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Hydrogen peroxide scavenging systems in vacuoles of mesophyll cells of Vicia faba
Phytochemistry, Vol. 3 1, No. 4, pp. 1127-l i 33, 1992 printed in Great Britain.
0
003i-9422/92 $5.00 + 0.00 1992 Pergamon Press pie
HYDROGEN PEROXIDE SCAVENGING SYSTEMS IN VACUOLES OF MESOPHYLL CELLS OF JfICIA F&3x4
UMEO TAKA~~M~
apartment
of Biology, Kyushu Dental College, K~taky~shu 803, Japan (Received 29 August 19911
Key Word Index-Viciafaba;
Leguminosae; ascorbate; DOPA; hydrogen peroxide; peroxidase; vacuoles.
A~~ct-Vacuoles isolated from mesophyll protoplasts of Vi& faba contaio~ ascorbate in addition to 3,4dihydroxyphenylalanine (DOPA) and peroxidase. When a low concentration of H,0,(0.25 mM) was added to the vacuole factions, a slow decrease in the level of ascorbate was observed. When 2.5 mM H,O, was added to the vacuole fractions, a rapid decrease in the levels of ascorbate and formation of dopachrome were observed. The oxidation of ascorbate and the formation of dopachrom~ were inhibited by NaNs (10 mM), KCN (1 mM) and stimulated by tropolone (5 mM), suggesting the participation of peroxidase but not phenol oxidase in the reactions. Vacuolar peroxidase isolated from leaves of V.faba oxidized DOPA and ascorbate in the presence of H,O,. The oxidation of DOPA was nearly completely inhibited by ascorbate but the oxidation of ascarbate was stimulated by DOPA, suggesting the reduction of an oxidized intermediate of DOPA to DOPA by ascorbate. In fact, ascorbate reduced dopaq~none, a two electron oxidized DOPA.
RESULTS AND DISCUSSION
Vacuoles of mesophyll cells of Vic~u~~~ucon~n peroxidase and phenolics which can donate electrons to the peroxidase in the presence of H,O, Cl]. If H,O, is added to vacuoles obtained from mesophyll cells, to mesophyll protoplasts or to leaves, DOPA is oxidized to melan~ via dopachrome, an orange-red compound [I, 21. Accompanying the formation of melanin, levels of DOPA decrease. However, if incubation of leaves in H,O, is continued, the levels ofDORA increase after the decrease, sugg~ting the synthesis of DOPA [2]. Vacuolar peroxidase isolated from leaves of I’.faba oxidized DOPA in the presence of H,Oa {3]. These results suggest the participation of vacuolar peroxidase in the oxidation of DOPA by H,O,. If the peroxidase functions to scavenge H,O, using DOPA as an &&on donor under normal conditions, there must be a regeneration of DOPA from its oxidized intermediate. In chloroplasts, if ascorbate is oxidized by ascorbate peroxidase, the oxidized product, dehydroascorbate, is reduced to ascorbate by giutathioue [4]. It has been reported than an oxidized intermediate of DOPA, dopaq~none, is reduced to DOPA by ascorbate [5,6]. As peroxidase possibly oxidizes DOPA to dop~hrome and melanin via dopaquinone [l, 7, 81, generation of DOPA from dopaquino~e is possible if ascorbate is present in vacuoles of mesophyll cells of I? $&a. Therefore, it is irn~~ant to know whether ascorbate is present in the vacuoles to elucidate H,Oz-scavenging function of vacuolar peroxidase. This paper deals with the presence of ascorbate in the vacuoles of leaves of P.faba, the reactivities of DOPA and aseorbate to the vacuolar peroxidase isolated from leaves of V. faba and the reduction of dopaquinone by aseorbate. Based on the data, a p~ysiolo~~~ function of the vaeuolar peroxidase is discussed.
A HPLC elution pattern of a metaphospho~c acid extract of vacuoles is shown in Fig. 1. There were four peaks: peaks A and I3 had R,s of 3.8 and 4.4 min and absorption maxima at 270 and 288 nm, respectively. These values were the same as those of authentic ascorbate and DOPA. Two peaks on HPLC had no distinct absorption peaks in the UV region. Typical values of the specific ~tivities of some enzyme and the amount of aseorbate in terms ofamount ofDOPA which is localized mainly in the vacuoles obtained from mesophyll cells of Y. fuba are shown in Table 1 [I]. In a given vacuole fraction, the relative activities of eatalase ~roxisomes) and glucose-6-phosphate dehy~ogen~ (cytoplasmic matrix) were lower than those in the corresponding protoplast fraction. Cytochrome c oxidase activity was not detected in the vacuole fraction. The contamiuation of the vacuole fraction by chloroplasts and protoplasts, respectively, was less than 10% when examined by a light microscope. However, the ratios of relative activities of peroxidase for DAB and a-mannosidase, which are mainly localized in the vacuoles [I], were much greater than those of other enzymes. These data suggest that the vacuole fraction contains mainly vacuoles although the vacuole fraction is co~t~inat~ by other cytoplas~c components. The ratio of the relative amount of ascorbate showed a moderate value, su~esting the loealization of ascorbate in vacuoles as well as in cytoplasm other than vacuoles. The ratio of the relative activity of ascorbate peroxidase, which is present in chloroplasts in addition to cytoplasm [4], also showed a moderate value. This is attributed to the ability of the peroxidase in vacuoles to utilize aseorbate as an electron donor (Fig, 2). The presence of ascorbate in vacuoles has been reported in other plants [9]+
1127
U. TAKAHAMA
1128
The HPLC elution patterns of a product obtained by the oxidation of DOPA by a I&O,-horseradish peroxidase system are shown in Fig. 3. The product (peak 2) had a retention time of 4.7 min and absorption maxima at 310 ,
0.4r \
0
220
300 260 Wavelength f nm)
340
o.2--
Time
(min)
Fig. 1. HPLC &&ion pattern of ascorbate and DOPA extracted from vacuoles with metaphosphoric acid (lower panel) and their absorption spectra (upper panel) using 20 4 extract. A, ascorbate; B. DOPA. Absorption spectra were measured in a mobile phase. methanol-KH,PO, (1:9).
and 480 nm and the generation of the product was stimulated by tropolone. The shape of absorption spectrum of peak 2 was similar to that of dopachrome [lO]. It has been reported that peroxidase oxidizes DOPA to dopachrome and the oxidation of DOPA producing dopachrome is stimulated by tropolone [lo, 111. These data suggest that the compound of peak 2 is dopachrome. As dopachrome can be separated by HPLC in this way, in the following, dopachrome was quantified as a measure of the oxidation of DOPA. The effects of some enzyme inhibits on N,O,dependent oxidation of ascorbate and DOPA in vacuoles and protoplasts are shown in Table 2. Tropolone, an inhibitor of phenol oxidase [ll], stimulated the oxidation of ascorbate as well as the formation of dopachrome. Azide and cyanide inhibited both oxidation reactions. From the data, vacuolar peroxidase but not phenol oxidase participates in the AEON-de~ndent oxidation of ascorbate and DOPA. Tropolone (5 mM) completely inhibits oxidation of DOPA by cell-free extracts of leaves of Kfaba in the absence of H,O, [Z], suggesting that the concentration of tropolone used functions to inhibit phenol oxidase. The time course of the oxidation of ascorbate and DOPA in vacuole and protoplast fractions in the presence of 0.25 or 2.5 mM H,O, is shown in Fig. 2. When 0.25 mM I-I,O, was added to a vacuole or protoplast fraction, slow decreases of the levels of ascorbate and DOPA were observed (Fig. 2A, C) and the levels of DOPA attained constant values within 2 min. The levels of ascorbate decrease gradually during incubation, especially in the vacuole fractions. There was no detectable formation of dopachrome. Part of the decrease of ascot= bate and DOPA might be due to spontaneous oxidation of the compounds in the absence of H,O, induced by transferring vacuoles and protoplasts from 0‘ to room temperature for experiments. When 2.5 mM H,O, was added to the fractions, the levels of ascorbate decreased rapidly. The degrees of the
Table I. Specific enzymatic activities and levels of DOPA and ascorbate of protoplast and vacuole fractions
DOPA (11mol mg-’ protein) a-Mannosidase (AA,,, mg-’ protein hr-‘) Ascorbate (p mol mg-’ protein) DAB peroxidase (AP&, mg-r protem mm -1) Ascorbate peroxidase (A&,, mg-’ protem min-t) Catalase (AA240mg-’ protein mm-‘) Glucose-6-P-dehydrogenase (AA340 mg- ’ protem hr-l)
Protoplasts (I)
Vacuoles (II)
(11)/(I)
2.3 (100)
34.3 (100)
1.00
2.0 (87)
24.6 (72)
0.83
0.6 (26)
6.0 (17)
0.65
30 (1304)
469 (1367)
1.05
2.8 (122)
16.4 (48)
0.39
3.0 (I 30)
3.0 (9)
0.07
6.6 (287)
3.6 (IO)
0.04
Values in parentheses are enzymatic activities in protoplast and vacuole fractions relative to the amount of DOPA in each fraction.
Hydrogen peroxide metabolism in vacuoles
1129
A
-5 2G
l-
0
IF-A-A-d---d
-___ A________&
g 50-
I\
-0
3 .I 5 5 z
-
!L
-5
3
-
s”
IoY: O-A--A-A-___A___A________A 1 I I I.
I
-0
I
0
10 Time
(min)
Fig. 2. H,O,-dependent changes of levels of ascorbate and DOPA and formation of dopachrome in protoplast (A and B) and vacuole (C and D) fractions. The initial amounts of DOPA and ascorbate in A, B, C and D were 46.7, 23.5, 13.2 and 31.1 nmol for DOPA and 16.1, 7.0, 2.6 and 6.3 nmol for ascorbate. Reactions were performed in 0.04 ml of reaction mixtures and were started by the addition of 0.25 mM H,O, (A and C) or 2.5 mM H,O, (B and D). After incubation, reactions were terminated by the addition of 0.08 ml of 2% metaphosphoric acid. Open circles, ascorbate; closed circles, DOPA, triangles, dopachrome.
Table 2. Effects of enzyme inhibitors on H,O,-dependent
Ascorbate (nmol) Vacuoles Protoplasts Dopachrome (relative) Vacuoles Protoplasts
oxidation of DOPA and ascorbate
Before addition of H,O,
+ H,O, (2.5 mM) No add.
+ tropolone (5 mM)
+NaN,
4.8 15.2
1.7 1.9
1.1 0.1
3.1 5.0
3.7 10.1
0 0
1.6 3.0
3.5 3.9
0.2 0
0 0
(10mM)
+KCN(lmM)
Reaction mixture (0.04 ml) contained vacuoles or protoplasts equivalent to 28.2 or 65.2 nmol DOPA, respectively. Vacuoles were suspended in a buffer solution that contained 0.4 M mannitol, 1 mM CaCl,, 1 mM EDTA and 50mM HEPES-KOH (PH 7.8). Protoplasts were suspended in a buffer solution that contained 0.55 M sorbitol and 5 mM MES-KOH (pH 5.8). Reactions were started by addition of H,O, and terminated at 1 min by adding 0.08ml 2% metaphosphoric acid.
decrease of the levels of ascorbate by 2.5 mM H,Oz were greater than those by 0.25 mM H,O*. In protoplasts, the levels of ascorbate increased after the decrease of the levels of ascorbate. During the decrease of the levels of ascorbate and DOPA, formation of dopachrome was observed. The maximal amount of dopachrome formed
was more than two-fold greater in vacuole fractions than in protoplast fractions, even when the initial level of DOPA in the protoplast
fraction was greater than that in
the vacuole fraction. In vacuoles, it took ca 4 min to attain a maximal level of dopachrome and the level decreased slowly. In protoplasts, the maximal level was
1130
U.
TAKAHAMA
attained at cu 1 min after the addition of H,Oz and the level decreased as the level of ascorbate increased. The difference in the time course of the changes of the levels of dopachrome may be due to the presence and absence of a system to regenerate ascorbate from dehydroascorbate. In protoplasts, if the levels of ascorbate decreases, the levels will be recovered and the formation of dopachrome will be inhibited. Thus, the data in Fig. 2 indicate that the formation of dopachrome becomes apparent when the levels of ascorbate in vacuoles and protoplasts decrease considerably. The incomplete recovery of the level of ascorbate may be, in part, due to the rupture of protoplasts during incubation, The decrease in the levels of dopachrome after its formation may be due to the transformation of dopachrome to melanin, as has been observed [ 11. Two mechanisms are considered for the appearance of dopachrome after the considerable decrease of the level of ascorbate in both vacuoles and protoplasts. (i) Competition of the following two reactions in vacuoles: ascorbate + H,O,zdehydroascorbate
0.8 I
,
Time
(mln) 1
f 2H,O Wavelength
(nm)
POX
DOPA + H,O,-----+dopaquinone
+ 2H,O.
Here, POX is peroxidase. If the ascorbate is a better electron donor than DOPA for the H,O,-vacuolar peroxidase system, the oxidation of DOPA producing dopachrome may occur after the decrease of the levels of ascorbate. Dopaquinone, a two-electron oxidized product of DOPA, is transformed to dopachrome non-enzymatically [7, S]. (ii) The reduction of the rate of the regeneration of DOPA from dopaquinone by ascorbate due to the decrease of the levels of ascorbate: POX
DOPA + H,O,----+dopaquinone dopaquinone
+ 2H,O
+ ascorbate-+ DOPA + dehydroascorbate.
The decrease of the levels of ascorbate results in the oxidation of dopaquinone to dopachrome. To distinguish the two mechanisms, the effects of ascorbate on the H,O,-dependent oxidation of DOPA and vice versa were examined using vacuolar peroxidase isolated from leaves of V. faba (Fig. 4). The concentrations of DOPA and ascorbate in the experiments in Fig. 4 were determined by considering the concentration of DOPA in vacuoles [3] and the ratio of DOPA to ascorbate in vacuoles (Table 2 and Fig. 2). When DOPA alone was added to a H,O,-peroxidase system, the decrease of level of DOPA and dopachrome formation were observed. When ascorbate alone was added to the system, slow oxidation of ascorbate was observed. In the presence of both DOPA and ascorbate in the H,O,-peroxidase system, the initial rate of oxidation of DOPA was smaller than in the presence of DOPA alone and the rate of oxidation of ascorbate was much greater than that in the presence of ascorbate alone as an electron donor in the system. As long as ascorbate remained in the reaction mixture, there was no formation of dopachrome. However, when ascorbate was eliminated from the reaction mixture, oxidation of DOPA and formation of dopachrome was observed. When 2 mM ascorbate was added together with 8 mM DOPA, no
Fig. 3. HPLC elution pattern of DOPA and dopachrome. DOPA was oxidized by a H,O,-horseradish peroxidase system m the presence of 3 Mughorseradish peroxidase, 1 mM DOPA and 1 mM H,O, m 3 ml of 50 mM Na-phosphate (pH 6). After incubation for 3 hr at about 18”, 20 pl was applied to HPLC column. Upper panel: A, incubated without horseradish peroxidase; 8, incubated in the above reaction mixture; C, incubated in the above reaction mixture but in the presence of 1 mM tropolone. 1, DOPA; 2, dopachrome. Lower panel: absorption spectra of peaks 1 (dashed line) and 2 (sohd Ime) in a mobile phase, methanol-KN,PO, (1: 9).
formation of dopachrome was observed and ascorbate was not oxidized completely during the incubation period for 20 min. The results suggest that the oxidized intermediate of DOPA can be reduced by ascorbate, thus suppressing dopachrome formation. To confirm this, the effects of ascorbate on the formation of dopaquinone were examined (Fig. 5). The addition of H,O, in the presence of DOPA and the vacuofar peroxidase resulted in the formation of a compound with an absorption maximum at ca 390 nm at first, and then a compound with an absorption maximum at ca NO nm. The former can be attributed to dopaquinone [12] and the latter to dopachrome. When ascorbate was added to the reaction mixture, the absorption intensity around 390 nm decreased and the formation of dopachrome was inhibited. The inset in Fig. 5 indicates the rapid absorption decrease at 390 nm by the addition of ascorbate. The data suggest the reduction of dopaquinone by ascorbate. It has been reported that dopaquinone is easily reducible to DOPA by ascorbate [S, 61. Based on the data, a scheme in Fig. 6 was proposed for the H,O,-dependent metabolism of DOPA and ascorbate in vacuoles of V.fubu leaves. If H,O, diffuses into vacuoles, peroxidase in vacuoles oxidizes DOPA to dopaquinone. The o-benzoquinone will be reduced to DOPA by ascorbate, producing DOPA and dehydroascorbate. Dehydroascorbate may be translocated from vacuole to cytoplasmic matrix and will be reduced to abcorbate by glutathione. Thus, the redox cycles to generate DOPA
Hydrogen peroxide metabolism in vacuoles
Time
1131
(min)
Fig. 4. H~O~d~~ndent oxidation of ascorbate and DOPA by peroxidase. The reaction mixture (0.5 mi) contained 0.05 ml of vacuolar peroxidase and 1.5 mM HzO, in 0.1 M citric acid-O.2 M Na2HP0, buffer (pH 5.4). Where indicated, 1 mM sodium ascorbate and/or 8 mM DOPA were added. Left panel: oxidation of ascorbate and DOPA when they were added separately to the above reaction mixture. Right panel: oxidation of ascorbate and DOPA when they were added together to the above reaction mixture. Reactions were started by the addition of H,O,. Open circles: DOPA; closed circles, dopachrome; triangles, ascorbate. The levels of dopachrome are relative
after its oxidation
0 A
I
I ,Asc
I
a04
I
a02
Wavelength
(nm)
Fig. 5. Effects of ascorbate on HOOK-indu~ abso~tion changes. The reaction mixture (1 ml) contained 0.05 ml of vacuolar peroxidase, 8mM DOPA, 1 mM H,O, in 0.1 M citric acid-O.2 M Na,HPO, buffer (pH 5). Upper panel: trace a, before addition of H,O,. Traces, b, c and d are 445 and 90 set after the addition of H,Oz, respectively. Lower panel: trace a, before the addition of H,O,; trace b, 0 set after the addition of H,O,. After recording trace b, 0.5 mM sodium ascorbate was added. Traces c and d are immediately and 45 set aRer the addition of ascorbate, respectively. Repeat scan was started from 600 nm and ended at 300 nm. The scan speed was 600 nm min- ‘. An inset shows a time course of A changes at 390 nm after the addition of 1 mM H,O, and 0.5 mM sodium ascorbate, successively.
by vacuolar
peroxidase
become
pos-
sible. An oxidized interm~iate of rutin, which is an odihydroxyphenoliG, is also reduced to rutin by ascorbate [133, suggesting that the occurrence of redox cycles similar to those in Fig. 6 is also possible when other o-hydroxyphenol& are present in vacuoles. Hehlhon and Kunert [14] and Peters et al. [lSJ have found that caffeic acid, an o-hydroxyphenol, increased the rate of oxidation of ascorbate by horseradish peroxidase and extracellular peroxidase in pinto bean leaves, respectively. The stimulation can be explained by the reduction of an obenzoquinone by ascorbate, which may be formed by the oxidation of caffeic acid by the peroxidases. If the level of ascorbate becomes lower in vacuoles, the reactions to produce melanin via dopachrome will predominate. The production of melanin will become apparent when the regeneration of ascorbate from dehydroascorbate is limited by i~ibition of the formation of NAD(P)H which can reduce dehydroascorbate via glutathione. This is confirmed by the fact that methyl viologen, which inhibits photoreduction of NADP+ in chloroplasts stimulating H,O, production [4], enhanced the formation of melanin by illumination 121.
EXPERIMENTAL Plot materials. Viciafabu (cv 6461, from Tohoku Utsunomiya, Japan) were grown in vermiculite in a growth chamber in which the temps by day (12 hr) and by night (12 hr) were 15 a and lo”, respectively, and seedlings were watered once a week with a 0.1% soln of Hyponex. The maximum intensity of light by day period was about 6000 Ix. The white light was obtained from day-light type fluorescent lamps (FL4OSD) from Nihon Denki Co (Tokyo). ~~puruti~n of ~sop~yl~ proto~l~s~s. Young, fully expanded leaves were used. They were harvested in the morning (ca 9:00 am) from plants grown for 1-3 months. Immediately after
U.
1132 b
---+
TAKAHAMA
--+
---+
Melanin
I+
Whrome
Asc
&
(Hz%- generating
systems)
Fig. 6. A proposed scheme of the H,O,-dependent metabolism of DOPA and ascorbate in vacuoles. POX, peroxidase; Asc, ascorbate; DHA, dehydroascorbate; GSH, ~lutathione; GSSG, oxidized form of glutathione. harvest, ca 20 leaves from which the lower epidermis has been partially removed were floated on a soln of enzymes which consisted of 550 mM sorbitol, 1% cellulase and 0.5% macerozyme in 5 mM MES-KOH (PI-I 5.8). The soln of enzymes was infiltrated into leaves zn WCUO,and the infiltrated leaves were incubated at 25 0 for 1 hr with gentle shaking under room light. Released protoplasts were collected by passage through 4 layers of gauze and centrifuged at IOOg for 2 min. Isolated protoplasts were suspended in 550 mM sorbitol in 5 mM MES-KOH (pH 5.8) and kept at 0” after two washings with the suspension medium. Frepura~~on ofvacuofes. Vacuoles were prepared as described previously El]. To release vacuoles from mesophyll protoplasts, suspensions of protoplasts (0.4 ml) equivalent to about 1 mg chlorophyl1 were added to 2 ml of a 0.1 M soln of K,HPO, adjusted to pH 8.5 with HCI. After 1-2 min at room temp. (ca 20 “), each suspension was loaded onto a discontinuous gradlent of Ficoll-400 in 50 mM HEPES-KOH (pH 7.8) which contained 0.4 M mannitol, 1 mM CaCl, and 1mM EDTA. The gradients were centrifuged at 3700 g for 15 min at 4 O.Vacuoles accumulatingat the interfaces between 1 and 3% and between 3 and 5% Ficoll were collected from the gradients with a Pasteur pipette and washed once by ~t~~tion (2OOg, 2min) in the buffer soln without Ficoll. The prepn of vacuoles contained a small number of protoplasts and chloroplasts when examined under a light microscope, and the ratio of vacuoles to protoplasts was more than 19:l. Isolated vacuoles were kept at 0”. The concn of protein was determined with a protein-assay reagent from BioRad with bovine serum albumin as standard. This assay is based on shift of A of an acidic soln of Coomassie Brilliant Blue when binding to protein. Assay of enzyme activities. Catalase activity was assayed following decrease in A at 240 nm in a reaction mixture (1 ml) of 1 mM H,O,, 1 mM CaCl,, 1 mM EDTA, 0.4M mannitoi and 5OmM HEPES-KOH @H 7.8). The activity of glucose-& phosphate dehydrogenase was followed at 340 nm in a reaction
mixture (1 ml) that contained 1 mM glucose-6-phosphate, 0.2 mM NADP+, 1 mM CaCl,, I mM EDTA, 0.4 M mannitol and 50 mM HEPES-KOH (pH 7.8). The activity of ascorbate peroxidase was followed at 290 nm in a reaction mixture (1 ml) which contained 1 mM Na ascorbate, 1 mM CaCI,, 1 mM EDTA, 0.4 M mannitol and 50 mM HEPES-KOH (pH 7.8). Diaminobenzldine peroxidase activity was followed at 460 nm in a reaction mixture (1 ml) which contained 1 mM diaminobenzidine, 0.55 M sorbitol and 5 mM MES--KOH (pH 5.8). The activity of r-mannosidase was followed in a reactlon mixture (I ml) which contamed 0.5 mM p-nitrophenol-~-mannopyranoside and 0.1 M citric acid- 0.2 M Na,HPO, (pH 5). After mcubatlon for 1 hr at ca 20 :, 1ml of 1 M Na,CO, was added and the A at 420 nm was determined. Blank values were taken into account in the calculation of the enzymatic activites. All reactions were started by the addition of vacuoles or protoplasts. Prior to the addition of vacuoles or protoplasts, they were disrupted by repeating suckmg and ejectIon using a microsyringe. Spectrophotometrlc measurements were carried out at room temp (ca 20”). The path length of the measurmg beam was 4 nm. Assay by HPLC. HPLC was performed using a Shimadzu CL6A pump combined with a Shim-pack CLC-C, column (15 cm x 6 mm i.d.) and a spectrophotometric detector with a photodiode array (Shimadzu SPD-MIA). The mobile phase was MeOHmM KH,PO, (1:9). The flow rate of the mobde phase was 1 ml min- ‘. Under the conditions ascorbate, DOPA and dopachrome were separated (Figs 1 and 2). Levels of the above compounds were determined from areas under the peaks on the chromatograms. H,O,-dependent
oxidation
of ascorbate
and DOPA. H,02-
dependent oxidation of ascorbate and DOPA in vacuoles and protoplasts was followed usmg vacuole and protoplast fractions eqmvalent to 13-48 and 2381 nmol of DOPA,respectively. The ratio of ascorbate to DOPA in the vacuole fractions was in the range of 1: 5 to 1:6 and that m protoplast fractions was 1: 3 to I :4.5. Vacuoles were suspended in 0.4 M mannitol, 1 mM CaCl,,
Hydrogen peroxide metabolism in vacuoles 1 mM EDTA and 50 mM HEPES-KOH (pH 7.8) and protoplasts were suspended in 0.5 M sorbitol and 50 mM MES-KOH (pH 5.8). Reactions were started by the addition of H,O, and terminated by the addition of 0.08 ml of 2% metaphosphoric acid to 0.04 ml of the above vacuole or protoplast suspension. After centrifugation at 1000 g for 5 min, the supematants (0.02 ml) were used for analysis by HPLC. Reactions were run at room temp. (ca 20 “). Vacuolar peroxidase-dependent oxidation of ascorbate and DOPA was followed in 0.5 ml of a buffer soln (0.1 M citric acid-O.2 M Na,HPO,, pH 5) which contained 0.05 ml of vacualar peroxidase, 1 mM Na ascorbate and/or 8 mM DOPA. Reactions were started by adding 1.5 mM H,O, at room temp. (ca 20”). The vacuolar peroxidase oxidized DOPA at 1.2 mM/5 min in the presence of 8 mM DGPA in the above reaction mixture. Vacuolar peroxidase was prepared by (NH&SO, pptn, cation exchange chromatography and gel filtration as had been reported [3]. Reagents. Macerozyme R-10 and cellulase “Onozuka” RS were obtained from Yakult Honsha Co., Ltd (Tokyo). DOPA and Na ascorbate were from Wako Pure Chemical Ind. Co. (Osaka). Tropolone and Ficoll-400 were from Tokyo Kasei Kogyo Co., Ltd (Tokyo) and Pharmacia, respectively. REFERENCES
1. Takahama, U. and Egashira, T. (1990) Plant Cell Physiol. 31, 539.
PHYTO
31:4-D
1133
2. Takahama, U. and Oniki, T. (1991) Plant Cell Physiol. 32, 745. 3. Takahama, U. and Egashiara, T. (1991) Phytochemistry 30, 73. 4. Asada, K. and Takahashi, M. (1987)in Photoinhibition (Kyle, D. J., Osmond, C. B. and Arntzen, C. J., eds), pp. 227-287. Elsevier, Amsterdam. 5. Fling, M., Horowitz, N. H. and Heinemann, S. F. (1963) J. Biol. Chem. 238, 2945. 6. Pomerantz, S. H. (1963) J. Biol. Chem. 238, 2351.
7. Young, T. E., Griswold, J. R. and Hulbert, M. H. (1974) J. Org. Chem. 39, 1980. 8. Gracia-Carmona, F., Gracia-Canovas, F., Iborra, J. L. and Lozano, J. A. (1982) Biochim. Biophys. Acta 717, 124. 9. Grob, K. and Matile, P. (1980) 2. Pjianzenphysiol. 98, 235. 10. Albrecht, C. and Kohlenbach, H. W. (1990) Protoplasma 154, 144. 11. Kahn, V. (1985) Phytochemistry 24, 915. 12. Pelizzetti, E., Mentasti, E., Pramauro, E. and Giraudi, G. (1976) Anal. Chim. Acta 85, 161. 13. Takahama, U. (1986) Biochim. Biophys. Acta 882, 445. 14. Mehlhorn, H. and Kunert, K. J. (1986) in Molecular and Physiological Aspects of Plant Peroxidases (Greppin, H., Penel, C. and Gaspar, Th., eds), pp. 4374. University of Geneve Press, Switzerland. 15. Peters, J. L., Castillo, F. J. and Heath, R. L. (1988) PIant Physiol. 89, 159.
0
003i-9422/92 $5.00 + 0.00 1992 Pergamon Press pie
HYDROGEN PEROXIDE SCAVENGING SYSTEMS IN VACUOLES OF MESOPHYLL CELLS OF JfICIA F&3x4
UMEO TAKA~~M~
apartment
of Biology, Kyushu Dental College, K~taky~shu 803, Japan (Received 29 August 19911
Key Word Index-Viciafaba;
Leguminosae; ascorbate; DOPA; hydrogen peroxide; peroxidase; vacuoles.
A~~ct-Vacuoles isolated from mesophyll protoplasts of Vi& faba contaio~ ascorbate in addition to 3,4dihydroxyphenylalanine (DOPA) and peroxidase. When a low concentration of H,0,(0.25 mM) was added to the vacuole factions, a slow decrease in the level of ascorbate was observed. When 2.5 mM H,O, was added to the vacuole fractions, a rapid decrease in the levels of ascorbate and formation of dopachrome were observed. The oxidation of ascorbate and the formation of dopachrom~ were inhibited by NaNs (10 mM), KCN (1 mM) and stimulated by tropolone (5 mM), suggesting the participation of peroxidase but not phenol oxidase in the reactions. Vacuolar peroxidase isolated from leaves of V.faba oxidized DOPA and ascorbate in the presence of H,O,. The oxidation of DOPA was nearly completely inhibited by ascorbate but the oxidation of ascarbate was stimulated by DOPA, suggesting the reduction of an oxidized intermediate of DOPA to DOPA by ascorbate. In fact, ascorbate reduced dopaq~none, a two electron oxidized DOPA.
RESULTS AND DISCUSSION
Vacuoles of mesophyll cells of Vic~u~~~ucon~n peroxidase and phenolics which can donate electrons to the peroxidase in the presence of H,O, Cl]. If H,O, is added to vacuoles obtained from mesophyll cells, to mesophyll protoplasts or to leaves, DOPA is oxidized to melan~ via dopachrome, an orange-red compound [I, 21. Accompanying the formation of melanin, levels of DOPA decrease. However, if incubation of leaves in H,O, is continued, the levels ofDORA increase after the decrease, sugg~ting the synthesis of DOPA [2]. Vacuolar peroxidase isolated from leaves of I’.faba oxidized DOPA in the presence of H,Oa {3]. These results suggest the participation of vacuolar peroxidase in the oxidation of DOPA by H,O,. If the peroxidase functions to scavenge H,O, using DOPA as an &&on donor under normal conditions, there must be a regeneration of DOPA from its oxidized intermediate. In chloroplasts, if ascorbate is oxidized by ascorbate peroxidase, the oxidized product, dehydroascorbate, is reduced to ascorbate by giutathioue [4]. It has been reported than an oxidized intermediate of DOPA, dopaq~none, is reduced to DOPA by ascorbate [5,6]. As peroxidase possibly oxidizes DOPA to dop~hrome and melanin via dopaquinone [l, 7, 81, generation of DOPA from dopaquino~e is possible if ascorbate is present in vacuoles of mesophyll cells of I? $&a. Therefore, it is irn~~ant to know whether ascorbate is present in the vacuoles to elucidate H,Oz-scavenging function of vacuolar peroxidase. This paper deals with the presence of ascorbate in the vacuoles of leaves of P.faba, the reactivities of DOPA and aseorbate to the vacuolar peroxidase isolated from leaves of V. faba and the reduction of dopaquinone by aseorbate. Based on the data, a p~ysiolo~~~ function of the vaeuolar peroxidase is discussed.
A HPLC elution pattern of a metaphospho~c acid extract of vacuoles is shown in Fig. 1. There were four peaks: peaks A and I3 had R,s of 3.8 and 4.4 min and absorption maxima at 270 and 288 nm, respectively. These values were the same as those of authentic ascorbate and DOPA. Two peaks on HPLC had no distinct absorption peaks in the UV region. Typical values of the specific ~tivities of some enzyme and the amount of aseorbate in terms ofamount ofDOPA which is localized mainly in the vacuoles obtained from mesophyll cells of Y. fuba are shown in Table 1 [I]. In a given vacuole fraction, the relative activities of eatalase ~roxisomes) and glucose-6-phosphate dehy~ogen~ (cytoplasmic matrix) were lower than those in the corresponding protoplast fraction. Cytochrome c oxidase activity was not detected in the vacuole fraction. The contamiuation of the vacuole fraction by chloroplasts and protoplasts, respectively, was less than 10% when examined by a light microscope. However, the ratios of relative activities of peroxidase for DAB and a-mannosidase, which are mainly localized in the vacuoles [I], were much greater than those of other enzymes. These data suggest that the vacuole fraction contains mainly vacuoles although the vacuole fraction is co~t~inat~ by other cytoplas~c components. The ratio of the relative amount of ascorbate showed a moderate value, su~esting the loealization of ascorbate in vacuoles as well as in cytoplasm other than vacuoles. The ratio of the relative activity of ascorbate peroxidase, which is present in chloroplasts in addition to cytoplasm [4], also showed a moderate value. This is attributed to the ability of the peroxidase in vacuoles to utilize aseorbate as an electron donor (Fig, 2). The presence of ascorbate in vacuoles has been reported in other plants [9]+
1127
U. TAKAHAMA
1128
The HPLC elution patterns of a product obtained by the oxidation of DOPA by a I&O,-horseradish peroxidase system are shown in Fig. 3. The product (peak 2) had a retention time of 4.7 min and absorption maxima at 310 ,
0.4r \
0
220
300 260 Wavelength f nm)
340
o.2--
Time
(min)
Fig. 1. HPLC &&ion pattern of ascorbate and DOPA extracted from vacuoles with metaphosphoric acid (lower panel) and their absorption spectra (upper panel) using 20 4 extract. A, ascorbate; B. DOPA. Absorption spectra were measured in a mobile phase. methanol-KH,PO, (1:9).
and 480 nm and the generation of the product was stimulated by tropolone. The shape of absorption spectrum of peak 2 was similar to that of dopachrome [lO]. It has been reported that peroxidase oxidizes DOPA to dopachrome and the oxidation of DOPA producing dopachrome is stimulated by tropolone [lo, 111. These data suggest that the compound of peak 2 is dopachrome. As dopachrome can be separated by HPLC in this way, in the following, dopachrome was quantified as a measure of the oxidation of DOPA. The effects of some enzyme inhibits on N,O,dependent oxidation of ascorbate and DOPA in vacuoles and protoplasts are shown in Table 2. Tropolone, an inhibitor of phenol oxidase [ll], stimulated the oxidation of ascorbate as well as the formation of dopachrome. Azide and cyanide inhibited both oxidation reactions. From the data, vacuolar peroxidase but not phenol oxidase participates in the AEON-de~ndent oxidation of ascorbate and DOPA. Tropolone (5 mM) completely inhibits oxidation of DOPA by cell-free extracts of leaves of Kfaba in the absence of H,O, [Z], suggesting that the concentration of tropolone used functions to inhibit phenol oxidase. The time course of the oxidation of ascorbate and DOPA in vacuole and protoplast fractions in the presence of 0.25 or 2.5 mM H,O, is shown in Fig. 2. When 0.25 mM I-I,O, was added to a vacuole or protoplast fraction, slow decreases of the levels of ascorbate and DOPA were observed (Fig. 2A, C) and the levels of DOPA attained constant values within 2 min. The levels of ascorbate decrease gradually during incubation, especially in the vacuole fractions. There was no detectable formation of dopachrome. Part of the decrease of ascot= bate and DOPA might be due to spontaneous oxidation of the compounds in the absence of H,O, induced by transferring vacuoles and protoplasts from 0‘ to room temperature for experiments. When 2.5 mM H,O, was added to the fractions, the levels of ascorbate decreased rapidly. The degrees of the
Table I. Specific enzymatic activities and levels of DOPA and ascorbate of protoplast and vacuole fractions
DOPA (11mol mg-’ protein) a-Mannosidase (AA,,, mg-’ protein hr-‘) Ascorbate (p mol mg-’ protein) DAB peroxidase (AP&, mg-r protem mm -1) Ascorbate peroxidase (A&,, mg-’ protem min-t) Catalase (AA240mg-’ protein mm-‘) Glucose-6-P-dehydrogenase (AA340 mg- ’ protem hr-l)
Protoplasts (I)
Vacuoles (II)
(11)/(I)
2.3 (100)
34.3 (100)
1.00
2.0 (87)
24.6 (72)
0.83
0.6 (26)
6.0 (17)
0.65
30 (1304)
469 (1367)
1.05
2.8 (122)
16.4 (48)
0.39
3.0 (I 30)
3.0 (9)
0.07
6.6 (287)
3.6 (IO)
0.04
Values in parentheses are enzymatic activities in protoplast and vacuole fractions relative to the amount of DOPA in each fraction.
Hydrogen peroxide metabolism in vacuoles
1129
A
-5 2G
l-
0
IF-A-A-d---d
-___ A________&
g 50-
I\
-0
3 .I 5 5 z
-
!L
-5
3
-
s”
IoY: O-A--A-A-___A___A________A 1 I I I.
I
-0
I
0
10 Time
(min)
Fig. 2. H,O,-dependent changes of levels of ascorbate and DOPA and formation of dopachrome in protoplast (A and B) and vacuole (C and D) fractions. The initial amounts of DOPA and ascorbate in A, B, C and D were 46.7, 23.5, 13.2 and 31.1 nmol for DOPA and 16.1, 7.0, 2.6 and 6.3 nmol for ascorbate. Reactions were performed in 0.04 ml of reaction mixtures and were started by the addition of 0.25 mM H,O, (A and C) or 2.5 mM H,O, (B and D). After incubation, reactions were terminated by the addition of 0.08 ml of 2% metaphosphoric acid. Open circles, ascorbate; closed circles, DOPA, triangles, dopachrome.
Table 2. Effects of enzyme inhibitors on H,O,-dependent
Ascorbate (nmol) Vacuoles Protoplasts Dopachrome (relative) Vacuoles Protoplasts
oxidation of DOPA and ascorbate
Before addition of H,O,
+ H,O, (2.5 mM) No add.
+ tropolone (5 mM)
+NaN,
4.8 15.2
1.7 1.9
1.1 0.1
3.1 5.0
3.7 10.1
0 0
1.6 3.0
3.5 3.9
0.2 0
0 0
(10mM)
+KCN(lmM)
Reaction mixture (0.04 ml) contained vacuoles or protoplasts equivalent to 28.2 or 65.2 nmol DOPA, respectively. Vacuoles were suspended in a buffer solution that contained 0.4 M mannitol, 1 mM CaCl,, 1 mM EDTA and 50mM HEPES-KOH (PH 7.8). Protoplasts were suspended in a buffer solution that contained 0.55 M sorbitol and 5 mM MES-KOH (pH 5.8). Reactions were started by addition of H,O, and terminated at 1 min by adding 0.08ml 2% metaphosphoric acid.
decrease of the levels of ascorbate by 2.5 mM H,Oz were greater than those by 0.25 mM H,O*. In protoplasts, the levels of ascorbate increased after the decrease of the levels of ascorbate. During the decrease of the levels of ascorbate and DOPA, formation of dopachrome was observed. The maximal amount of dopachrome formed
was more than two-fold greater in vacuole fractions than in protoplast fractions, even when the initial level of DOPA in the protoplast
fraction was greater than that in
the vacuole fraction. In vacuoles, it took ca 4 min to attain a maximal level of dopachrome and the level decreased slowly. In protoplasts, the maximal level was
1130
U.
TAKAHAMA
attained at cu 1 min after the addition of H,Oz and the level decreased as the level of ascorbate increased. The difference in the time course of the changes of the levels of dopachrome may be due to the presence and absence of a system to regenerate ascorbate from dehydroascorbate. In protoplasts, if the levels of ascorbate decreases, the levels will be recovered and the formation of dopachrome will be inhibited. Thus, the data in Fig. 2 indicate that the formation of dopachrome becomes apparent when the levels of ascorbate in vacuoles and protoplasts decrease considerably. The incomplete recovery of the level of ascorbate may be, in part, due to the rupture of protoplasts during incubation, The decrease in the levels of dopachrome after its formation may be due to the transformation of dopachrome to melanin, as has been observed [ 11. Two mechanisms are considered for the appearance of dopachrome after the considerable decrease of the level of ascorbate in both vacuoles and protoplasts. (i) Competition of the following two reactions in vacuoles: ascorbate + H,O,zdehydroascorbate
0.8 I
,
Time
(mln) 1
f 2H,O Wavelength
(nm)
POX
DOPA + H,O,-----+dopaquinone
+ 2H,O.
Here, POX is peroxidase. If the ascorbate is a better electron donor than DOPA for the H,O,-vacuolar peroxidase system, the oxidation of DOPA producing dopachrome may occur after the decrease of the levels of ascorbate. Dopaquinone, a two-electron oxidized product of DOPA, is transformed to dopachrome non-enzymatically [7, S]. (ii) The reduction of the rate of the regeneration of DOPA from dopaquinone by ascorbate due to the decrease of the levels of ascorbate: POX
DOPA + H,O,----+dopaquinone dopaquinone
+ 2H,O
+ ascorbate-+ DOPA + dehydroascorbate.
The decrease of the levels of ascorbate results in the oxidation of dopaquinone to dopachrome. To distinguish the two mechanisms, the effects of ascorbate on the H,O,-dependent oxidation of DOPA and vice versa were examined using vacuolar peroxidase isolated from leaves of V. faba (Fig. 4). The concentrations of DOPA and ascorbate in the experiments in Fig. 4 were determined by considering the concentration of DOPA in vacuoles [3] and the ratio of DOPA to ascorbate in vacuoles (Table 2 and Fig. 2). When DOPA alone was added to a H,O,-peroxidase system, the decrease of level of DOPA and dopachrome formation were observed. When ascorbate alone was added to the system, slow oxidation of ascorbate was observed. In the presence of both DOPA and ascorbate in the H,O,-peroxidase system, the initial rate of oxidation of DOPA was smaller than in the presence of DOPA alone and the rate of oxidation of ascorbate was much greater than that in the presence of ascorbate alone as an electron donor in the system. As long as ascorbate remained in the reaction mixture, there was no formation of dopachrome. However, when ascorbate was eliminated from the reaction mixture, oxidation of DOPA and formation of dopachrome was observed. When 2 mM ascorbate was added together with 8 mM DOPA, no
Fig. 3. HPLC elution pattern of DOPA and dopachrome. DOPA was oxidized by a H,O,-horseradish peroxidase system m the presence of 3 Mughorseradish peroxidase, 1 mM DOPA and 1 mM H,O, m 3 ml of 50 mM Na-phosphate (pH 6). After incubation for 3 hr at about 18”, 20 pl was applied to HPLC column. Upper panel: A, incubated without horseradish peroxidase; 8, incubated in the above reaction mixture; C, incubated in the above reaction mixture but in the presence of 1 mM tropolone. 1, DOPA; 2, dopachrome. Lower panel: absorption spectra of peaks 1 (dashed line) and 2 (sohd Ime) in a mobile phase, methanol-KN,PO, (1: 9).
formation of dopachrome was observed and ascorbate was not oxidized completely during the incubation period for 20 min. The results suggest that the oxidized intermediate of DOPA can be reduced by ascorbate, thus suppressing dopachrome formation. To confirm this, the effects of ascorbate on the formation of dopaquinone were examined (Fig. 5). The addition of H,O, in the presence of DOPA and the vacuofar peroxidase resulted in the formation of a compound with an absorption maximum at ca 390 nm at first, and then a compound with an absorption maximum at ca NO nm. The former can be attributed to dopaquinone [12] and the latter to dopachrome. When ascorbate was added to the reaction mixture, the absorption intensity around 390 nm decreased and the formation of dopachrome was inhibited. The inset in Fig. 5 indicates the rapid absorption decrease at 390 nm by the addition of ascorbate. The data suggest the reduction of dopaquinone by ascorbate. It has been reported that dopaquinone is easily reducible to DOPA by ascorbate [S, 61. Based on the data, a scheme in Fig. 6 was proposed for the H,O,-dependent metabolism of DOPA and ascorbate in vacuoles of V.fubu leaves. If H,O, diffuses into vacuoles, peroxidase in vacuoles oxidizes DOPA to dopaquinone. The o-benzoquinone will be reduced to DOPA by ascorbate, producing DOPA and dehydroascorbate. Dehydroascorbate may be translocated from vacuole to cytoplasmic matrix and will be reduced to abcorbate by glutathione. Thus, the redox cycles to generate DOPA
Hydrogen peroxide metabolism in vacuoles
Time
1131
(min)
Fig. 4. H~O~d~~ndent oxidation of ascorbate and DOPA by peroxidase. The reaction mixture (0.5 mi) contained 0.05 ml of vacuolar peroxidase and 1.5 mM HzO, in 0.1 M citric acid-O.2 M Na2HP0, buffer (pH 5.4). Where indicated, 1 mM sodium ascorbate and/or 8 mM DOPA were added. Left panel: oxidation of ascorbate and DOPA when they were added separately to the above reaction mixture. Right panel: oxidation of ascorbate and DOPA when they were added together to the above reaction mixture. Reactions were started by the addition of H,O,. Open circles: DOPA; closed circles, dopachrome; triangles, ascorbate. The levels of dopachrome are relative
after its oxidation
0 A
I
I ,Asc
I
a04
I
a02
Wavelength
(nm)
Fig. 5. Effects of ascorbate on HOOK-indu~ abso~tion changes. The reaction mixture (1 ml) contained 0.05 ml of vacuolar peroxidase, 8mM DOPA, 1 mM H,O, in 0.1 M citric acid-O.2 M Na,HPO, buffer (pH 5). Upper panel: trace a, before addition of H,O,. Traces, b, c and d are 445 and 90 set after the addition of H,Oz, respectively. Lower panel: trace a, before the addition of H,O,; trace b, 0 set after the addition of H,O,. After recording trace b, 0.5 mM sodium ascorbate was added. Traces c and d are immediately and 45 set aRer the addition of ascorbate, respectively. Repeat scan was started from 600 nm and ended at 300 nm. The scan speed was 600 nm min- ‘. An inset shows a time course of A changes at 390 nm after the addition of 1 mM H,O, and 0.5 mM sodium ascorbate, successively.
by vacuolar
peroxidase
become
pos-
sible. An oxidized interm~iate of rutin, which is an odihydroxyphenoliG, is also reduced to rutin by ascorbate [133, suggesting that the occurrence of redox cycles similar to those in Fig. 6 is also possible when other o-hydroxyphenol& are present in vacuoles. Hehlhon and Kunert [14] and Peters et al. [lSJ have found that caffeic acid, an o-hydroxyphenol, increased the rate of oxidation of ascorbate by horseradish peroxidase and extracellular peroxidase in pinto bean leaves, respectively. The stimulation can be explained by the reduction of an obenzoquinone by ascorbate, which may be formed by the oxidation of caffeic acid by the peroxidases. If the level of ascorbate becomes lower in vacuoles, the reactions to produce melanin via dopachrome will predominate. The production of melanin will become apparent when the regeneration of ascorbate from dehydroascorbate is limited by i~ibition of the formation of NAD(P)H which can reduce dehydroascorbate via glutathione. This is confirmed by the fact that methyl viologen, which inhibits photoreduction of NADP+ in chloroplasts stimulating H,O, production [4], enhanced the formation of melanin by illumination 121.
EXPERIMENTAL Plot materials. Viciafabu (cv 6461, from Tohoku Utsunomiya, Japan) were grown in vermiculite in a growth chamber in which the temps by day (12 hr) and by night (12 hr) were 15 a and lo”, respectively, and seedlings were watered once a week with a 0.1% soln of Hyponex. The maximum intensity of light by day period was about 6000 Ix. The white light was obtained from day-light type fluorescent lamps (FL4OSD) from Nihon Denki Co (Tokyo). ~~puruti~n of ~sop~yl~ proto~l~s~s. Young, fully expanded leaves were used. They were harvested in the morning (ca 9:00 am) from plants grown for 1-3 months. Immediately after
U.
1132 b
---+
TAKAHAMA
--+
---+
Melanin
I+
Whrome
Asc
&
(Hz%- generating
systems)
Fig. 6. A proposed scheme of the H,O,-dependent metabolism of DOPA and ascorbate in vacuoles. POX, peroxidase; Asc, ascorbate; DHA, dehydroascorbate; GSH, ~lutathione; GSSG, oxidized form of glutathione. harvest, ca 20 leaves from which the lower epidermis has been partially removed were floated on a soln of enzymes which consisted of 550 mM sorbitol, 1% cellulase and 0.5% macerozyme in 5 mM MES-KOH (PI-I 5.8). The soln of enzymes was infiltrated into leaves zn WCUO,and the infiltrated leaves were incubated at 25 0 for 1 hr with gentle shaking under room light. Released protoplasts were collected by passage through 4 layers of gauze and centrifuged at IOOg for 2 min. Isolated protoplasts were suspended in 550 mM sorbitol in 5 mM MES-KOH (pH 5.8) and kept at 0” after two washings with the suspension medium. Frepura~~on ofvacuofes. Vacuoles were prepared as described previously El]. To release vacuoles from mesophyll protoplasts, suspensions of protoplasts (0.4 ml) equivalent to about 1 mg chlorophyl1 were added to 2 ml of a 0.1 M soln of K,HPO, adjusted to pH 8.5 with HCI. After 1-2 min at room temp. (ca 20 “), each suspension was loaded onto a discontinuous gradlent of Ficoll-400 in 50 mM HEPES-KOH (pH 7.8) which contained 0.4 M mannitol, 1 mM CaCl, and 1mM EDTA. The gradients were centrifuged at 3700 g for 15 min at 4 O.Vacuoles accumulatingat the interfaces between 1 and 3% and between 3 and 5% Ficoll were collected from the gradients with a Pasteur pipette and washed once by ~t~~tion (2OOg, 2min) in the buffer soln without Ficoll. The prepn of vacuoles contained a small number of protoplasts and chloroplasts when examined under a light microscope, and the ratio of vacuoles to protoplasts was more than 19:l. Isolated vacuoles were kept at 0”. The concn of protein was determined with a protein-assay reagent from BioRad with bovine serum albumin as standard. This assay is based on shift of A of an acidic soln of Coomassie Brilliant Blue when binding to protein. Assay of enzyme activities. Catalase activity was assayed following decrease in A at 240 nm in a reaction mixture (1 ml) of 1 mM H,O,, 1 mM CaCl,, 1 mM EDTA, 0.4M mannitoi and 5OmM HEPES-KOH @H 7.8). The activity of glucose-& phosphate dehydrogenase was followed at 340 nm in a reaction
mixture (1 ml) that contained 1 mM glucose-6-phosphate, 0.2 mM NADP+, 1 mM CaCl,, I mM EDTA, 0.4 M mannitol and 50 mM HEPES-KOH (pH 7.8). The activity of ascorbate peroxidase was followed at 290 nm in a reaction mixture (1 ml) which contained 1 mM Na ascorbate, 1 mM CaCI,, 1 mM EDTA, 0.4 M mannitol and 50 mM HEPES-KOH (pH 7.8). Diaminobenzldine peroxidase activity was followed at 460 nm in a reaction mixture (1 ml) which contained 1 mM diaminobenzidine, 0.55 M sorbitol and 5 mM MES--KOH (pH 5.8). The activity of r-mannosidase was followed in a reactlon mixture (I ml) which contamed 0.5 mM p-nitrophenol-~-mannopyranoside and 0.1 M citric acid- 0.2 M Na,HPO, (pH 5). After mcubatlon for 1 hr at ca 20 :, 1ml of 1 M Na,CO, was added and the A at 420 nm was determined. Blank values were taken into account in the calculation of the enzymatic activites. All reactions were started by the addition of vacuoles or protoplasts. Prior to the addition of vacuoles or protoplasts, they were disrupted by repeating suckmg and ejectIon using a microsyringe. Spectrophotometrlc measurements were carried out at room temp (ca 20”). The path length of the measurmg beam was 4 nm. Assay by HPLC. HPLC was performed using a Shimadzu CL6A pump combined with a Shim-pack CLC-C, column (15 cm x 6 mm i.d.) and a spectrophotometric detector with a photodiode array (Shimadzu SPD-MIA). The mobile phase was MeOHmM KH,PO, (1:9). The flow rate of the mobde phase was 1 ml min- ‘. Under the conditions ascorbate, DOPA and dopachrome were separated (Figs 1 and 2). Levels of the above compounds were determined from areas under the peaks on the chromatograms. H,O,-dependent
oxidation
of ascorbate
and DOPA. H,02-
dependent oxidation of ascorbate and DOPA in vacuoles and protoplasts was followed usmg vacuole and protoplast fractions eqmvalent to 13-48 and 2381 nmol of DOPA,respectively. The ratio of ascorbate to DOPA in the vacuole fractions was in the range of 1: 5 to 1:6 and that m protoplast fractions was 1: 3 to I :4.5. Vacuoles were suspended in 0.4 M mannitol, 1 mM CaCl,,
Hydrogen peroxide metabolism in vacuoles 1 mM EDTA and 50 mM HEPES-KOH (pH 7.8) and protoplasts were suspended in 0.5 M sorbitol and 50 mM MES-KOH (pH 5.8). Reactions were started by the addition of H,O, and terminated by the addition of 0.08 ml of 2% metaphosphoric acid to 0.04 ml of the above vacuole or protoplast suspension. After centrifugation at 1000 g for 5 min, the supematants (0.02 ml) were used for analysis by HPLC. Reactions were run at room temp. (ca 20 “). Vacuolar peroxidase-dependent oxidation of ascorbate and DOPA was followed in 0.5 ml of a buffer soln (0.1 M citric acid-O.2 M Na,HPO,, pH 5) which contained 0.05 ml of vacualar peroxidase, 1 mM Na ascorbate and/or 8 mM DOPA. Reactions were started by adding 1.5 mM H,O, at room temp. (ca 20”). The vacuolar peroxidase oxidized DOPA at 1.2 mM/5 min in the presence of 8 mM DGPA in the above reaction mixture. Vacuolar peroxidase was prepared by (NH&SO, pptn, cation exchange chromatography and gel filtration as had been reported [3]. Reagents. Macerozyme R-10 and cellulase “Onozuka” RS were obtained from Yakult Honsha Co., Ltd (Tokyo). DOPA and Na ascorbate were from Wako Pure Chemical Ind. Co. (Osaka). Tropolone and Ficoll-400 were from Tokyo Kasei Kogyo Co., Ltd (Tokyo) and Pharmacia, respectively. REFERENCES
1. Takahama, U. and Egashira, T. (1990) Plant Cell Physiol. 31, 539.
PHYTO
31:4-D
1133
2. Takahama, U. and Oniki, T. (1991) Plant Cell Physiol. 32, 745. 3. Takahama, U. and Egashiara, T. (1991) Phytochemistry 30, 73. 4. Asada, K. and Takahashi, M. (1987)in Photoinhibition (Kyle, D. J., Osmond, C. B. and Arntzen, C. J., eds), pp. 227-287. Elsevier, Amsterdam. 5. Fling, M., Horowitz, N. H. and Heinemann, S. F. (1963) J. Biol. Chem. 238, 2945. 6. Pomerantz, S. H. (1963) J. Biol. Chem. 238, 2351.
7. Young, T. E., Griswold, J. R. and Hulbert, M. H. (1974) J. Org. Chem. 39, 1980. 8. Gracia-Carmona, F., Gracia-Canovas, F., Iborra, J. L. and Lozano, J. A. (1982) Biochim. Biophys. Acta 717, 124. 9. Grob, K. and Matile, P. (1980) 2. Pjianzenphysiol. 98, 235. 10. Albrecht, C. and Kohlenbach, H. W. (1990) Protoplasma 154, 144. 11. Kahn, V. (1985) Phytochemistry 24, 915. 12. Pelizzetti, E., Mentasti, E., Pramauro, E. and Giraudi, G. (1976) Anal. Chim. Acta 85, 161. 13. Takahama, U. (1986) Biochim. Biophys. Acta 882, 445. 14. Mehlhorn, H. and Kunert, K. J. (1986) in Molecular and Physiological Aspects of Plant Peroxidases (Greppin, H., Penel, C. and Gaspar, Th., eds), pp. 4374. University of Geneve Press, Switzerland. 15. Peters, J. L., Castillo, F. J. and Heath, R. L. (1988) PIant Physiol. 89, 159.