Polyphenol oxidase of bird cherry-oat aphid

Aphids and Other Homopterous Insects

2001, vol. 8: 55-64

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Polyphenol oxidase of bird cherry-oat aphid

Anna Urbańska, Magdalena Waluk, Henryk Matok, Bogumił Leszczyński

Department of Biochemistry, University of Podlasie, ul. B. Prusa 12, Siedlce, Poland

Introduction

Polyphenol oxidase (EC 1.10.3.1 o-diphenol : O2 oxidoreductase), commonly known

as o-diphenol oxidase, catechol oxidase, tyrosinase and PPO is the enzyme widely

distributed in nature, which catalyses two different reaction: (1) the oxidation of o-

diphenols (diphenol oxidase activity), and (2) the hydroxylation of monophenols e.g.

tyrosine to o-diphenols (monophenol oxidase or tyrosinase activity) [22]. In insects

PPO was generally considered as responsible for the melanization and sclerotization

of cuticle [1]. Neverthless, it has been detecting amongst plant-sucking insects

containing soft cuticle, particularly in Aphididae [11,18]. In that case, Miles

[13,14,15,16] propounded that PPO because of catalysis of the rapid conversion of o-

diphenols via quinones finally to non - toxic polymers might enable the aphids to

overcome defensive phenolic monomers present in their host plant tissues.

Consequently, a lot of research has been doing on PPO of Aphididae in this aspect

[8,19,20,21,24,25,26,27]. Yet, according to recent Miles’ résumé [18] on aphid

saliva, PPO implication in detoxication of the plant phenolics persists still rather

hypothetical.

In the present study we re-examined the aphid PPO, in particular: 1) occurrence in the

saliva fractions, salivary glands and midgut, 2) the activity distribution amongst

tissues of the aphid body; 3) electrophoretic separation of the isoenzymes.

Material and methods

Aphids

Apterous adults of the bird cherry oat aphid Rhopalosiphum padi (L.) were collected

from the stock culture, reared on seedlings of Polish winter wheat cultivar Sakwa, at L

16 : D8 photoperiod and 22oC, at the Department of Biochemistry of the University of

Podlasie.

Aphids and Other Homopterous Insects, vol. 8

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Detection of PPO in the gelling saliva

Darkening of stylet sheaths formed through Parafilm M membrane into agarose (1,25%) - sucrose

(30%) gel in 0.1 M Na-phosphate buffer, pH 7.4, containing 0.1% L-DOPA (3,4-

dihydroxyphenylalanine was detected. Control gel incorporated 0.005M phenylthiourea (PTU) [27].

Detection of PPO in the watery saliva

The analysis was done on the basis of the method of Miles [12]. The aphids punctured a

very thin layer of 80% sucrose syrup with 0.1% L-DOPA at pH 7.4 placed on the

Parafilm M membrane stretched across the plastic ring and injected on upper surface the

watery saliva fraction visible as dark spots [27].

Determination of PPO in the salivary glands and midgut

Aphids were left without food overnight. The freshly dissected tissues were incubated in

0.1% DOPA in 0.1M Na-phosphate buffer pH 7.4 for 4h at 30oC and their darkening was

used as indicator of PPO activity areas [21,26].

Measurements of PPO activity within tissues of the aphid body

One hundred of whole aphids, pairs of salivary glands and alimentary canals were taken

to assess the activity. After homogenization in 0.1M Na-phosphate buffer, pH 7.4 and

centrifugation at 3000 x g, the colorimetric assay was carried out with 0.1% DOPA and

increase of A at 460 nm after 30 min was measured [20].

Protein assay was done by the method of Bradford [2].

PPO isoenzymes polymorphism

Around one thousand of whole aphids, pairs of salivary glands and alimentary canals was

homogenised in 15% (w/v) sucrose, pH 7.0 and centrifuged at 3000xg [5]. SDS - PAGE

electrophoresis was used as described by Gillespie et al. [4]. The electrophoresis was

performed on slab gels of 100 x 60 mm. The separating slab gel contained 12.5%

acrylamide, pH 8.8 and the stacking gel contained 3.0% 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 16 mA/gel. When the separation

was completed, the gels were incubated in 1.0% L-DOPA in 0.1M phosphate buffer, pH

7.4 at room temperature for 2 hrs. The PPO appeared as dark bands on a clear

background. Moreover, the staining for the protein fractions was performed with 0.1%

Commasie Brillant Blue R-250.


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Results

Occurrence of PPO in the aphid

The aphids produced almost black salivary sheaths into the agarose-sucrose gels

containing L-DOPA (Fig. 1a) In the presence of PTU within the gel the sheaths were

overall uncolored (Fig. 1b). Activity extent of PPO was evident not only within

salivary sheath and also around it and was assessed for a single aphid at value of 30-60

µm (Fig. 1c). Next, when the aphids probed shallow layers of the sucrose - DOPA

syrup the black colored spots of up to 0.05-0.1 mm in diameter were noticed; they

evidenced the PPO presence in the watery saliva (Fig. 1d).

Fig. 1. Occurrence of PPO in the saliva of R.padi: a) salivary sheath within agarose-

sucrose gel with 0.1% DOPA. 400; b) the sheath in the presence of PTU. 400; c)

‘halo’ surrounding the sheath inserted perpendicularly inside the gel. 400; d) dark

spots of the watery saliva on surface of sucrose syrup with L-DOPA 10.


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After incubation in L-DOPA due to PPO action a major part of the glands and content

of midgut, especially the stomach and initial part of ‘intestine’ stained almost black

(Fig. 2).

Fig. 2. Areas of PPO activity within salivary glands and midgut revealed by

incubation in L-DOPA. 50.

‘Division’ of PPO amongst tissues of the aphid body

The following order of the activity was registered: salivary glands < alimentary canal < all

tissues of the aphid body remained after removal of salivary glands and alimentary canal.

As much as sixty per cent of PPO activity belongs to salivary glands and midgut together

(Fig. 3,4).


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Fig. 3. The activity of PPO extracted from R. padi tissues: 1,2 - whole aphid body; 3,4 -

salivary glands, 5,6 - alimentary canal

Fig. 4. ‘Division’ of PPO action amongst tissues of the aphid body; a) salivary glands; b)

midgut; c) tissues without s. glands and midgut

PPO activity in electrophoresis gel

Two fractions of protein of whole aphid body, salivary glands and alimentary canal

separated within the SDS-PAGE gel matrix indicating PPO activity after incubation with

L-DOPA solution were found (Fig. 5).

0

0,1

0,2

0,3

0,4

0,5

0,6

1 2 3 4 5 6

A/ 30 min / 100 aphids

A/ 30 min / mg protein

a)

b)

c)


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Fig. 5. Results of staining SDS-PAGE gels, first for PPO activity with L-DOPA and

subsequently for total fraction of proteins using Coomassie Brillant Blue

R-250 reagent; (a) salivary glands (b) midgut; (c) whole aphid body.

Discussion and conclusions

The results demonstrate that PPO is widely distributed within the bird cherry-oat aphid

body and that the aphid tissues are in general rich in this enzyme; as an example it is

easily detectible in the saliva, salivary glands and midgut of a single aphid. The

presence of PPO both in the gelling saliva (salivary sheath) which is secreted during

the stylet penetration between cells of the external plant tissues, and in watery saliva

injected predominantly into phloem [25,27], seems to signify for the aphid - host plant

interaction. There was observed that the gelling and watery saliva apparently diffused

into the agarose gel and sucrose syrup, respectively. Extrapolation to the plant, the

o-dihydroxyphenolics in the intercellular fluid of mesophyll [10] will be exposed to

rapid oxidation by the PPO of gelling saliva, and next PPO of the watery saliva will

oxidase o-diphenolics translocated in phloem [3,10]. There was already observed that


61

aphid saliva infiltrates tissues surrounding the feeding puncture and also reduces

concentrations of phenolic monomers in tissues sap. Moreover, increase in oxidative

activity of tissues around the feeding site and a deposition of phenolic polymers were

noticed [9,17]. Oxidation of wide range of phenolic compounds by the bird cherry -

oat aphid salivary secretions was detected using liquid diets [24]. Products of catechin

oxidation catalysed by PPO of the salivary glands and gut of the rose aphid became

more acceptable and even phagostimulant to the aphids [17,19,20,21]. Our earlier study

showed that PPO of the grain aphid was specific to spectrum of phenolic

compounds, e.g. gallic acid, caffeic acid, (+) catechin, quercetin, procatechuic acid

and chlorogenic acid [8,26,27] that are known to disturb seriously aphid biology and

thereby to determine a degree of resistance of winter wheat cultivars to the cereal

aphids, also to R.padi [6,7,8,23].

Occurrence of PPO in the midgut not seems to be pointless, as there was indicated that

the saliva while Aphididae are ingesting phloem sap is sucked back. Therefore, it is

difficult to escape the conclusion that oxidation of o-diphenolics takes place within the

midgut of R. padi. Amongst Aphididae as yet, PPO was detected in the midgut tissues

of the rose aphid and the grain aphid; it was able to oxidase many plant o-

dihydroxyphenolic compounds [21,26]. Unquestionably a majority of PPO of R. padi

is focused in salivary glands and alimentary canal, around sixty per cent together but

quite a few activity of this enzyme, nearly forty per cent has been recorded for the

tissues of the aphid body after taking out of the salivary glands and alimentary canal;

thus o-diphenolic oxidation seems to be continued outside of the midgut. There is

commonly known that hemolymph is rich in PPO.

The whole aphid body, salivary glands and alimentary canal PPO of R. padi was

composed of two fractions. The same result was obtained for homogenates from

whole S. avenae (Urbanska et al. unpublished data). On the other hand, Madhusudhan

& Miles [9] for salivary secretions of pea aphid and spotted alfalfa aphid injected into

water, presented three fractions of this enzyme, when in potato aphid only one protein

band showed PPO activity [18]; thus this turned out to be debatable subject.

In conclusion, it seems probable that PPO of R. padi oxidises / detoxifies

o-dihydroxyphenolics both without and within its organism, namely: (1) during stylet

penetration across of epidermal and mesophyll cells and phloem reaching, due to the

action of the gelling and watery saliva enzyme, respectively; (2) in the midgut while


62

transportation of ingested food and also in hemolymph and other tissues. The results

suggest that a majority of the aphid PPO is concentrated equally in the salivary glands

and midgut.

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Oksydaza polifenolowa mszycy czeremchowo-zbożowej

Streszczenie

Badano oksydazę polifenolową (PPO) mszycy czeremchowo-zbożowej

Rhopalosiphum padi L. w następujących aspektach: 1) lokalizacja w wydzielinach

ślinowych, oraz tkankach i narządach; 2) dystrybucja aktywności w organizmie; 3) skład

izoenzymatyczny. PPO zlokalizowano w żelującej i wodnistej frakcji śliny, gruczołach

ślinowych oraz jelicie środkowym mszycy. Zarejestrowano następujący szereg

aktywności PPO: gruczoły ślinowe przewód pokarmowy < tkanki/narządy, oprócz

gruczołów ślinowych i przewodu pokarmowego. W całych mszycach, gruczołach

ślinowych i przewodach pokarmowych zidentyfikowano dwa izoenzymy PPO. Badania

sugerują, że R. padi może utlenianiać/inaktywować związki fenolowe przy udziale PPO

w: 1)tkance rośliny żywicielskiej, z udziałem enzymu obydwu frakcji śliny; 2)

przewodzie pokarmowym, w pobranym pokarmie; 3) w hemolimfie i innych tkankach

organizmu, po ich przeniknięciu do wnętrza komórek.

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Polyphenol oxidase of bird cherry-oat aphid