441
Ph;'siological and .liolecular Plant Pathology \ 1993) 42, 441--454
Detection of several enzymatic activities in leaf prints of
cucumber plants
S. A. AvmusHKO,
X.
S. YE and J.
Kuc*
Department of Plant Pathology, Uni1'ersity of /,_-entuck)', Lexington, /,_-entuckr 40546, l'.S.A.
(Accepted for p11blication Atay 1993)
Leaf printing directly onto nitroce!!ulosc mcn1branes under high pressure was adapted for
screening activities of defence-related enzymes after inoculation of cucumber plants with
Colletotrichum lagenarium, tobacco necrosis virus �TN\') or treatment with dipotassium phosphate
and after inoculation of tobacco plants with tobacco mosaic virus ·:T'.\1\'). Increased activities of
peroxidase, polyphenol oxidase, lipoxygenase, chitinase and a-glucosidase were detected in
cucumber leaves in the vicinity of lesions caused by pathogens or phosphate application. The
activity of fi-glucosidases was increased in cucumber leaves by TN\', to a lesser extent with C.
lagenarium and was not altered by phosphate treatment. Galactosidasc activity was not induced in
cucumber leaves. Low activity of ,B-1,3-glucanase in cucumber precluded the detection of this
enzyme by the tissue printing technique. However, an increase of /J-1,3-glucanase activity was
detected around lesions caused by TivIV in tobacco. Peroxidase, polyphcnol oxidasc, a- and P­
glucosidases and galactosidase were detected with specific substrates on the 1nembranes after leaf
printing. Activities of chitinase and /J-1,3-glucanase were determined as lytic zones in substrate­
containing overlaying gels incubated with leaf prints on the membranes and lipoxygenase was
detected on the membranes by the reaction with specific antibodies. The leaf printing technique
is applicable to the determination of the spalial distribution of various defence-related enzymatic
activities and their rcle\'ance to disease resistance in plants.
INTRODUCTION
Tissue printing is a technique for the detection of metabolites and their distribution in
tissues. It has been widely used for the detection of R�A [19] and proteins using
specific antibodies [14, 23] or for assays for enzymatic activities [25]. The localization
of pathogenesis-related (PR) proteins in the vicinity of virus-induced lesions was shown
for tobacco leaves inoculated with tobacco mosaic virus (TMV) [13]. Leaf blotting
techniques provide sufficient resolution for demonstration of non-uniform distribution
of viral proteins in the whole leaf [15, 21]. The efficiency of the transfer of proteins to
nitrocellulose membranes was increased by abrasion of the epidermis and application
of high pressure [13].
Inoculation of cucumber plants with Colletotrichum lagenarium (5], tobacco necrosis
virus (TNV) [ 12] or treatment with dipotassium or disodium phosphate induces
*
To whom correspondence should be addressed.
Abbreviations used in the text: PR, pathogenesis-related; T1.1\r, tobacco mosaic virus; TNV, tobacco
necrosis virus; DOPA, 3,4-dihydroxyphenylalanine; RBB, Remazol brilliant blue.
0885 5765/93/060441+14 $08.00/0
© 1993 Academic Press Limited
442
S. A.
Avdiushko
et al.
systemic resistance [8, fl]. Induced resistance is associated \vith locally and
systemically increased activities of peroxidase and chitinase [11] and lipoxygenase
[1] in the leaves of cucumber plants. The experiments reported in this paper were
conducted to develop a whole leaf printing technique to determine the spatial
distribution of defence-related enzymes in cucumber leaves infected by or treated with
inducing agents.
MATERIALS AND METHODS
Biological material
Cucumber plants (Cucumis sativus L.) cv. Wisconsin SMR-58 were grown in the
greenhouse at 23-33 °C with a 14 h photoperiod as previously described [5].
Colletotrichum lagenarium (Pass.) Ell. and Halst. race 1 was maintained on green bean
juice agar at 24 °C in the dark. Conidial suspensions were prepared from cultures
which were 6-8 days old [5].
Plants, with the first true leaf (leaf l ) fully expanded and leaf 2 one-half expanded,
were used for induction of systemic resistance. A conidial suspension ( 10 µI) of C.
lagenarium (5 x 10,. conidia ml-1) or water (controls) was infiltrated at 30 sites into the
lower surface of leaf l using an Eppendorf repeating pipette. Resistance was also
induced by inoculation of!eaf l with TNV [12] or spraying the lower leaf surface with
50 mM dipotassium phosphate [JJ). To determine the level of induced resistance, plants
were challenged 7 days after induction by inoculation of leaf 2 with 20 10-µl drops of
a conidial suspension (5 x 10,. conidia ml-1) of C. lagenarium on the upper surface.
Disease caused by C. lagenarium was assessed 7 days after challenge by determining the
number and size of lesions.
Burley tobacco plants (Nicotiana tabacum L. cv Ky 14) were grown in the greenhouse
at 25-30 °C with a 14 h photoperiod. Purified tobacco mosaic virus (TMV), Ul strain
in water was kindly provided by Dr J. Shaw, Department of Plant Pathology,
University of Kentucky. Tobacco plants at the 9-10 leaf stage were inoculated with
25 µg ml-1 of TMV on three or four lower leaves in the growth room with a 14 h
photoperiod under white fluorescent and incandescent light at 23 °C (28].
Leaf blotting
The procedure of blotting was based on the technique described by Jung and Hahne
[13]. Leaf I was detached 7 days after treatment and soaked in 70 % ethanol for 30 s.
The lo\.ver epidermis was abraided by gently rubbing the leaf after dusting the surface
with carborundum. After blotting dry with absorbent paper, the leaves were placed on a
sheet of nitrocellulose membrane (pore size 0·45 µm), sandwiched between Whatman
3 MM paper and two blocks of plexiglass and pressed in a hydraulic press for 5 min at
45 kg cm-2• Proteins blotted to the nitrocellulose membrane were either stained for
2 min with O· l 0/0 amido black in an aqueous solution containing 45 % methanol and
IO °;S acetic acid, detected immunologically or assayed for enzymatic activities.
Experiments were repeated three times with one leaf per treatment per experiment.
Patterns of enzyme distribution were similar for each experiment.
Enzymatic activities in leaf prints of cucumber
443
Immunological detection of lipoxygenase
Lipoxygenascs \Vere detected by modifying a standard method employed in VVestern
blotting [27] using rabbit polyclonal antibodies raised against soybean leaf I and 3
lipoxygenase isozymes. Goat anti-rabbit IgG conjugated vvith alkaline phosphatase at
a dilution of l: 5000 was used to visualize lipoxygenase proteins.
Detection of poiyphenol oxidase
Leaf prints on nitrocellulose were incubated in O· l M sodium phosphate buffer, pH 6·5,
containing O· l M catechol [18]. After the development of intense yellow staining, blots
were washed with distilled water and dried. An additional substrate, 20 mM 3,4dihydroxyphenylalanine (DOPA) [16], also was used for the detection of polyphenol
oxidase activity.
Detectiurt of endonucleajej
Leaf prints on nitrocellulose were incubated for 30 min in O·O l M Tris-HCI buffer and
blotted to 10% native polyacrylamide gels (1-5 mm thick) containing 0·3 mg ml-1 of
either denatured calf thymus DNA or Torula yeast type VI RNA [2]. Gels and prints
on nitrocellulose were sandwiched between t\VO glass plates, incubated at room
temperature for 6 h to allow enzymatic degradation of embedded substrate and the gel
was stained with l 0/0 Toluidine blue in \vater [2].
Peroxidase determination
Leaf prints on nitrocellulose were incubated for 10 min in 10 mM sodium phosphate
buffer, pH 6·0. Peroxidase activity \Vas detected according to the procedure of Ye et al.
[29]. The prints were incubated on a shaker (70 r min-1) for 10 min in phosphate buffer
containing 0·6 mg of 4-chloro-1-naphthol per ml and 0· l M H202•
Detection of chitinase activity
Leaf prints on nitrocellulose were incubated in 150 mM sodium acetate buffer, pH 5·0
for 5 min followed by blotting of the prints to the gel (0·75 mm thick) containing
chitinase substrate [26]: 7·5 % (w /v) polyacrylamide gel containing O·O1 �o glycol
chitin in 10 mM sodium acetate buffer, pH 5·0. Prints on nitrocellulose and gels \Vere
sandvviched between two glass plates and incubated under moist conditions at 37 °C for
I h in a plastic container. Gels \Vere incubated with freshly prepared 0·01°0 Calcofluor
white M2R in 500 mM Tris-HCl buffer, pH 8·9 for 5 min followed by 1 h incubation
in djstilled water at room temperature. Lyric zones \Vere visualized under u. \'. light on
a transilluminator and photographed using orange filters.
Detection of /3-1,3-glucanases
The activity of /3-1,3-glucanases was determined in a buffered agar gel containing a
soluble substrate with a covalently-linked dye (laminarin linked to Remazol brilliant
blue (RBB)). Conjugated RBB substrate and substrate containing gel were prepared
according to Sock et al. [24]. Laminarin-RBB (8 1ng 1n\-1) and agar {25 tng 1111-1) \Vere
dissolved by boiling in I M sodium acetate buffer, pH 5·2. Gels \Vere cast by pouring the
hot mixture betvveen t\VO glass plates assembled \Vith l ·5 rn1n spacers. Leaf prints on
444
S. A Avdiushko
a
b
I
.
,.
d
c
l'rc. I. For legend see opposite.
et al.
Enzymatic activities in leaf prints of cucumber
445
nitrocellulose and laminarin-RBB-containing gels \Vere sandwiched between two glass
plates and incubated under moist conditions for 5 h at 37 °C. After incubation, the
agar gel \Vas destained overnight in 95 °·0 ethanol. Gel areas with enzyme activity
appeared almost colourless on a dark blue background. Gels were photographed using
an orange filter.
Detection ef a- and P-glucosidase and galactosidase
Leaf prints on nitrocellulose \Vere incubated for 10 min in 100 mM sodium phosphate
buffer, pH 6·0, followed by 4 h incubation in the same buffer containing the
appropriate substrates at 3 mg ml- 1 [25]: 6-bromo-2-naphthyl-IX- and P-o-gluco­
pyranoside for IX- and P-glucosidases, respectively, and 6-bromo-2-naphthyl-P-o­
galactopyranoside for galactosidase. Enzymatic activities were visualized by incubating
the membranes wlth the solution of Fast Blue B salt at 1 mg ml ·1 for 4 min,
RESULTS
The inoculation of the first true leaf of cucumber \Vith C. lagenarium or TNV or
treatment \Vith phosphate resulted in the enhancement of several enzymatic activities.
Peroxidase activity \Vas markedly increased in the vicinity of lesions caused by
inoculation or treatment with phosphate (Fig. 1). Peroxidase activity was detected in
the control plants [Fig. I (a)], but inoculation [Fig. I (b), (c)] or phosphate treatment
[Fig. l (d)J markedly enhanced peroxidase activity. The areas on the leaf prints where
peroxidase activity \Vas detected corresponded to the sites of lesion development (Fig.
2). The pattern of total protein distribution in the leaf prints on nitrocellulose was
similar both in the control and treated leaves as judged by amido black staining of the
blotted proteins (Fig. 3). Thus, it appears that the difference in the distribution of
enzymatic activities jn the leaf tissues upon treatment \Vith pathogens or phosphate
reflects the actual alterations occurring in vivo.
Polyphenol oxidase activity \vas also increased in the vicinity of lesions caused by C.
lagenarium, TN\1 or phosphate (Fig. 4). Of the two polyphenol oxidase substrates used
in the experiments, DOPA \Vas more sensitive, and its application was not accompanied
with che development of a dark background as was observed for catechol. The
distribution pattern of polyphenol oxidase activity (Fig. 4) was similar to that of
peroxidase (Fig. I ) .
A similar pattern of distribution \vas also observed for lipoxygenase and chitinase
using immunological visualization or glycol chitin-containing overlay gels (Figs. 5, 6).
The accumulation of the enzymes \vas associated primarily \Vith the sites of lesion
development caused by C. lagenarium, T:\\' or dipotassium phosphate.
Several other enzymatic activities \'\'ere altered in the first true leaves of cucumber
plants inoculated \vith C. lagenarium, TN\1 or treated with dipotassium phosphate. All
F1G. l. Peroxidase activity, detected \Vith 0·6 mg ml -l of4-ehloro- l -naphthol, on leaf prints of
cucun1ber plants on nitrocellulose filters. First true leaves of control plants 1 a) or those inoculated
\vith C. lagenarium (b), T�\' (c) or sprayed \Vith 50 mM dipotassium pho�phate '.d}. Peroxidase
activity increased markedly around k�ions caused by inoculation with pathogens or phosphate
treatment.
S. A. Avdiushko
446
b
a
c
d
et al.
Enzymatic activities in leaf prints of cucumber
447
the treatments caused an increase in a-glucosidase activity (data not shown). On the
other hand, the expression ofP-glucosidase \Vas markedly increased by inoculation with
TNV and to a lesser extent by inoculation with C. lagenarium (Fig. 7), and was not
altered after spraying with phosphate (data not shown). Galactosidase activity \vas
similarly expressed both in the induced and control plants (Fig. 8). P- 1,3-glucanase
activity could not be detected in the glucanase substrate-containing gels incubated
with the nitrocellulose prints of cucumber leaves. This result may be explained by the
low P-1,3-glucanase activity in cucumber leaf tissues [11]. In order to determine
whether the technique employed would be effective for the detection of the spatial
distribution of P- 1,3-glucanase, we used a different model system. Tobacco leaves
containing the N gene for resistance to T.MV were inoculated with TMV. Such leaves
contain high levels of /l- 1,3-glucanase activity [20, 28]. /l- 1,3-glucanase activity was
detected on leafprints of tobacco using a gel containing remazol - laminarin conjugate
and activity increased in the vicinity of the lesions caused by T�V development as
compared to control leaves (Fig. 9).
We were able to detect the association ofDNase and RNase activities with the lesions
caused by C. lagenarium, TNV or dipotassium phosphate. The areas of elevated R:-.Jase
or DNase activities were visible as pale spots against a blue background on the gels and
corresponded to the lesions caused by pathogens or phosphate treatment. However, the
difference in the colours of the background and the areas of enzymatic activities did not
produce good photo images (data not shown).
DISCUSSION
Inoculation of the cucumber plants with C. legenarium, TN\' or treatment with
phosphate induces defence-related reactions in the leaves such as lignification [9] or
increased peroxidase activity [JO]. Localized infection with necrotrophic pathogens in
general results in the appearance of new pathogenesis-related (PR) proteins that have
been found in many plant species including tobacco [6], bean [22], tomato [4],
co\vpea [3] and cucumber [7]. In cucumber plants1 one of the PR-proteins \Vas
characterized as chitinase [17]. In tobacco plants, one of the major PR-proteins was
characterized as a P- 1,3-glucanase [20].
In the present paper we describe the application of the leaf printing technique to the
detection of enzymatic activities in the pathogen-infected or chemically-treated
cucumber and tobacco leaves either by immunological visualization or by incubation
with substrates. The combination of these techniques (which have not been reported
previously for the detection of chitinase, nucleases, P-1,3-glucanase and lipoxygenase)
provides information about the spatial distribution of defense-related enzymatic
activities in leaf tissues and their relevance to plant resistance. Three methods were
used for the detection of enzyme activities on leaf prints. Lipoxygenase was visual­
ized after incubation of the prints on nitrocellulose vvith specific antibodies (Fig. 5).
FIG. 2. First true leave� of control cucumber plant (a) or those inoculated with C. lagenarium (b),
TNV (c) or sprayed with 50 mM dipotassium phosphate 1d) after printing on nitrocellulose
mcn1brancs used for peroxidase detection (see Fig. I). The necrotic lesions caused by inoculation
with pathogens or phosphate treatment are still visible on leaves after transfer to nitrocellulose and
can be aligned with peroxidase activity.
448
S. A. Avdiushko
et al.
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.
or those inoculated with C. lagen"rium (b), or TNV (c) or sprayed with 50 nlM dipo1assiu111
phosphate (d) arc shown. Proteins were similarly transferred 10 nitr<Kcllulosc.
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449
Enzymatic activities in leaf prints of cucumber
b
a
.·
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,
c
Fie. 4.
Polyphenol oxidasc activity, detected with
d
20 mM DOPA, on kaf prints of cucumber
plants. First true leaves of comrol plants :a) or those inoculated with C. lagmarium (b), TNV (c)
or sprayed with 50 mM dipotassium phosphate !d� are shown. Polyphenol oxidase activity
increased around lesions caused by inocul at i on with pathogens or phosphate treatment.
Peroxidase, polyphenol oxidase, a- and P-glucosidase and galactosidase were directly
detected on nitrocellulose prints after incubation with specific substrates (Figs 1, 4, 7,
8). Chitinasc, P-1,3-glucanase and endonucleases were detected in the specific
substrate-containing overlay gels which were incubated with prints on nitrocellulose
(Figs 6, 9).
The accumulation of most of the enzymes reported in this paper was associated with
lesions caused by pathogens or chemical treatment. However, P-glucosidase activity
450
S. A. Avdiushko
et al.
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Fm. 5. Immunological visualization oflipoxygenasc on leaf prints of cucumber plants. First true
leaves of control plants (a) or those inoculated with C. iagenarium (b) or TNV (c) or sprayed with
50 mM dipotassium phosphate (d) are shown. Lipoxygenase accumulation was increased around
lesions caused by inoculation with pathogens (indicated by arrows for C. lagenarium) or phosphate
treatment.
was highly increased in cucumber leaves after TNV infection, less increased by
inoculation with C. lagenarium (Fig. 7) and was not altered by phosphate treatment.
Galactosidase activity was not induced in cucumber leaves by pathogens or phosphate
(Fig. 8). The pattern of spatial distribution of enzymatic activities in the leaf cannot
be obtained by enzyme assays of leaf homogenates, but can be provided by the leaf
printing technique.
The procedures described in this paper may be applicable for the detection of spatial
distribution not only of the above mentioned enzymes, but also of other enzymes.
Immunological visualization is limited only by availability of the specific antibodies of
interest. The criterion for the detection of enzymatic activities on the leaf prints on
nitrocellulose either directly or after incubation with substrate-containing overlay gels
is to find a proper substrate that forms a coloured product on the membrane or
traceable zones on the gels. This method can provide useful information about the
451
Enzymatic activities in leaf prints of cucumber
b
a
d
c
FIG. 6. Chitinase act1v1ty, detected after leaf prints of rm·umber plants on nitrocellulose
membranes were blotted to a gel containing
O·Ol 00 of glycol chitin. Lytic zones were visualized
0· l 0 0 Calcofluor white M2R. First true leaves of
C. lagenariwn ib:, T�\" ;c; or sprayed with 50 mM
under u.v. light after incubation of the gel with
control plants
(a)
or those inoculated with
dipotassium phosphate (d) are shown. Chitinase activity increased around lesions caused by
inoculation with pathogens or phosphate treatment.
452
S. A Avdiushko et al.
b
a
c
·.
FrG. 7. P-Glucosidasc activity, detected with 3 mg m1-1 6-bromo-2-naphthyl-,B-o-gluco­
pyranosidc and virnalized with l mg ml-1 of Fast Blue B, on leaf prints of cucumber plants. First
true leaves of control plants (a) or those inoculated with C. lagmarium (b) or with TNV (c) arc
shown. The activity of ,8-glucosidasc incre,tscd markedly around lesions caused by inoculation
with TNV and to a lesser extent around lesions caused by C. lagenarium.
F1G. 8. Galactosidasc activity, detected with 3 rng mi-• 6-bromo-2-naphthyl-ft-n-galacto­
pyranosidc and visualized with l mg rnl-1 of Fast Blue B, on leaf prints of cucumber plants. First
true leaves of control plants (a) or those inoculated with C. lagerwrium (b). TNV (c) or sprayed
wirh 50 mM dipotassium phosphate (d) are shown. Galactosidase activity was similarly distributed
in control or induced lcaYcs.
453
Enzymatic activities in leaf prints of cucumber
a
b
FIG. 9. fi-1,3-Glucanase activity in kaws from control tnhacrn plants a) or those inoculated
with Tl\1V (b). Leaves wt·rc printed onto nitrocellulose membranes, and the prints were blotted
to agar gels containing laminarin-RBB conjugate at 8 mg ml -1. Lytic zones corresponding to /J1,3-gluranasc activity were detected around necrotic lrsions cami-d by T�I\'.
association of certain enzymes with disease developmtn t and resistance response of
different plant species.
The authors thank Dr David Hildebrand, Agronomy Department, University of
Kentucky, for the kind gift of lipoxygenase antiserum. The research reported in this
paper was supported in part by grants from the R.j. Reynolds Tobacco Company and
Cooperative Agreement 43YK-5-0030 of the USDA-A.RS.Journal paper 92- 1 1-176 of
the Kentucky Agricultural Experimental Station, Lexington, KY 40546.
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