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The Effects of Phenolic Compounds on Peroxidase and Polyphenol Oxidase in Dwarf Pea (Pisum sativum var. «Little Marvel») Tissue
Department of Biological Sciences, Oakland University, Rochester, Michigan 48063, U. S. A.
The Effects of Phenolic Compounds on Peroxidase and Polyphenol Oxidase in Dwarf Pea (Pisum saiivum var. «Little Marvel») Tissue EGBERT
W.
HENRY, JAMES M. DEMoRROW
and
LAWRENCE
B.
RICHARD
With 6 figures Received June 6, 1978 . Accepted November 8, 1978
Summary Ethrel (3 ppm) and ultraviolet irradiation (UV), separate and in combination with visible or dark, were applied to seeds of dwarf pea (Pisum sativum, var. «Little Marvel,,) to determine their effect in the presence of sixteen different substrates, on polyphenol oxidase and peroxidase activity in 20 day-old distal root, root tip, leaf, and proximal stem tissues respectively. In terms of polyphenol oxidase activity, the enzyme is light dependent and activity is highest in visible light-exposed tissue, followed by UV plus Ethrel-treated tissues in comparison to untreated controls. Distal root and root tip tissues generally contained higher PPO activity than did the leaf or proximal stem tissues. Hydroxyanthranilic acid, catechol, and pyrogallol appear to be the most reactive substrates for PPO activity. Ethrel caused an increase in peroxidase activity in root tip and proximal stem tissues and depressed enzymic activity in distal root tissue. Leaf tissue had the highest level of peroxidase activity and etiolated tissue demonstrated higher levels of peroxidase activity than did visible lighttreated tissue. In addition to showing high specificity with the three aforementioned substrates that were cited as being specific for PPO, peroxidase activity was also specific with guaiacol and p-phenylene diamine.
Key words: polyphenol oxidase, peroxidase, ethrel, UV irradiation, dwarf pea.
Introduction
It has been proposed that a close affinity exists between peroxidase, indoleacetic acid oxidase (IAA oxidase), and polyphenol oxidase (PPO), (SHEEN, 1969; PILET et al., 1970; HOYLE, 1972; SRIVASTAVA and VAN HUYSTEE, 1973). An association among polyphenol oxidase and peroxidase has been reported (SHEEN, 1969); however, there are counter-proposals to this idea (VAN LOON, 1971; STAFFORD and GALSTON, 1970). HINMAN and LANG (1965) identified the intermediates resulting from the IAA oxidation due to horseradish peroxidase. Subsequently, RICARD and JOB (1974) proposed a reaction mechanism for the degradation of indole-3-acetate by peroxidase. Z. Pflanzenphysiol. Bd. 92. S. 221-240. 1979.
222
EGBERT W. HENRY, JAMES M. DEMoRROW and LAWRENCE B. RICHARD
However, other investigators question these reactions (SEQUEIRA and MINEO, 1966; RUBERY, 1972). It has recently been suggested that PPO and IAA oxidase share the same active site on the apoenzyme (SRIVASTAVA and VAN HUYSTEE, 1977). Polyphenol oxidase is a copper-containing enzyme that can catalyze two types of reactions involving the molecular form of oxygen (HENRY and JORDAN, 1977). The first type of reaction concerns the hydroxylation of monophenols, with the subsequent formation of o-dihydroxy compounds to quinones. PPO in some plants can undergo both of these reactions (RIVAS and WHITAKER, 1973). PPO may be present, in a latent form, in all plant chloroplasts (TOLBERT, 1973). It appears that PPO activity is light-dependent (TOLBERT, 1973). The ability of low light intensity to cause PPO activation in chloroplasts supports the suggestion that a configurational change and not electron transport, may be critical in PPO light activation of chloroplasts (TOLBERT, 1973). Many studies have been done on the damaging effects of ultraviolet (UV) irradiation on higher plants (KELNER, 1949; SINGER and AMES, 1970; EL-MANSY and SALISBURY, 1971; HENRY et al., 1977; DEMoRROW and HENRY, 1978). UV irradiation effects on plants may be via dimerization of thymine (WACKER et al., 1962; WITKIN, 1966); the breaking of the sugar phosphate bonds in the DNA double helix and the hydration of cytosine (BOLLUM and SETLOW, 1963; SETLOW et al., 1963; SINGER and AMES, 1970). It has been suggested that UV irradiation may cause photochemical responses, such as enhanced hydrogen peroxide production, thus causing plant tissue injury (LOCKHART and BRODUHRER-FRANZGROTE, 1961). Some plants, when treated with ethylene gas, exhibit an enhancement in peroxidase activity (RIDGE and OSBORNE, 1971). In addition, the observed increase in peroxidase activity may be due to de novo synthesis of the enzyme (RIDGE and OSBORNE, 1971). Ethrel: CICH 2CH2PO aH 2, undergoes an endogenous conversion to ethylene via base-catalyzed reaction, attributable to the action of hydrolytic enzymes (COOKE and RANDALL, 1968). If ethylene is applied to pea stem sections, there is a reduction of polar auxin transport (BURG and BURG, 1967). In addition, ethylene inhibits lateral IAA movement in isolated pea stem segments (BURG and BURG, 1968). Since Ethrel (a mixture of 2-chlorethanephosphoric acid and its ethyl ester) has similar effects to ethylene (MORGAN, 1969), we investigated the effects of UV irradiation, with and without Ethrel, on both light and darkgrown «Little Marvel» dwarf pea (Pisum sativum) seedlings, with respect to peroxidase and PPO activity vs. sixteen phenolic substrates. Materials and Methods Dwarf pea (Pisum sativum, var. «Little Marvel») seeds were sterilized with 1 % sodium hypochlorite, with subsequent rinsing in distilled water. The seeds were then separated into control and experimental (treated) lots. The experimental seeds were soaked for 24 h in a solution of 3 ppm Ethrel, with the non-Ethrel-treated control seeds being soaked in distilled
z. PJlanzenphysiol. Bd. 92. s. 221-240.1979.
Peroxidase and polyphenol oxidase activity
223
water. Both control and treated seeds were separated into three lots of fifty seeds each respectively and germinated for 20 days in growth chambers at a temperature of 23°C, under the following conditions: 1. complete darkness; 2. visible light; 3. UV irradiation. The growth chambers were equipped with banks of fluorescent tubes and incandescent bulbs, providing a light intensity of 3000 ft-C. For UV irradiation, four 18 in. (45-cm) germicidal tubes (Sylvania, low pressure mercury are, 15 W) were used to provide a dosage level of 1-10 callcm2 (HENRY et al., 1977; DEMoRROW and HENRY, 1978). Ten-mm sections of tissue (distal root, root tip, leaf, and proximal stem) were selected from the control and treated seedlings. After excision of the four separate tissues, each sample was weighed and immediately put into pre-chilled mortars at 4 0c. The tissue samples were ground at 4 °c for 5 min in 4-ml of 0.01 M phosphate buffer, pH 6.0. The respective samples were then centrifuged (15,000 g) at 4°C for 15 min to remove cellular debris. The respective supernatants were used for the following assays: Peroxidase: Spectrophotometric determinations were measured at a wavelength of 460 nm (HENRY et al., 1977). Polyphenol oxidase: Readings were taken at 475 nm (HENRY and JORDAN, 1977). Protein: Protein determinations were measured spectrophotometrically, with bovine serum albumin being used as a standard (LOWRY et al., 1951).
Key for 16 substrates used in Assay: Substrate and equivalent number Number used in text 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13. 14. 15. 16.
Name Phenol 3-Hydroxyanthranilic acid 3,4-Dihydroxyphenylalamine Hydroquinone Resorcinol Orcinol Catechol Caffeic acid Ferulic acid Chlorogenic acid Pyrogallol Phloroglucinol p-Phenylene diamine I-Phenylene diamine Diethyldithio carbamic acid Guaiacol
Results
Polyphenol oxidase: Dark, Dark plus Ethrel, Visible, Visible plus Ethrel (Figs. 1-3). a) Dark plus Ethrel- depressed PPO activity. b) Visible plus Ethrel- enhanced PPO activity. c) Visible - highest PPO activity vs. control. Z. PJlanzenphysiol. Bd. 92. S. 221-240. 1979.
EGBERT W. HENRY, JAMES M. DEMoRROW and LAWRENCE B. RICHARD
224
PPO ACTIVITY DARK I(J DARK DISTAL
2.0
+
1A
ETHREL
ROOT
. 1.8 1.8 1.4
,
ci>
1.2
>-
1.0
.~
;:;
<
.8
n. n.
.8
o
A
•2
"I
II 2
3
4
5
.-
8
I I.
7
8
.•.1 II
10
11
11.11• 12
13
..
14
,III 15
18
Substrates PPO ACflVITY DARK Vj DARK ROOT
2.0
+
18
ETHREL
TIP
1.8 1.8 1.4 Q
1.2
f
1.0
!;! o
n. n.
.8
.8 .4 .2
,. 2
3
4
5
8
7
,-
,_ III
8
9
10
11_ .• 11
12
13
14
15
18
Substrates
Figs. 1-3: Polyphenol oxidase (PPO): Dark (1 A-D), Dark plus Ethrel (1 A-D), Visible (2 A-D), Visbile plus Ethrel (2 A-D), and UV (3 A-D) vs. UV plus Ethrel (3 A-D). PPO activity is shown for distal root (1 A, 2 A, 3 A), root tip (1 B, 2 B, 3 B), leaf (1 C, 2 C, 3 C) and proximal stem (1 D, 2 D, 3 D), using substrates # 1-16 (.) vs. the corresponding control
(I D.
Z. Pjlanzenphysiol. Bd. 92. S. 221-240.1979.
225
Peroxidase and polyphenol oxidase activity PPO ACTIVITY DARK'!l' DARK
+
ETHREL
Ie
LEAF
1. distal root and root tip - highest levels of PPO activity (Figs. 1-3). 2. leaf and proximal stem - lower relative levels of PPO activity (Figs. 1-3). 3. most active substrates: 2, 7 and 11 for distal root, root tip, leaf and proximal stem tissues (Figs. 1-3). UV vs. UV plus Ethrel (Figs. 1-3). Z. PJlanzenphysiol. Bd. 92. S. 221-240. 1979.
226
EGBERT
W.
HENRY, JAMES M. DEMoRROW PPO VIS.
LIGHT '
2. 0
and
LAWRENCE
B.
RICHARD
ACTIVITY
VIS.
LIGHT
DISTAL
ROOT
+
2A
ETHREL
1.8 1.6 .c
""
~
1.4
,C>
1. 2
:: .~
1 .0
.u o
""-
8 6
4
11II
2 I
I
I
2
3
4
5
6
7
8
I 10
9
11
I I.
12
13
I 14
I
I
15
16
Substrates ACTIVITY
PPO VIS.
LIGHT \(§
VIS.
ROOT
2.0
LIGHT
+
28
ETHREL
TIP
1.8
i: .c
""
~
1.6 1.4
ci>
1.2
?:-
1.0
..u .~
0
"0..
.8 .6 A .2
I 2
3
4
5
III
6
7
8
9
10
11
III 12
13
.1
14
15
16
Substrates
Distal Root: a) Substrates: 2 and 11 - UV vs. UV plus Ethrel-treated tissues have equally high levels. b) Substrate: 7-UV has higher PPO level than UV plus Ethrel. c) Substrate: 8-UV - treated tissue has a much lower level of PPO activity than does UV + Ethrel. Z. P/lanzenphysiol. Bd. 92. S. 221-240. 1979.
227
Peroxidase and polyphenol oxidase activity ppo VIS.
lIG H T '!!i
ACTIVITY
VIS.
+
LIGHT
2C
ETHREL
LEAF
2.0 1.8
~
1.6
~
1.4
" ,0>
~
:: .~
;:;
« 0
Q. Q.
1.2 1.0
.8 .6 .4 .2 13
14
15
16
Substrates ACTIVITY
PPO VIS.
LIGHT v.1
VIS.
LIGHT
PROXIMAL
2.0
+
2D
ETHREL
STE M
1.8 1.6 1.4 0>
1.2
.~
1.0
«
.8
;:;
o
Q. Q.
.6 .4
.2
1 2
~
3
I_III 4
5
,I
6
I
7
8
9
I
10
11
II. 12
I
13
II. 14
11_11_ 15
16
Substrates
d) Substrates: 9 and 15 - UV plus Ethrel-treated tissue has higher level of PPO activity t-an does UV alone (Fig. 3 A). Root Tip: a) Substrates: 2 and 11 - UV vs. UV PPO levels.
+ Ethrel-treated
tissues have equally high
Z. Pjlanzenphysiol. Bd. 92. S. 221-240. 1979.
228
EGBERT
W.
HENRY, JAMES M. DEMoRROW PPO UV ~
and
LAWRENCE
B.
RICHARD
ACTI V I TY
+
UV
DIST~
L
3A
ETHREL ROOT
2.0 1.8
ii ~
1.6
u:"
1.4
ci>
1.2
,., ~
v « 0
""-
1.0 .8 .6 .4 .2 2
7
8
Subslrates PPO UV v,}
ACTIVITY
+
UV ROOT
2.0
9
38
ETHREL TIP
1.8 ~
1.6
'"'" u:'"
1.4
ci>
1.2
:!"
1.. 0
~
.8
0-
.6
.~
o0-
A
2
,I 2
3
II.ii_ 4
5
III
7
6
I 8
IIII 9
10
11
12
13
II
14
I
I
15
16
Substrates
b) Substrates: 3, 8, 15, 16 and 13 - UV of PPO than does UV alone.
+ Ethrel-treated tissue has a higher level
c) Substrate: 4-UV - treated tissue has more PPO activity than does UV (Fig. 3 B).
Z. Pjlanzenphysiol. Bd. 92. S. 221-240. 1979.
+ Ethrel
229
Peroxidase and polyphenol oxidase activity PPO UV ~
ACTIVITY
UV
+
ETHREL
3C
LEAF
2.0 1.8 ~ -;:;
1.6
u:
1.4
'"
1.2
?:
1.0
Q)
>
U
«
.8
0
11. 11.
.6 .4 .2
Substrates
PPO UV
v.}
UV
ACTIVITY
+
ETHREL
3D
PROXIMAL STEM
2.0 1.8 ~ -;:;
u:
Q)
1.6 1.4
'"
1.2
>
1.0
~ « ,0 11. 11.
.8 .6 A
.2 2 Substrates
Leaf: a) Substrates: 2, 7 and 11 - UV-treated tissue has higher level of PPO activity than does UV + Ethrel. b) Substrates: 1,3, 8, 9, 10, 12, 15 and 16 - UV-treated tissue has a higher level of PPO activity than does UV alone (Fig. 3 C).
Z. Pjlanzenphysiol. Bd. 92. S. 221-240. 1979.
230
EGBERT W. HENRY, JAMES M. DEMoRROW and LAWRENCE B. RICHARD
4A
16
48
Figs. 4-6: Peroxidase: Dark (4 A-D), Dark plus Ethrel (4 A-D, Visible (5 A-D), Visible plus Ethrel (5 A-D), and UV (6 A-D) vs. UV plus Ethrel (6 A-D). Peroxidase activity is shown for distal root (4 A, 5 A, 6 A), root tip (4 B, 5 B, 6 B). leaf (4 C, 5 C, 6 C) and proximal stem (4 D, 5 D, 6 D), using substrates # 1-16 (.) vs. the corresponding control
(II)·
Z. PJlanzenphysiol. Bd. 92. S. 221-240. 1979.
Proximal Stem: a) Substrates: 2, 7 and 11 - UV -treated tissue has a higher level of PPO activity than does UV + Ethrel. b) Substrates: 3, 5, 8 and 9 = UV + Ethrel-treated tissue has a higher level of PPO activity than does UV alone (Fig. 3 D).
z. Pjlanzenphysiol. Bd. 92. s. 221-240. 1979.
232
EGBERT
W.
HENRY, JAMES M. DEMoRROW PEROX IDASE VIS.
LIGHT '(}
VIS.
DISTAL
2.0
and
LAWRENCE
B.
RICHARD
ACTIVITY LIGH T T
5A
ETHREL
ROOT
1.8 ~
1.6
~
1.4
.::" 0>
"-
.;
.
U ~
1.2 1.0 .8
'"x
.6
"
.4
~
f'
a.
.2
IIJ 2
3
4
6
5
7
8
9
Substrales PEROXIDASE VIS.
2.0
LIGHT
v~
VIS.
ROOT
10
11lJ •
ACTIVITY LIGHT T
ETHREL
13
1-
14
15
16
58
TIP
~ .t::
...!" cio
1.2
:::
1.0
.
~
U
. .
~
~
.8
.6
K
~
.4
Q.
.2
15
16
Substrates
Peroxidase:
Dark, Dark + Ethrel, Visible, Visible + Ethrel (Figs. 4-6). a) leaf - most enhancement of peroxidase activity (Fig. 4 C, 5 C, 6 C). b) Proximal stem - slight increase in peroxidase activity under all light conditions (Fig. 4 D). Z. PJlanzenphysiol. Bd. 92. S. 221-240. 1979.
Peroxidase and polyphenol oxidase activity PEROXIDASE VIS
LIGHT It}
VIS
ACTIVITY
5C
+ ETHREL
LIGHT
233
LEAF
2.0 1.8
1.6 1.4
'"
1.2
.~
1.0
,..
U
«
.8
.2
2
3
4
5
6
7
8
9
16
10
Substrates PEROXIDASE VIS.
2.0
LIGHT It}
V'S
PROXIMAL
ACTIVITY LIGHT
+
50
ETHREL
STEM
1.8
1.6 1.4
'"
1.2
;
1.0
U
«
., ~
~ o
Q;
Q.
.8 .6 .4
9
10
11
12
13
14
15
16
Substrates
c) Ethrel - depressed peroxidase activity under all light conditions (Figs. 4-6). d) Etiolated vs. light - etiolated tissues show a higher level of peroxidase activity than tissues exposed to visible light (Figs. 4-5). 1. Most active substrates: 2, 7, 13 and 16 (Figs. 4-5). Z. Pjlanzenphysiol. Bd. 92. S. 221-240. 1979.
234
EGBERT
W.
HENRY, ]AMES M. DEMoRROW
+
UV
LAWRENCE
B.
RICHARD
ACTIVI TV
PEROXIDASE UV Y.j
and
6A
ETHREL
ROOT
DISTAL
2.0 1.8
3
1.6
.;:"
"
1.4
'"
1.2
?:
1.0
s:;
~
U
«
..'"
.8
;;;
.4
"0
g
a.
.6
2
3
4
5
7
6
8
Substrates PEROXIDASE
uv
v~
uv +
10
11
AC rlVITY
12
13
14
15
16
68
ETHREL
TIP
ROOT
2.0
9
1.8
3
..
1.6
.;.
1.2
?:
1.0
u «
.8
s:;
.::
:~
'..."
"0
1.4
.6
;<
0
;;
A
Q.
.2 2
3
4
5
6
7
8
9
10
11
16
Substrates
Distal Root: UV vs. UV + Ethrel (Figs. 6 A). a) Substrates: 2 and 15 - UV vs. UV + Ethrel-equally high levels of peroxidase activity. b) Substrates: 1, 5,6, 7, 8, 9, 10 and 16 - UV-treated has higher peroxidase level than UV + Ethrel.
z. Pflanzenphysiol. Bd. 92. s. 221-240. 1979.
Peroxidase and polyphenol oxidase activity PEROXIDASE
uv
~
6e
ACTIVITY
uv +
235
ETHREL
LEAF
2.0 1.8 ~ ~
.::" ,
Cl
,.
"
\;
«
.. ~
'2 0
Oi
n.
1.6 1.4 1.2 1.0 .8 .6 .4 .2
2
3
4
5
10
11
12
II
13
14
15
16
Substrates PEROXIDASE UV V.Ji
2.0
9
8
uv +
PROXIMAL
ACTIVITY
60
ETHREL STEM
1.8
~ ~
1.6
'"
1.4
u:" Cl
1.2
?:
1.0
;:; «
.8
.~
.~
.'2 0
Q;
""
.6
A .2
7
8
9
10
11
16
Substrates
c) Substrates: 3, 12 and 13 - UV + Ethrel has higher level of peroxidase than does UV -treated alone (Fig. 6 A). Root Tip: a) Substrates: 2 and 15 - UV vs. UV activity.
+ Ethrel
equally high levels of peroxidase
z. Pjlanzenphysiol. Bd. 92. s. 221-240. 1979.
236
EGBERT
W.
HENRY, JAMES M. DE MORROW
and
LAWRENCE
B.
RICHARD
b) Substrates: 7, 11, 16 and 3 - UV-treated tissue has higher peroxidase activity than UV + Ethrel. c) Substrates: 1, 5, 6, 8, 9, 10, 12 and 13 - UV + Ethrel has higher peroxidase activity than does UV alone (Fig. 6 B). Leaf: a) Substrates: 2, 3, 7, 11, 15 and 16 - UV vs. UV + Ethrel - equally higher in peroxidase activity. b) Substrates: 1, 4, 5, 6, 10 and 14 - UV-treated tissue has higher level of peroxidase activity than does UV + Ethrel. c) Substrates: 8, 9 and 13 - UV + Ethrel - treated tissue has higher level of peroxidase activity than does UV alone (Fig. 6 C). Proximal Stem:
a) Substrates: 2 and 15 - UV vs. UV + Ethrel - equally high levels of peroxidase activity. b) Substrates: 10 and 11 - UV-treated tissue has higher levels of peroxidase activity than UV + Ethrel. c) Substrates: 1, 3, 4, 5, 7, 8, 9, 12, 13, 14 and 16 - UV + Ethrel-treated tissue has a higher level of peroxidase activity than does UV alone (Fig. 6 D). Discussion The largest increase in PPO activity occurs in the various visible light treatments (Fig. 2). These results suggest a light-dependent requirement for PPO activity which agrees with earlier observations of TOLBERT (1973). In the present studies, PPO activity was depressed in Dark + Ethrel-treated tissues (Fig. 1). While UV + Ethreltreated tissue showed enhanced PPO activity, the enzyme activity was highest in Visible Light-treated tissues (Fig. 3). With respect to substrate specificity, hydroxyanthranilic acid, catechol, and pyrogallol appear to be the most reactive with dwarf pea tissue (Figs. 1-3). In terms of tissues, higher levels of PPO activity was observed in distal root and root tip than in leaf or proximal stem samples (Figs. 1-3). Under all light conditions, Ethrel caused an increase in peroxidase activity in root tip tissues compared to untreated controls (Fig. 4-6). Conversely, Ethrel caused lowered enzymic activity in distal root tissue under similar light conditions (Figs. 4-6). Proximal stem tissue showed a relatively small increase in peroxidase activity under all light conditions compared to untreated controls (Figs. 4 D, 5 D, 6 D). Etiolated tissues demonstrated higher levels of PPO activity than did light exposed seedlings (Figs. 4-5). The leaf tissue exhibited the highest degree of PPO enhancement in light-treated tissues compared to untreated controls (Figs. 4 C, 5 C, 6 C). Z. Pflanzenphysiol. Bd. 92. S. 221-240.1979.
Peroxidase and polyphenol oxidase activity
237
It is well known that ethylene induces peroxidase activity in plant tissues (SHANNON et al., 1971 MORGAN, 1969; HENRY et al., 1977). The effect of UV irradiation on causing an increase in peroxidase activity in several plant tissues may well be due to its effect on mediating ethylene production (ENDO, 1967; SREBO et al., 1972). In the present studies, Ethrel caused a greater enhancement in PPO activity than in peroxidase activity (Figs. 1-6). It has recently been proposed that a single protein molecule may possess three enzyme activities: peroxidase, PPO and IAA oxidase (SRIVASTAVA and VAN HUYSTEE, 1977). There are other examples of a single protein molecule having more than one enzyme activity: 1. the dual enzyme capability of certain plant phenolases (CHALICE and WILLIAMS, 1970); and 2. the evidence that IAA oxidase activity and guaiacol peroxidase activity may be due to the dual catalytic function of one enzyme (HOYLE, 1972). The present studies indicate that the monohydroxy, dihydroxy and trihydroxy compounds (substrates: 2, 7 and 11 respectively) are specific for both PPO and peroxidase, while guaiacol and p-phenylene diamine are also specific for peroxidase. However, it remains an open question as to the number of active sites on the protein molecule responsible for either PPO or peroxidase activities. As PPO is present in very immature chloroplast lamellae (HENRY, 1976) and light-dependent (TOLBERT, 1973) it is possible that it may be somewhat different as a protein molecule - from the peroxidase moiety. It has been suggested that a specific function of peroxidase in germinating plant cells may be correlated with the existence of a specific molecular configuration of the protein (GORDON and ALLDRIDGE, 1971). The possibility of enzymes such as PPO and peroxidase having more than one reactive site may, in part, explain their apparent differential activities with respect to the sixteen substrates assayed in the present study. Acknowledgements We thank Ms. MARY N. O'CONNOR for skilled technical assistance. References BOLLUM, F. J. and R. B. SETLOW: Ultraviolet inactivation of DNA primer activity. I. Effect of different wavelengths and doses. Biochem. Biophys. Acta 68, 599-607 (1963). BURG, S. P. and E. A. BURG: Molecular requirements for the biological activity of ethylene. Plant Physiol. 42,144-152 (1967). - - Ethylene formation in pea seedlings. Its relation to the inhibition of bud growth caused by indole-3-acetic acid. Plant Physiol. 43, 1069-1074 (1968). CHALICE, J. S. and A. H. WILLIAMS: A comparative biochemical study of phenolase apecificity in Malus, Pyrus and other plants. Phytochem. 9, 1261-1265 (1970). COOKE, A. R. and D. I. RANDALL: Properties of phenoloxidase in Mycobacterium leprae. Nature 218, 973-974 (1968). DEMoRROW, J. M. and E. W. HENRY: Substrate comparability vs. polyphenol oxidase activity in Ethrel and ultraviolet-treated «Little Marvel" dwarf Pea (Pisum sativum) tissue. Z. Pflanzenphysiol. 86, 353-361 (1978).
z. Pjlanzenphysiol. Bd. 92. s. 221-240. 1979.
238
EGBERT W. HENRY, JAMES M. DEMoRROW and LAWRENCE B. RICHARD
ENDO, T.: Comparison of effects of gamme-rays and maleic hydrazide on enzyme systems of maize seeds. Radiat. Bot. 7, 35-40 (1967). EL-MANSY, H. I. and F. B. SALISBURY: Biochemical responses of Xanthium leaves to ultraviolet radiation. Radiation Botany 11, 325-328 (1971). GORDON, A. R. and N. A. ALLDRIDGE: Cytochemical localization of peroxidase A' in developing stem tissues of extreme dwarf tomato. Can. J. Bot. 49,1487-1496 (1971). HENRY, E. W.: The ultrastructural localization of polyphenol oxidase in chloroplasts of Brassica napus cv. ZEPHYR. Z. Pflanzenphysiol. 78,446-452 (1976). HENRY, E. W., C. NCHEKWUBE, and J. Russo: Physiological responses of dwarf pea (Pisum sativum, var. «Little Marvel») seedlings to Ethrel and UV irradiation. Z. Pflanzenphysiol. 81,459-465 (1977). HENRY, E. W. and W. JORDAN, III: The enzymic response of pea (Pisum sativum) stem sections to applied indoleacetic acid, gibberellic acid and Ethrel: Catalase, peroxidase and polyphenol oxidase. Z. Pflanzenphysiol. 84, 321-327 (1977). HINMAN, R. L. and J. LANG: Peroxidase catalyzed oxidation of indole-3-acetic acid. Biochern. 4, 144-158 (1965). HOYLE, M. c.: Indole acetic acid oxidase. A dual catalytic enzyme. Plant Physiol. 50, 15-18 (1972). KELNER, A.: Effect of visible light on the recovery of Streptomyces griseus conidia from ultraviolet irradiation injury. Proc. Nat. Acad. Sci. 35, 73-79 (1949). LOCKHARD, J. and U. BRODUHRER-FRANZGROTE: The effect of ultraviolet radiation on plants. In: W. RUHLAND (Ed.), Encyclopedia of Plant Physiology XVI., pp. 532-554. SpringerVerlag, Berlin, 1961. LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL: Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193, 265-275 (1951). MORGAN, P. W.: Stimulation of ethylene evolution and bascission in cotton by 2-chlorethanephosphonic acid. Plant Physiol. 44, 337-341 (1969). PILET, P. E., P. LAVANCHY, and S. SEVHONKIAN: Interactions between peroxidase, polyphenol oxidase and auxin oxidase. Physiol. Plant 23,800-804 (1970). RICARD, J. and D. JOB: Reaction mechanism of indole 3-acetate degradation by peroxidases. A stop flow low temperature spectroscopy study. Eur. J. Biochem. 44, 359-374 (1974). RIDGE, I. and D. J. OSBORNE: Role of peroxidase when hydroxy-proline-rich protein in plant cell walls is increased by ethylene. Nature New BioI. 229, 205-209 (1971). RIVAS, N. D. and J. R. WHITAKER: Purification and some properties of two polyphenol oxidases from bartlett pears. Plant Physiol. 52, 501-507 (1973). RUBRY, P. H.: Studies in indole acetic acid oxidation by liquid medium from crown gall tissue culture cells; the role of malic acid and related compounds. Biochim. Biophys. Acta 261,21-34 (1972). SEQUEIRA, L. V. and L. MINEO: Partial purification and kinetics of indole acetic acid oxidase from tobacco roots. Plant Physiol. 41,1200-1208 (1966). SETLOW, R. B., P. A. SWENSON, and W. L. CARRIER: Thymidine dimers and inhibition of DNA synthesis by ultraviolet irradiation of cells. Science 142, 1464-1466 (1963). SHEEN, S. J.: The distribution of polyphenols, chlorogenic acid oxidase and peroxidase III different parts of tobacco, Nicotiana tabacum. Phytochem. 8, 1839-1847 (1969). SINGER, C. E. and B. M. AMES: Sunlight ultraviolet and bacterial DNA base ratios. Science, 170, 822-826 (1970). SREBO, Z., H. LACH, and E. SZIRMAI: A novel aspect of the response of the central nervous system to irradiation: The peroxidase-positive Gilia. Radiation Research 50,65-72 (1972). SRIVASTAVA, O. P. and R. B. VAN HUYSTEE: Evidence for close association of peroxidase, polyphenol oxidase, and IAA oxidase isozymes of peanut cell suspension culture medium. Can. J. Bot. 51,2207-2215 (1977).
Z. Pflanzenphysiol. Bd. 92. S. 221-240. 1979.
Peroxidase and polyphenol oxidase activity
239
STAFFORD, H. A. and A. W. GALSTON: Ontogeny and hormonal control of polyphenol oxidase isozymes in tobacco pith. Plant Physiol. 46, 763-767 (1970). TOLBERT, N. E.: Activation of polyphenol oxidase of chloroplasts. Plant Physiol. 51, 234-244 (1973). VAN LOON, L. c.: Tobacco polyphenol oxidase: A specific staining method indicating nonidentity with peroxidase. Phytochem. 10, 503-507 (1971). WACKER, A., H. DELLWEG und D. JACHERTS: Thymindimerisierung und Dberlebensrate bei Bakterien. J. Mol. BioI. 4, 410-412 (1962). WITKIN, E. M.: Radiation-induced mutations and their repair. Science 152, 1345-1353 (1966). Dr. EGBERT W. HENRY, Department of Biological Sciences, Oakland University, Rochester, Michigan 48063 U. S. A.
z. PJlanzenphysiol. Bd. 92. s. 221-240. 1979.
The Effects of Phenolic Compounds on Peroxidase and Polyphenol Oxidase in Dwarf Pea (Pisum saiivum var. «Little Marvel») Tissue EGBERT
W.
HENRY, JAMES M. DEMoRROW
and
LAWRENCE
B.
RICHARD
With 6 figures Received June 6, 1978 . Accepted November 8, 1978
Summary Ethrel (3 ppm) and ultraviolet irradiation (UV), separate and in combination with visible or dark, were applied to seeds of dwarf pea (Pisum sativum, var. «Little Marvel,,) to determine their effect in the presence of sixteen different substrates, on polyphenol oxidase and peroxidase activity in 20 day-old distal root, root tip, leaf, and proximal stem tissues respectively. In terms of polyphenol oxidase activity, the enzyme is light dependent and activity is highest in visible light-exposed tissue, followed by UV plus Ethrel-treated tissues in comparison to untreated controls. Distal root and root tip tissues generally contained higher PPO activity than did the leaf or proximal stem tissues. Hydroxyanthranilic acid, catechol, and pyrogallol appear to be the most reactive substrates for PPO activity. Ethrel caused an increase in peroxidase activity in root tip and proximal stem tissues and depressed enzymic activity in distal root tissue. Leaf tissue had the highest level of peroxidase activity and etiolated tissue demonstrated higher levels of peroxidase activity than did visible lighttreated tissue. In addition to showing high specificity with the three aforementioned substrates that were cited as being specific for PPO, peroxidase activity was also specific with guaiacol and p-phenylene diamine.
Key words: polyphenol oxidase, peroxidase, ethrel, UV irradiation, dwarf pea.
Introduction
It has been proposed that a close affinity exists between peroxidase, indoleacetic acid oxidase (IAA oxidase), and polyphenol oxidase (PPO), (SHEEN, 1969; PILET et al., 1970; HOYLE, 1972; SRIVASTAVA and VAN HUYSTEE, 1973). An association among polyphenol oxidase and peroxidase has been reported (SHEEN, 1969); however, there are counter-proposals to this idea (VAN LOON, 1971; STAFFORD and GALSTON, 1970). HINMAN and LANG (1965) identified the intermediates resulting from the IAA oxidation due to horseradish peroxidase. Subsequently, RICARD and JOB (1974) proposed a reaction mechanism for the degradation of indole-3-acetate by peroxidase. Z. Pflanzenphysiol. Bd. 92. S. 221-240. 1979.
222
EGBERT W. HENRY, JAMES M. DEMoRROW and LAWRENCE B. RICHARD
However, other investigators question these reactions (SEQUEIRA and MINEO, 1966; RUBERY, 1972). It has recently been suggested that PPO and IAA oxidase share the same active site on the apoenzyme (SRIVASTAVA and VAN HUYSTEE, 1977). Polyphenol oxidase is a copper-containing enzyme that can catalyze two types of reactions involving the molecular form of oxygen (HENRY and JORDAN, 1977). The first type of reaction concerns the hydroxylation of monophenols, with the subsequent formation of o-dihydroxy compounds to quinones. PPO in some plants can undergo both of these reactions (RIVAS and WHITAKER, 1973). PPO may be present, in a latent form, in all plant chloroplasts (TOLBERT, 1973). It appears that PPO activity is light-dependent (TOLBERT, 1973). The ability of low light intensity to cause PPO activation in chloroplasts supports the suggestion that a configurational change and not electron transport, may be critical in PPO light activation of chloroplasts (TOLBERT, 1973). Many studies have been done on the damaging effects of ultraviolet (UV) irradiation on higher plants (KELNER, 1949; SINGER and AMES, 1970; EL-MANSY and SALISBURY, 1971; HENRY et al., 1977; DEMoRROW and HENRY, 1978). UV irradiation effects on plants may be via dimerization of thymine (WACKER et al., 1962; WITKIN, 1966); the breaking of the sugar phosphate bonds in the DNA double helix and the hydration of cytosine (BOLLUM and SETLOW, 1963; SETLOW et al., 1963; SINGER and AMES, 1970). It has been suggested that UV irradiation may cause photochemical responses, such as enhanced hydrogen peroxide production, thus causing plant tissue injury (LOCKHART and BRODUHRER-FRANZGROTE, 1961). Some plants, when treated with ethylene gas, exhibit an enhancement in peroxidase activity (RIDGE and OSBORNE, 1971). In addition, the observed increase in peroxidase activity may be due to de novo synthesis of the enzyme (RIDGE and OSBORNE, 1971). Ethrel: CICH 2CH2PO aH 2, undergoes an endogenous conversion to ethylene via base-catalyzed reaction, attributable to the action of hydrolytic enzymes (COOKE and RANDALL, 1968). If ethylene is applied to pea stem sections, there is a reduction of polar auxin transport (BURG and BURG, 1967). In addition, ethylene inhibits lateral IAA movement in isolated pea stem segments (BURG and BURG, 1968). Since Ethrel (a mixture of 2-chlorethanephosphoric acid and its ethyl ester) has similar effects to ethylene (MORGAN, 1969), we investigated the effects of UV irradiation, with and without Ethrel, on both light and darkgrown «Little Marvel» dwarf pea (Pisum sativum) seedlings, with respect to peroxidase and PPO activity vs. sixteen phenolic substrates. Materials and Methods Dwarf pea (Pisum sativum, var. «Little Marvel») seeds were sterilized with 1 % sodium hypochlorite, with subsequent rinsing in distilled water. The seeds were then separated into control and experimental (treated) lots. The experimental seeds were soaked for 24 h in a solution of 3 ppm Ethrel, with the non-Ethrel-treated control seeds being soaked in distilled
z. PJlanzenphysiol. Bd. 92. s. 221-240.1979.
Peroxidase and polyphenol oxidase activity
223
water. Both control and treated seeds were separated into three lots of fifty seeds each respectively and germinated for 20 days in growth chambers at a temperature of 23°C, under the following conditions: 1. complete darkness; 2. visible light; 3. UV irradiation. The growth chambers were equipped with banks of fluorescent tubes and incandescent bulbs, providing a light intensity of 3000 ft-C. For UV irradiation, four 18 in. (45-cm) germicidal tubes (Sylvania, low pressure mercury are, 15 W) were used to provide a dosage level of 1-10 callcm2 (HENRY et al., 1977; DEMoRROW and HENRY, 1978). Ten-mm sections of tissue (distal root, root tip, leaf, and proximal stem) were selected from the control and treated seedlings. After excision of the four separate tissues, each sample was weighed and immediately put into pre-chilled mortars at 4 0c. The tissue samples were ground at 4 °c for 5 min in 4-ml of 0.01 M phosphate buffer, pH 6.0. The respective samples were then centrifuged (15,000 g) at 4°C for 15 min to remove cellular debris. The respective supernatants were used for the following assays: Peroxidase: Spectrophotometric determinations were measured at a wavelength of 460 nm (HENRY et al., 1977). Polyphenol oxidase: Readings were taken at 475 nm (HENRY and JORDAN, 1977). Protein: Protein determinations were measured spectrophotometrically, with bovine serum albumin being used as a standard (LOWRY et al., 1951).
Key for 16 substrates used in Assay: Substrate and equivalent number Number used in text 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.
13. 14. 15. 16.
Name Phenol 3-Hydroxyanthranilic acid 3,4-Dihydroxyphenylalamine Hydroquinone Resorcinol Orcinol Catechol Caffeic acid Ferulic acid Chlorogenic acid Pyrogallol Phloroglucinol p-Phenylene diamine I-Phenylene diamine Diethyldithio carbamic acid Guaiacol
Results
Polyphenol oxidase: Dark, Dark plus Ethrel, Visible, Visible plus Ethrel (Figs. 1-3). a) Dark plus Ethrel- depressed PPO activity. b) Visible plus Ethrel- enhanced PPO activity. c) Visible - highest PPO activity vs. control. Z. PJlanzenphysiol. Bd. 92. S. 221-240. 1979.
EGBERT W. HENRY, JAMES M. DEMoRROW and LAWRENCE B. RICHARD
224
PPO ACTIVITY DARK I(J DARK DISTAL
2.0
+
1A
ETHREL
ROOT
. 1.8 1.8 1.4
,
ci>
1.2
>-
1.0
.~
;:;
<
.8
n. n.
.8
o
A
•2
"I
II 2
3
4
5
.-
8
I I.
7
8
.•.1 II
10
11
11.11• 12
13
..
14
,III 15
18
Substrates PPO ACflVITY DARK Vj DARK ROOT
2.0
+
18
ETHREL
TIP
1.8 1.8 1.4 Q
1.2
f
1.0
!;! o
n. n.
.8
.8 .4 .2
,. 2
3
4
5
8
7
,-
,_ III
8
9
10
11_ .• 11
12
13
14
15
18
Substrates
Figs. 1-3: Polyphenol oxidase (PPO): Dark (1 A-D), Dark plus Ethrel (1 A-D), Visible (2 A-D), Visbile plus Ethrel (2 A-D), and UV (3 A-D) vs. UV plus Ethrel (3 A-D). PPO activity is shown for distal root (1 A, 2 A, 3 A), root tip (1 B, 2 B, 3 B), leaf (1 C, 2 C, 3 C) and proximal stem (1 D, 2 D, 3 D), using substrates # 1-16 (.) vs. the corresponding control
(I D.
Z. Pjlanzenphysiol. Bd. 92. S. 221-240.1979.
225
Peroxidase and polyphenol oxidase activity PPO ACTIVITY DARK'!l' DARK
+
ETHREL
Ie
LEAF
1. distal root and root tip - highest levels of PPO activity (Figs. 1-3). 2. leaf and proximal stem - lower relative levels of PPO activity (Figs. 1-3). 3. most active substrates: 2, 7 and 11 for distal root, root tip, leaf and proximal stem tissues (Figs. 1-3). UV vs. UV plus Ethrel (Figs. 1-3). Z. PJlanzenphysiol. Bd. 92. S. 221-240. 1979.
226
EGBERT
W.
HENRY, JAMES M. DEMoRROW PPO VIS.
LIGHT '
2. 0
and
LAWRENCE
B.
RICHARD
ACTIVITY
VIS.
LIGHT
DISTAL
ROOT
+
2A
ETHREL
1.8 1.6 .c
""
~
1.4
,C>
1. 2
:: .~
1 .0
.u o
""-
8 6
4
11II
2 I
I
I
2
3
4
5
6
7
8
I 10
9
11
I I.
12
13
I 14
I
I
15
16
Substrates ACTIVITY
PPO VIS.
LIGHT \(§
VIS.
ROOT
2.0
LIGHT
+
28
ETHREL
TIP
1.8
i: .c
""
~
1.6 1.4
ci>
1.2
?:-
1.0
..u .~
0
"0..
.8 .6 A .2
I 2
3
4
5
III
6
7
8
9
10
11
III 12
13
.1
14
15
16
Substrates
Distal Root: a) Substrates: 2 and 11 - UV vs. UV plus Ethrel-treated tissues have equally high levels. b) Substrate: 7-UV has higher PPO level than UV plus Ethrel. c) Substrate: 8-UV - treated tissue has a much lower level of PPO activity than does UV + Ethrel. Z. P/lanzenphysiol. Bd. 92. S. 221-240. 1979.
227
Peroxidase and polyphenol oxidase activity ppo VIS.
lIG H T '!!i
ACTIVITY
VIS.
+
LIGHT
2C
ETHREL
LEAF
2.0 1.8
~
1.6
~
1.4
" ,0>
~
:: .~
;:;
« 0
Q. Q.
1.2 1.0
.8 .6 .4 .2 13
14
15
16
Substrates ACTIVITY
PPO VIS.
LIGHT v.1
VIS.
LIGHT
PROXIMAL
2.0
+
2D
ETHREL
STE M
1.8 1.6 1.4 0>
1.2
.~
1.0
«
.8
;:;
o
Q. Q.
.6 .4
.2
1 2
~
3
I_III 4
5
,I
6
I
7
8
9
I
10
11
II. 12
I
13
II. 14
11_11_ 15
16
Substrates
d) Substrates: 9 and 15 - UV plus Ethrel-treated tissue has higher level of PPO activity t-an does UV alone (Fig. 3 A). Root Tip: a) Substrates: 2 and 11 - UV vs. UV PPO levels.
+ Ethrel-treated
tissues have equally high
Z. Pjlanzenphysiol. Bd. 92. S. 221-240. 1979.
228
EGBERT
W.
HENRY, JAMES M. DEMoRROW PPO UV ~
and
LAWRENCE
B.
RICHARD
ACTI V I TY
+
UV
DIST~
L
3A
ETHREL ROOT
2.0 1.8
ii ~
1.6
u:"
1.4
ci>
1.2
,., ~
v « 0
""-
1.0 .8 .6 .4 .2 2
7
8
Subslrates PPO UV v,}
ACTIVITY
+
UV ROOT
2.0
9
38
ETHREL TIP
1.8 ~
1.6
'"'" u:'"
1.4
ci>
1.2
:!"
1.. 0
~
.8
0-
.6
.~
o0-
A
2
,I 2
3
II.ii_ 4
5
III
7
6
I 8
IIII 9
10
11
12
13
II
14
I
I
15
16
Substrates
b) Substrates: 3, 8, 15, 16 and 13 - UV of PPO than does UV alone.
+ Ethrel-treated tissue has a higher level
c) Substrate: 4-UV - treated tissue has more PPO activity than does UV (Fig. 3 B).
Z. Pjlanzenphysiol. Bd. 92. S. 221-240. 1979.
+ Ethrel
229
Peroxidase and polyphenol oxidase activity PPO UV ~
ACTIVITY
UV
+
ETHREL
3C
LEAF
2.0 1.8 ~ -;:;
1.6
u:
1.4
'"
1.2
?:
1.0
Q)
>
U
«
.8
0
11. 11.
.6 .4 .2
Substrates
PPO UV
v.}
UV
ACTIVITY
+
ETHREL
3D
PROXIMAL STEM
2.0 1.8 ~ -;:;
u:
Q)
1.6 1.4
'"
1.2
>
1.0
~ « ,0 11. 11.
.8 .6 A
.2 2 Substrates
Leaf: a) Substrates: 2, 7 and 11 - UV-treated tissue has higher level of PPO activity than does UV + Ethrel. b) Substrates: 1,3, 8, 9, 10, 12, 15 and 16 - UV-treated tissue has a higher level of PPO activity than does UV alone (Fig. 3 C).
Z. Pjlanzenphysiol. Bd. 92. S. 221-240. 1979.
230
EGBERT W. HENRY, JAMES M. DEMoRROW and LAWRENCE B. RICHARD
4A
16
48
Figs. 4-6: Peroxidase: Dark (4 A-D), Dark plus Ethrel (4 A-D, Visible (5 A-D), Visible plus Ethrel (5 A-D), and UV (6 A-D) vs. UV plus Ethrel (6 A-D). Peroxidase activity is shown for distal root (4 A, 5 A, 6 A), root tip (4 B, 5 B, 6 B). leaf (4 C, 5 C, 6 C) and proximal stem (4 D, 5 D, 6 D), using substrates # 1-16 (.) vs. the corresponding control
(II)·
Z. PJlanzenphysiol. Bd. 92. S. 221-240. 1979.
Proximal Stem: a) Substrates: 2, 7 and 11 - UV -treated tissue has a higher level of PPO activity than does UV + Ethrel. b) Substrates: 3, 5, 8 and 9 = UV + Ethrel-treated tissue has a higher level of PPO activity than does UV alone (Fig. 3 D).
z. Pjlanzenphysiol. Bd. 92. s. 221-240. 1979.
232
EGBERT
W.
HENRY, JAMES M. DEMoRROW PEROX IDASE VIS.
LIGHT '(}
VIS.
DISTAL
2.0
and
LAWRENCE
B.
RICHARD
ACTIVITY LIGH T T
5A
ETHREL
ROOT
1.8 ~
1.6
~
1.4
.::" 0>
"-
.;
.
U ~
1.2 1.0 .8
'"x
.6
"
.4
~
f'
a.
.2
IIJ 2
3
4
6
5
7
8
9
Substrales PEROXIDASE VIS.
2.0
LIGHT
v~
VIS.
ROOT
10
11lJ •
ACTIVITY LIGHT T
ETHREL
13
1-
14
15
16
58
TIP
~ .t::
...!" cio
1.2
:::
1.0
.
~
U
. .
~
~
.8
.6
K
~
.4
Q.
.2
15
16
Substrates
Peroxidase:
Dark, Dark + Ethrel, Visible, Visible + Ethrel (Figs. 4-6). a) leaf - most enhancement of peroxidase activity (Fig. 4 C, 5 C, 6 C). b) Proximal stem - slight increase in peroxidase activity under all light conditions (Fig. 4 D). Z. PJlanzenphysiol. Bd. 92. S. 221-240. 1979.
Peroxidase and polyphenol oxidase activity PEROXIDASE VIS
LIGHT It}
VIS
ACTIVITY
5C
+ ETHREL
LIGHT
233
LEAF
2.0 1.8
1.6 1.4
'"
1.2
.~
1.0
,..
U
«
.8
.2
2
3
4
5
6
7
8
9
16
10
Substrates PEROXIDASE VIS.
2.0
LIGHT It}
V'S
PROXIMAL
ACTIVITY LIGHT
+
50
ETHREL
STEM
1.8
1.6 1.4
'"
1.2
;
1.0
U
«
., ~
~ o
Q;
Q.
.8 .6 .4
9
10
11
12
13
14
15
16
Substrates
c) Ethrel - depressed peroxidase activity under all light conditions (Figs. 4-6). d) Etiolated vs. light - etiolated tissues show a higher level of peroxidase activity than tissues exposed to visible light (Figs. 4-5). 1. Most active substrates: 2, 7, 13 and 16 (Figs. 4-5). Z. Pjlanzenphysiol. Bd. 92. S. 221-240. 1979.
234
EGBERT
W.
HENRY, ]AMES M. DEMoRROW
+
UV
LAWRENCE
B.
RICHARD
ACTIVI TV
PEROXIDASE UV Y.j
and
6A
ETHREL
ROOT
DISTAL
2.0 1.8
3
1.6
.;:"
"
1.4
'"
1.2
?:
1.0
s:;
~
U
«
..'"
.8
;;;
.4
"0
g
a.
.6
2
3
4
5
7
6
8
Substrates PEROXIDASE
uv
v~
uv +
10
11
AC rlVITY
12
13
14
15
16
68
ETHREL
TIP
ROOT
2.0
9
1.8
3
..
1.6
.;.
1.2
?:
1.0
u «
.8
s:;
.::
:~
'..."
"0
1.4
.6
;<
0
;;
A
Q.
.2 2
3
4
5
6
7
8
9
10
11
16
Substrates
Distal Root: UV vs. UV + Ethrel (Figs. 6 A). a) Substrates: 2 and 15 - UV vs. UV + Ethrel-equally high levels of peroxidase activity. b) Substrates: 1, 5,6, 7, 8, 9, 10 and 16 - UV-treated has higher peroxidase level than UV + Ethrel.
z. Pflanzenphysiol. Bd. 92. s. 221-240. 1979.
Peroxidase and polyphenol oxidase activity PEROXIDASE
uv
~
6e
ACTIVITY
uv +
235
ETHREL
LEAF
2.0 1.8 ~ ~
.::" ,
Cl
,.
"
\;
«
.. ~
'2 0
Oi
n.
1.6 1.4 1.2 1.0 .8 .6 .4 .2
2
3
4
5
10
11
12
II
13
14
15
16
Substrates PEROXIDASE UV V.Ji
2.0
9
8
uv +
PROXIMAL
ACTIVITY
60
ETHREL STEM
1.8
~ ~
1.6
'"
1.4
u:" Cl
1.2
?:
1.0
;:; «
.8
.~
.~
.'2 0
Q;
""
.6
A .2
7
8
9
10
11
16
Substrates
c) Substrates: 3, 12 and 13 - UV + Ethrel has higher level of peroxidase than does UV -treated alone (Fig. 6 A). Root Tip: a) Substrates: 2 and 15 - UV vs. UV activity.
+ Ethrel
equally high levels of peroxidase
z. Pjlanzenphysiol. Bd. 92. s. 221-240. 1979.
236
EGBERT
W.
HENRY, JAMES M. DE MORROW
and
LAWRENCE
B.
RICHARD
b) Substrates: 7, 11, 16 and 3 - UV-treated tissue has higher peroxidase activity than UV + Ethrel. c) Substrates: 1, 5, 6, 8, 9, 10, 12 and 13 - UV + Ethrel has higher peroxidase activity than does UV alone (Fig. 6 B). Leaf: a) Substrates: 2, 3, 7, 11, 15 and 16 - UV vs. UV + Ethrel - equally higher in peroxidase activity. b) Substrates: 1, 4, 5, 6, 10 and 14 - UV-treated tissue has higher level of peroxidase activity than does UV + Ethrel. c) Substrates: 8, 9 and 13 - UV + Ethrel - treated tissue has higher level of peroxidase activity than does UV alone (Fig. 6 C). Proximal Stem:
a) Substrates: 2 and 15 - UV vs. UV + Ethrel - equally high levels of peroxidase activity. b) Substrates: 10 and 11 - UV-treated tissue has higher levels of peroxidase activity than UV + Ethrel. c) Substrates: 1, 3, 4, 5, 7, 8, 9, 12, 13, 14 and 16 - UV + Ethrel-treated tissue has a higher level of peroxidase activity than does UV alone (Fig. 6 D). Discussion The largest increase in PPO activity occurs in the various visible light treatments (Fig. 2). These results suggest a light-dependent requirement for PPO activity which agrees with earlier observations of TOLBERT (1973). In the present studies, PPO activity was depressed in Dark + Ethrel-treated tissues (Fig. 1). While UV + Ethreltreated tissue showed enhanced PPO activity, the enzyme activity was highest in Visible Light-treated tissues (Fig. 3). With respect to substrate specificity, hydroxyanthranilic acid, catechol, and pyrogallol appear to be the most reactive with dwarf pea tissue (Figs. 1-3). In terms of tissues, higher levels of PPO activity was observed in distal root and root tip than in leaf or proximal stem samples (Figs. 1-3). Under all light conditions, Ethrel caused an increase in peroxidase activity in root tip tissues compared to untreated controls (Fig. 4-6). Conversely, Ethrel caused lowered enzymic activity in distal root tissue under similar light conditions (Figs. 4-6). Proximal stem tissue showed a relatively small increase in peroxidase activity under all light conditions compared to untreated controls (Figs. 4 D, 5 D, 6 D). Etiolated tissues demonstrated higher levels of PPO activity than did light exposed seedlings (Figs. 4-5). The leaf tissue exhibited the highest degree of PPO enhancement in light-treated tissues compared to untreated controls (Figs. 4 C, 5 C, 6 C). Z. Pflanzenphysiol. Bd. 92. S. 221-240.1979.
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It is well known that ethylene induces peroxidase activity in plant tissues (SHANNON et al., 1971 MORGAN, 1969; HENRY et al., 1977). The effect of UV irradiation on causing an increase in peroxidase activity in several plant tissues may well be due to its effect on mediating ethylene production (ENDO, 1967; SREBO et al., 1972). In the present studies, Ethrel caused a greater enhancement in PPO activity than in peroxidase activity (Figs. 1-6). It has recently been proposed that a single protein molecule may possess three enzyme activities: peroxidase, PPO and IAA oxidase (SRIVASTAVA and VAN HUYSTEE, 1977). There are other examples of a single protein molecule having more than one enzyme activity: 1. the dual enzyme capability of certain plant phenolases (CHALICE and WILLIAMS, 1970); and 2. the evidence that IAA oxidase activity and guaiacol peroxidase activity may be due to the dual catalytic function of one enzyme (HOYLE, 1972). The present studies indicate that the monohydroxy, dihydroxy and trihydroxy compounds (substrates: 2, 7 and 11 respectively) are specific for both PPO and peroxidase, while guaiacol and p-phenylene diamine are also specific for peroxidase. However, it remains an open question as to the number of active sites on the protein molecule responsible for either PPO or peroxidase activities. As PPO is present in very immature chloroplast lamellae (HENRY, 1976) and light-dependent (TOLBERT, 1973) it is possible that it may be somewhat different as a protein molecule - from the peroxidase moiety. It has been suggested that a specific function of peroxidase in germinating plant cells may be correlated with the existence of a specific molecular configuration of the protein (GORDON and ALLDRIDGE, 1971). The possibility of enzymes such as PPO and peroxidase having more than one reactive site may, in part, explain their apparent differential activities with respect to the sixteen substrates assayed in the present study. Acknowledgements We thank Ms. MARY N. O'CONNOR for skilled technical assistance. References BOLLUM, F. J. and R. B. SETLOW: Ultraviolet inactivation of DNA primer activity. I. Effect of different wavelengths and doses. Biochem. Biophys. Acta 68, 599-607 (1963). BURG, S. P. and E. A. BURG: Molecular requirements for the biological activity of ethylene. Plant Physiol. 42,144-152 (1967). - - Ethylene formation in pea seedlings. Its relation to the inhibition of bud growth caused by indole-3-acetic acid. Plant Physiol. 43, 1069-1074 (1968). CHALICE, J. S. and A. H. WILLIAMS: A comparative biochemical study of phenolase apecificity in Malus, Pyrus and other plants. Phytochem. 9, 1261-1265 (1970). COOKE, A. R. and D. I. RANDALL: Properties of phenoloxidase in Mycobacterium leprae. Nature 218, 973-974 (1968). DEMoRROW, J. M. and E. W. HENRY: Substrate comparability vs. polyphenol oxidase activity in Ethrel and ultraviolet-treated «Little Marvel" dwarf Pea (Pisum sativum) tissue. Z. Pflanzenphysiol. 86, 353-361 (1978).
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ENDO, T.: Comparison of effects of gamme-rays and maleic hydrazide on enzyme systems of maize seeds. Radiat. Bot. 7, 35-40 (1967). EL-MANSY, H. I. and F. B. SALISBURY: Biochemical responses of Xanthium leaves to ultraviolet radiation. Radiation Botany 11, 325-328 (1971). GORDON, A. R. and N. A. ALLDRIDGE: Cytochemical localization of peroxidase A' in developing stem tissues of extreme dwarf tomato. Can. J. Bot. 49,1487-1496 (1971). HENRY, E. W.: The ultrastructural localization of polyphenol oxidase in chloroplasts of Brassica napus cv. ZEPHYR. Z. Pflanzenphysiol. 78,446-452 (1976). HENRY, E. W., C. NCHEKWUBE, and J. Russo: Physiological responses of dwarf pea (Pisum sativum, var. «Little Marvel») seedlings to Ethrel and UV irradiation. Z. Pflanzenphysiol. 81,459-465 (1977). HENRY, E. W. and W. JORDAN, III: The enzymic response of pea (Pisum sativum) stem sections to applied indoleacetic acid, gibberellic acid and Ethrel: Catalase, peroxidase and polyphenol oxidase. Z. Pflanzenphysiol. 84, 321-327 (1977). HINMAN, R. L. and J. LANG: Peroxidase catalyzed oxidation of indole-3-acetic acid. Biochern. 4, 144-158 (1965). HOYLE, M. c.: Indole acetic acid oxidase. A dual catalytic enzyme. Plant Physiol. 50, 15-18 (1972). KELNER, A.: Effect of visible light on the recovery of Streptomyces griseus conidia from ultraviolet irradiation injury. Proc. Nat. Acad. Sci. 35, 73-79 (1949). LOCKHARD, J. and U. BRODUHRER-FRANZGROTE: The effect of ultraviolet radiation on plants. In: W. RUHLAND (Ed.), Encyclopedia of Plant Physiology XVI., pp. 532-554. SpringerVerlag, Berlin, 1961. LOWRY, O. H., N. J. ROSEBROUGH, A. L. FARR, and R. J. RANDALL: Protein measurement with the Folin phenol reagent. J. BioI. Chern. 193, 265-275 (1951). MORGAN, P. W.: Stimulation of ethylene evolution and bascission in cotton by 2-chlorethanephosphonic acid. Plant Physiol. 44, 337-341 (1969). PILET, P. E., P. LAVANCHY, and S. SEVHONKIAN: Interactions between peroxidase, polyphenol oxidase and auxin oxidase. Physiol. Plant 23,800-804 (1970). RICARD, J. and D. JOB: Reaction mechanism of indole 3-acetate degradation by peroxidases. A stop flow low temperature spectroscopy study. Eur. J. Biochem. 44, 359-374 (1974). RIDGE, I. and D. J. OSBORNE: Role of peroxidase when hydroxy-proline-rich protein in plant cell walls is increased by ethylene. Nature New BioI. 229, 205-209 (1971). RIVAS, N. D. and J. R. WHITAKER: Purification and some properties of two polyphenol oxidases from bartlett pears. Plant Physiol. 52, 501-507 (1973). RUBRY, P. H.: Studies in indole acetic acid oxidation by liquid medium from crown gall tissue culture cells; the role of malic acid and related compounds. Biochim. Biophys. Acta 261,21-34 (1972). SEQUEIRA, L. V. and L. MINEO: Partial purification and kinetics of indole acetic acid oxidase from tobacco roots. Plant Physiol. 41,1200-1208 (1966). SETLOW, R. B., P. A. SWENSON, and W. L. CARRIER: Thymidine dimers and inhibition of DNA synthesis by ultraviolet irradiation of cells. Science 142, 1464-1466 (1963). SHEEN, S. J.: The distribution of polyphenols, chlorogenic acid oxidase and peroxidase III different parts of tobacco, Nicotiana tabacum. Phytochem. 8, 1839-1847 (1969). SINGER, C. E. and B. M. AMES: Sunlight ultraviolet and bacterial DNA base ratios. Science, 170, 822-826 (1970). SREBO, Z., H. LACH, and E. SZIRMAI: A novel aspect of the response of the central nervous system to irradiation: The peroxidase-positive Gilia. Radiation Research 50,65-72 (1972). SRIVASTAVA, O. P. and R. B. VAN HUYSTEE: Evidence for close association of peroxidase, polyphenol oxidase, and IAA oxidase isozymes of peanut cell suspension culture medium. Can. J. Bot. 51,2207-2215 (1977).
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STAFFORD, H. A. and A. W. GALSTON: Ontogeny and hormonal control of polyphenol oxidase isozymes in tobacco pith. Plant Physiol. 46, 763-767 (1970). TOLBERT, N. E.: Activation of polyphenol oxidase of chloroplasts. Plant Physiol. 51, 234-244 (1973). VAN LOON, L. c.: Tobacco polyphenol oxidase: A specific staining method indicating nonidentity with peroxidase. Phytochem. 10, 503-507 (1971). WACKER, A., H. DELLWEG und D. JACHERTS: Thymindimerisierung und Dberlebensrate bei Bakterien. J. Mol. BioI. 4, 410-412 (1962). WITKIN, E. M.: Radiation-induced mutations and their repair. Science 152, 1345-1353 (1966). Dr. EGBERT W. HENRY, Department of Biological Sciences, Oakland University, Rochester, Michigan 48063 U. S. A.
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