the POD activity. The molecular weights of these bands were determined to be approximately 17 kDa and 160 kDa. For aphids starved for 24 hrs were estimated also two bands, POD – positive (Fig. 3).
There were no differences between S. avenae and R. padi for this aspect of the studies.
The present studies showed POD presence in the salivary glands and consequently in the gelling saliva (the stylet sheath). In contrast, POD lack was found for salivary secretions diffusing from salivary sheat material. Moreover, no activity of POD was visible/occurred in the watery saliva. These results are pro et contra the POD findings for the salivary secretions of aphids, published up till now [1,8,11,13,14,15,16,17,29,30], and seem to be substantial evidence to distinguish real function of aphid POD which remains debatable and even controversial still. In résumé on aphid saliva Miles  asked 'Why aphids secrete their own POD and apparently increase the formation of toxic/or deterrent compounds, in particular quinines that may be/are known to be toxic to insects due to their ability to copolymerize with proteins'. According to the obtained results the POD action/activity of the aphid salivary secretions is 'closed' within the stylet sheath. This location may limite effectively the potenciality of production of toxic quinones by the oxidation of phenols/polyphenols that occure within/away of plant cells/tissues which the aphid penetrates.
Then, what for aphid possesses POD in stylet sheath. The first suggested function for aphid salivary oxidoreductases was that they served to oxidize precursors in the formation of the stylet sheath [13, 15]. POD appears to be such oxidoreductase. Namely, POD in conjunction with H2O2 catalyses the oxidation of phenols/polyphenols to quinoines, that condense with unoxidized phenols spontaneously and are covalently bind to amino and thiol groups of proteins. POD may cross-link proteins by formation of dityrosine bridges; furthermore, POD is known to catalyse the cross-linking of hydroxyproline – rich glycoproteins [3,9,10].
The stylet sheath of tested aphids is protein-rich and POD – positive and as a result of those it seems to be able to absorb and therefore immobilize/deactivate defensive phenolic chemicals. Miles  described salivary sheat material/stylet sheath from plant cell with brown/dark colouration, typical for phenolic oxidation products.
The occurrence of POD in the midgut is most likely a biochemical adaptation of aphid for 'overcoming' toxicity of dietary H2O2. The source of H2O2 in the midgut are phenolic chemicals, in particular diphenols (DP) and polyphenols (PP) i.e. caffeic acid, chlorogenic acid and (+)-catechin. H2O2 in vivo is generated via superoxide anion (O2∸) and is by-product of oxidative enzyme reactions e.g. the oxidation of DP and PP by the enzyme, polyphenol oxidase (PPO) [5,25,26,27]. H2O2 is required for phenol/polyphenol oxidation by POD and is simultaneously eliminated in this reaction. Just POD in aphid midgut seems to be 'useful' to eliminate H2O2. This function of POD in aphids is likely due to acid pH of their midgut; namely just under acid conditions phenols oxidized by POD do not bind covalently to proteins, but instead polymerize forming non toxic polymers [15,28].
When the S. avenae and R. padi were examined for POD isoenzyme polymorphism, for salivary glands two bands were apparent. Aphid digestive system and whole organism displayed two bands, distinctly defined as POD bands and one weakly staining band, that can be defined as the catalase (CAT) band. CAT has been found in aphid midgut . CAT activity converts H2O2 to H2O and may show also the peroxidative activity.
Aphids reared on resistant and susceptible winter wheat varieties,
both fed and starved, showed constantly a double band/isoenzyme, major slow band
with a molecular weight of approximately 160 kDa, and minor fast band with mol.wt.
of approx. 17 kDa. These findings suggest firstly that POD is a monomer that
is susceptible to denaturation under the electrophoretic technique conoditions,
while secondly, that POD of the studies aphids is dimer enzyme. The obtained
results showed, that the ingestion of the phenol-rich phloem, of the winter wheat
varieties foliage did not induce of POD isoenzyme polymorphism. Then, aphid POD
seems to be constitutive oxidoreductase. Madhusudhan & Miles  found three
POD isoenzymes in saliva injected into water, indicating mol. wt. of approx.
20, 90, and 200 kDa for Therioaphis trifolii maculata and mol.
wt. approx. 120, 180 and 300 kDa for Acyrthosiphon pisum. Clones of the Sitobion and Rhopalosiphum species
had single or double POD isoenzyme . It is known, that individual enzymes
may differ between species in their molecular weight. Yet, isoenzymes of Aphididae seem
to be debatable and poorly examined subject, still.
The salivary POD of aphid is 'focused' within the stylet sheath; real chemical function of POD may be that it acts as a 'immobilizer' of compounds i.e. phenols/polyphenols and proteins, close by aphid stylet.
The midgut POD appears to be a 'scavenger' of H2O2 produced in vivo via superoxide anion as a result of polyphenol oxidase activity towards DP/PP.
Generally, two a constant isoenzymes/fractions are characteristic
for aphid POD; moreover, aphid POD appears as uninduced enzyme by dietary phenols/polyphenols.
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Accepted for print: 17.12.2008
Department of Biochemistry and Molecular Biology,
University of Natural Sciences and Humanities in Siedlce, Poland
B. Prusa 12, 08-110 Siedlce, Poland
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