Plant Physiol. (1997) 115: 1405-1 41 2
Flavonoid-Peroxidase Reaction as a Detoxification Mechanism of Plant Cells against H 2 0 2 H i d e o Yamasaki*, Yasuko Sakihama, and N o r i k a t s u l k e h a r a
Laboratory of Cell and Functional Biology, College of Science, University of the Ryukyus, Nishihara, Okinawa 903-01, Japan vonoids in the scavenging of 'O, and/or oxygen radicals (0,- and .OH) may be limited to nearby generating sites, i.e. only in the vacuoles and partly in the cell wall. However, unlike other active oxygen species, H,O, is stable and able to diffuse across membranes. The toxicity of H,O, itself is relatively weak compared with that of other active oxygen species, but in the presence of O,-, H,O, can generate highly reactive hydroxyl radicais via the metal-catalyzed Haber-Weiss reaction. Thus, the scavenging of H,O, in cells is critica1to avoid oxidative damage. Plant cells are prone to produce H,O, not only under stressed conditions, but also from regular metabolism. Accumulation of flavonoids seems to be pronounced in tissues under such conditions. Food chemical studies have revealed that flavonols can act as electron donors for peroxidase (Miller and Schreier, 1985; Schreier and Miller, 1985). Takahama (198813, 1989) has proposed that this flavonol-peroxidase reaction may function as an H,O,scavenging system in vivo as well as in vitro. In contrast to our understanding of pharmacological properties of flavonoid pigments, however, there is little information on whether flavonols can contribute to the scavenging of H,O, in vivo. In this study the participation of a flavonol-peroxidase system in H,O, detoxification is examined in contrast to the well-established ascorbate-APX system in plants. Results obtained from the field-grown tropical plants further support the H,O,-scavenging function of flavonoids.
Recent studies have revealed that dietary flavonoids are potent radical scavengers, acting in a manner similar to ascorbate and a-tocopherol. However, it is still not clear whether flavonoids have a similar antioxidative function in plants. We examined the possibility that flavonoids could function as stress protectants in plant cells by scavenging H,O,. Two major flavonoids, quercetin and kaempferol glycosides, were isolatedfrom leaves of the tropical tree Schefflera arboricola Hayata. Both glycosides and aglycones of isolated flavonols were oxidized by H,O, in the presence of horseradish peroxidase and/or in a soluble fraction of S. arboricola leaf extract. The rates of oxidation were in the order quercetin > kaempferol > quercetin glycoside >> kaempferol glycoside. Judging from the effects of inhibitorssuch as KCN, pchloromercuribenzoate, and 3-amino-1H-l,2,4-triazole, we conclude that guaiacol peroxidase in the soluble fraction catalyzes H,O,-dependent oxidation of flavonols. In the flavonol-guaiacol peroxidase reaction, ascorbate had the potential to regenerate flavonols by reducing the oxidized product. These results provide further evidence that the flavonoidperoxidase reaction can function as a mechanism for H,O, scavenging in plants. ~
Flavonoids are the most common secondary metabolites in vascular plants, with the exception of betalain in a few families (Stafford, 1994).As components in the human diet, flavonoids have recently received considerable attention as efficient antioxidants, in addition to ascorbate, a-tocopherol, and carotenoids. Numerous in vitro studies have shown that flavonoids can directly scavenge molecular species of active oxygen: superoxide (O,-), hydrogen peroxide (H202),hydroxyl radical (.OH),singlet oxygen ('O,), or peroxyl radical (Bors et al., 1990, 1994; Yamasaki et al., 1996). In plants the photosynthetic electron transport system is the major source of active oxygen species. To avoid oxygen-mediated toxicity, chloroplasts have evolved a highly developed detoxification system, which has been termed the ascorbate-glutathione cycle (Foyer, 1993).It had been proposed that flavonoids scavenge active oxygen species, such as O,- or 'O,, photoproduced in the chloroplast in a manner similar to a-tocopherol and carotenoids. However, this is unlikely in vivo because flavonoids are largely localized in vacuoles (Charriere-Ladreix and Tissut, 1981) and radicals cannot readily diffuse into vacuoles from chloroplasts. Therefore, in plant cells the participation of fla-
MATERIALS A N D M E T H O D S
Leaves of the tropical tree Sckefflera arboricola Hayata were harvested from a field on Okinawa island, a subtropical region of Japan. The age of the plants was between 10 and 15 years. The sampling period was the summer season, from July to October. A11 materials analyzed were harvested from the canopy of the trees, where the light environment was nearly constant (maximum 2200 pmol quanta . m-'s-') lsolation of Flavonols
Leaves with a removed midrib were homogenized for 30 s with a 3-fold volume of 80% MeOH at room temper~
* Corresponding author; e-mail
[email protected]; fax 81-98-895-5376.
Abbreviations: APX, ascorbate peroxidase; GuPX, guaiacol peroxidase; MeOH, methanol; SF, Schefflera arboricola flavonoid. 1405
1406
Yamasaki et al.
ature. The homogenate was filtered through eight layers of gauze and further through a paper filter (No. 2, Advantec, Tokyo, Japan) in vacuo to remove cellular debris. To exclude contaminants of photosynthetic pigments, such as chlorophylls or carotenoids, the MeOH extract was passed through a Sep-Pak C,, filter cartridge (Waters). Subsequently, the same volume of 100% acetone was added to the filtrate (final ratio, acetone:MeOH:H,O, 5:4:1, v/v). The precipitates caused by acetone were removed by centrifugation (5000g for 3 min). This step efficiently separated flavonols from contaminants in the MeOH extract. Afterward, a 3-fold volume of diethyl ether was added to the supernatant. Phase separation was carried out by centrifugation at 20009 for 3 min. A brownish precipitate was obtained and washed again with diethyl ether. The concentrate was diluted with 50% MeOH and subjected to reverse-phase column chromatography (Lobar RP-18, Merck, Darmstadt, Germany). The concentrate was eluted with acetic acid:MeOH:H,O (5:25:75, v/v) at a detection wavelength of 346 nm. The peak fractions were collected, and flavonoid glycosides were adsorbed to a Sep-Pak C, filter cartridge (Waters). After rinsing with distilled water, the adsorbed flavonol glycosides were eluted with a small volume of MeOH (100%) and used for subsequent biochemical assays. HPLC and TLC Analysis
HPLC analysis was carried out with a reverse-phase C,, column (SC,,AR, 4.6 X 50 mm; Nacalai tesque, Kyoto, Japan). The solvent for elution contained acetic acid: MeOH:H,O in a ratio of 5:25:75 (v/v) for 10 min, 5:51:49 for a subsequent 8 min, and 5:90:10 for 7 min at a flow rate of 1.0 mL min-l. Peak areas were monitored at 370 nm with a Chromatopac integrator (C-R3A, Shimadzu, Kyoto, Japan). The identification of flavonoids was confirmed by cellulose TLC (Funacel SF, Funakoshi, Tokyo, Japan) as previously described (Yamasaki et al., 1995a). Spectroscopic Analysis
Determination of chlorophyll, total flavonoids, and ascorbate were carried out with a UV-160A spectrophotometer (Shimadzu). Leaves with midribs removed were divided into halves. Halves of leaves were cut into small pieces and were homogenized for 30 s with a known volume of solvent (80% acetone for chlorophylls and 100% MeOH for flavonoids) at room temperature. The supernatant obtained by centrifugation was used for spectrophotometric determination. To entirely exclude chlorophylls and carotenoids, the supernatant was passed through a Sep-Pak C,, cartridge before the measurement of flavonoids. This preparation was also used for the HPLC analysis. The sample preparation for the determination of ascorbate content was similar to that described above. The homogenization was carried out with a 0.1 M phosphate buffer (pH 6.8) at 0°C. The homogenate was filtered through four layers of gauze. The supernatant obtained by centrifugation (20,0009 for 2 min at OOC) was passed
Plant Physiol. Vol. 115, 1997
through a cellulose nitrate filter (DISMIC 25, Advantec) to remove proteins, and further through a Sep-Pak C, filter cartridge to exclude UV-absorbing compounds such as polyphenols. The ascorbate content was determined with the enzymatic method (Takahama, 1992). DTT at 100 p~ was added before the measurement to reduce dehydroascorbate. Measurement of the Peroxidase Activity
For sample preparation leaves were homogenized with a 10-fold volume of buffer containing 5% (w/v) polyvinyl polypyrrolidone, 1 mM EDTA, 10 mM sodium ascorbate, and 50 mM potassium phosphate (pH 7.0). The supernatant obtained by centrifugation (20,00Og, 2 min at OOC) was passed through a cellulose acetate filter (DISMIC 25) to exclude membrane fractions. After addition of sorbitol (20% at final [w/v]) for protection of the APX, the soluble fraction obtained was applied to a gel-filtration column to remove low-molecular-interference compounds, using an Econopak 10 DG column (Bio-Rad)equilibrated with buffer containing 1 mM EDTA, 20% sorbitol (w/v), and 10 mM potassium phosphate (pH 7.0). Enzyme activity was measured as described previously (Amako et al., 1994). The kinetics of flavonol oxidation were monitored at 380 and 360 nm for aglycones and glycosides, respectively. Ascorbate oxidation in the presence of flavonol was monitored at 265 nm, at which leve1 there was no significant overlap of the absorption changes to cause difficulties in interpretation (Takahama and Oniki, 1992).Kinetics parameters were determined from the absorbance decrease using reported extinction coefficients (Takahama and Egashira, 1991). RESULTS Quercetin and Kaempferol Clycosides as the Major Flavonoids in S. arboricola Leaves
In S. arboricola leaves two major flavonoids were identified as yellow fluorescent spots by two-dimensional TLC under UV light (not shown). We have isolated these flavonoids with C,, reverse-phase column chromatography (Fig. 1).Judging from the ratio of A346to A,, as a measure of purity, fractions showing a value below 0.9 were collected and concentrated to use as the flavonoid preparation for the subsequent assays. Each preparation was resolved as a single peak upon retesting by HPLC. The spectral profile of the isolated flavonoids showed two characteristic absorption maxima in the UV range, corresponding to bands I and I1 of flavone/flavonols (Markham, 1993). For convenience, the flavonoids isolated here are tentatively designated as SF1 and SF2 in elution order. The absorption maxima of SF1 in 100% MeOH were 258 and 359 nm, and those of SF2 were 266 and 346 nm (Fig. 1, insets). The R, value of the aglycones obtained after acidhydrolysis treatment of SFs corresponded to that of authentic quercetin (0.62) and kaempferol (0.47) on cellulose TLC (Fig. 2), indicating that SF1 is a quercetin glycoside and that SF2 is a kaempferol glycoside. This was confirmed by
1407
H2O2-Scavenging Function of Flavonoids
shown in Table I suggest that the three positions of both quercetin and kaempferol are glycosylated in SF1 and SF2. These results are consistent with the identification of the major leaf flavonoids contained in S. arboricola as flavonol glycosides, namely quercetin-3-glycoside (SF1) and kaempferol-3-glycoside (SF2). H2O2-lnduced Oxidation of Flavonols in the Presence of o VI
Horseradish Peroxidase
kaempferol > SF1 » SF2. These results clearly show that SF1 and SF2 can also act as electron donors for the peroxidase reaction in addition to the corresponding aglycones. As previously noted in the case of quercetin and its glycosides (Takahama, 1986), the reactivity of SF glycosides was considerably lower than that of their aglycones.
quercelin (3. 3', 4'-OH)
myricetin (3. 3', 4' 5'-OH) Table I. UV spectral data of the flavonoid glycosides isolated from S. arboricola Figure 2. TLC of the aglycone moiety of the flavonoids isolated from S. arboricola. The aglycones were obtained after acid hydrolysis of isolated SFs 1 and 2. Forestal (acetic acid:HCI:H2O, 30:3:10, v/v) was used as the developer. Lane 1, Authentic aglycones (apigenin, kaempferol, quercetin, and myricetin); lane 2, SF1 aglycone; and lane 3, SF2 aglycone. The chemical structure of flavone/flavonol is represented on the right. Among these, only apigenin, lacking 3-OH, is a flavone and the others are flavonols. The number of OH groups of the B ring represents the only structural difference between kaempferol, quercetin, and myricetin.
Absorption Maxima Flavonoid MeOH
NaOMe
AICI3
AlCU/HCI
nm
SF1 SF2
359, 303sh'' 258 346, 302sh 266
" sh, Shoulder.
402, 273 396, 325 274
417, 303 271 398, 346 306, 276
400, 303, 397, 306,
358 269 346 276
1408
Yamasaki et al.
-E 0.2 A -4
-HRP
0.2
O
W
c a, o
c
P O
0.1
o. 1
+ HRP
v)
9 n
“o IC
1
4
2
0
1
O
2
-HRP
E
o 0.08 -
0.08
a
(3
Y
a,
o C
([I
Plant Physiol. Vol. 115, 1997
cells contain two molecular families of peroxidases: GuPXs, such as horseradish peroxidase, and APXs. Both activities are inhibited by KCN, but p-chloromercuribenzoic acid inhibits only APXs (Amako et al., 1994). Table I1 shows the effect of inhibitors on the peroxidase-mediated oxidation of flavonols by H,O, in the presence of the leaf extract. The specificity of inhibitors was clearly observed with a soluble fraction of leaf extract when pyrogallol and ascorbate were used as electron donors for the peroxidase reactions. KCN virtually completely suppressed the oxidation of SF1, suggesting the participation of peroxidase in the soluble fraction. The oxidation of SF1 was not prevented by pchloromercuribenzoic acid at 200 p ~ whereas , ascorbate oxidation was completely inhibited. Another possibility is that catalase, which is known to exhibit peroxidase activity under certain conditions, was responsible for the oxidation of flavonol glycosides. However, we eliminated this possibility, because the oxidation of SF1 was not affected by 10 mM aminotriazole, an inhibitor of catalase (data not shown). These overall data suggest that SFs can be oxidized by GuPXs rather than APXs in leaves.
Q
Ô 0.04-
+ HRP
8 a
01 o
1
2
I O
B
5
10
0.04
‘o
15
Time (min) Figure 3. Oxidation of flavonols by H,O, in conjunction with horseradish peroxidase. A, Quercetin; 6, kaempferol; C, SF1 (quercetin3-glycoside);and D, SF2 (kaempferol-3-glycoside). The reaction medium (1 mL) contained 50 mM potassium phosphate (pH 6.5) and 30 p~ flavonol. H,O, was added to reaction medium at the times indicated by the arrows (final concentration, 30 p ~ ) -HRP, . In the
absence of horseradish peroxidase; +HRP, horseradish peroxidase (final, 30 milliunits mL-’) was present before adding H,O,.
This decrease in reactivity was most pronounced in SF2; the oxidation rate of SF1 was decreased to 20% of quercetin by glycosylation, whereas it was decreased to 2% of kaempferol in the case of SF2. The relative K , values for quercetin, kaempferol, SF1, and SF2 were 28 2 5, 14 2 4, 180 ? 36, and 1080 2 271 p ~respectively, , in the presence of 1 mM H20,. The relative V,,, for the oxidation of quercetin, kaempferol, SF1, and SF2 were 16, 7, 2, and 0.7 mmol mg-’ protein min-l, respectively, in the presence of 1mM H,O,. Because a similar decrease of the reactivity was also observed in the case of commercially available kaempferol-3-rutinoside (not shown), the decrease may be ascribed to the significance of the 3-OH substitution pattern (Takahama, 1986).
Cooperative Function of Ascorbate to the Flavonol-Peroxidase Reaction
Figure 4 shows the effect of ascorbate on the oxidation of flavonols by peroxidase. Under the same conditions to those in Figure 3, ascorbate in the reaction medium completely suppressed the apparent H,O,-induced oxidation of SF1 even in the presence of horseradish peroxidase (Fig. 4A, top trace). One explanation for thís result would be the preferential oxidation of ascorbate by peroxidase instead of SF1, as proposed by Mehlhorn et al. (1996). However, as shown in Figure 4B (top trace), the activity of ascorbate in the GuPX reaction is very low. Nevertheless, H,O, caused rapid oxidation of ascorbate when SF1 was also present in the reaction medium (Fig. 4B, bottom trace). A similar phenomenon was also observed in the case of SF2 (not shown). Obviously, ascorbate does not act as a primary
Table II. Effects of peroxidase inhibitors on the rate of oxidation of
flavonols by leaf extract A soluble extract was prepared from leaves as described in “Materials and Methods.” A reaction mixture contained 50 mM potassium phosphate (pH 7.0), 0.1 mM H,O,, and 50 FL of soluble extract in a total volume of 1 mL. As the electron donor, 40 p~ SF1, 20 mM pyrogallol, or 1 mM ascorbate was present in a reaction mixture. Values are the means ? so of three measurements. The control rates were 0.021, 1.37, and 2.1 7 pmol min-’ mg-’ protein in oxidation of SF1, pyrogallol, and ascorbate, respectively. Relative Activity
Inh ibitor
Oxidation of Flavonols by H,O, in Conjunction with the Soluble Fraction of Leaf Extract
Similar to the results obtained with horseradish peroxidase, H,O, oxidized quercetin, kaempferol, SF1, and SF2 in the presence of the soluble fraction of leaf extract. Plant
SF1
Pvroeallol
Ascorbate
%
1 O0
Control 200
/.LM
1 O0
p-chloromercuribenzoate 64 f 1 O 122 f 11
1 mM KCN
5?4
1+-1
1 O0 823 1152
H,O,-Scavenging Function of Flavonoids
s
-
0.10
(*,
0.08
C
O
0.06
E X o 0.04
f .$ .-5
a 0.16 0.12 0.08
c a, O
go
G v)
2
0.02
0.04
O O
-
0-
120 180 Time (s)
60
-SFI
electron donor for the peroxidase reaction, but may be consumed to reduce the oxidized product(s) of flavonols.
clearly shows the close relationship between photosynthetic and detoxification capacity in leaves. Because the number of chloroplasts in the mesophyll cell did not change during leaf development (data not shown), the age-dependent changes in ascorbate content can be attributed to an increase in antioxidative capacity concomitant with the development of thylakoid lamella. In contrast to these metabolites, flavonoid levels estimated from the A,,, of MeOH extracts decreased with leaf area, showing a negative correlation to chlorophyll ( r = -0.61) and ascorbate (r = -0.61). Similar results were obtained by HPLC analysis when flavonoid contents were compared between young and mature leaves (Table 111). In particular, the content of SF1 (quercetin glycoside) was strongly dependent on the leaf age, but SF2 (kaempferol glycoside) was less dependent. The content ratio of SF1 to SF2 decreased during leaf development (Table 111). These age-dependent changes in flavonoid content were less apparent in shade leaves, which experienced maximum irradiances of approximately 200 pmol m-’ s-’ at noon and in which the flavonoid levels were low, even in young leaves (data not shown).
Quantitative Relationship between Flavonoids and Ascorbate in Leaves during Leaf Development
It is generally accepted that the ascorbate-APX reaction is the most significant H,O,-scavenging mechanism in plant cells (Asada, 1992). Thus, plant cells contain high concentrations of ascorbate, which serves as the electron donor for this reaction (Foyer, 1993). This is particularly pronounced in leaves because of a high requirement for detoxification of active oxygens produced during photosynthesis (Grace and Logan, 1996). Figure 5 shows the changes in chlorophyll and ascorbate content during leaf development. The leaf area of S. arboricola increased significantly with developmental age. A rate of increase in leaf area was approximately 1 cm2 d-l under field conditions. Concomitantly, the apparent leaf color changed from yellow-green to deepgreen during development. Chlorophyll content was well correlated with leaf area ( r = 0.72), and ascorbate pool size, calculated from the amount of ascorbate plus dehydroascorbate, also increased with leaf area (Y = 0.67). This 100 I
o
t
0.12
Nf -E,O
0.1
I
1
0 O
10
20
a,
w m
e 8 9
0 .
30
40
Leaf area (cm2)
&- 0 . 4 1
6
2
v
0.04
0.3
v VI
2
0.06 -
e
0.2
9m
-
o -
o
g 0.0%-
i’ 0
:‘=
Figure 4. Cooperative effect of ascorbate on the peroxidase-dependent oxidation of flavonol by H,O,. A, Suppression of the flavonol oxidation by ascorbate. The oxidation of SF1 was monitored at 360 nm in the presence (+ASA) or absence (-ASA) of 50 /.LM ascorbate. B, Stimulation of ascorbate oxidation by flavonol. The oxidation of ascorbate (initial concentration, 50 p ~ was ) monitored at 265 nm in the presence ( + S F l ) or absence (-SFl) of 30 /.LM SF1. The experimental conditions of the top trace in A and the bottom trace in B were identical except for the monitoring wavelength. Other experimental conditions were similar to those in Figure 3 (H,O,, 30 ~LM;horseradish peroxidase, 30 milliunits mL-’).
y
(\1
-\
(o
-1
2 0.20 -
+ ASA
1409
ii
0.1
’
o
50
0.02 10
I
I
20
I
I
I
30
Leaf area (cm2)
40
0 O
10
20
30
40
50
Leaf area (cm2)
Figure 5. Correlation between ascorbate and flavonoid contents during leaf development. Chlorophyll content is represented by chlorophyll a + b, and ascorbate i s represented by ascorbate + dehydroascorbate. Flavonoid levels were estimated from A,,, of the MeOH extract.
Plant Physiol. Vol. 11 5, 1997
Yamasaki et al.
1410
Table 111. Changes in flavonoid contenfs during leaf development in S. arboricola Values are the means ? SD of four to six measurements. Leaf area was used as a measure of leaf age. Leaf Area
Young (2.5 -+ 0.5) Middle (10.9 2 1.1) Mature (26.3 ? 1.7)
S F2
SF1 pmol g-
cmz
4.24 2 2.58 2.96 2 0.90 1.29 2 0.27
SFl/SF2
~
’ fresh wt
2.02 2 0.07 3.37 ? 0.66 2.35 ? 0.45
are 100 times higher than the K, values for the vacuolar peroxidase, they have suggested that the flavonols in vacuoles can function as electron donors to vacuolar peroxidase in vivo (Takahama and Egashira, 1991). Results obtained from S. arboricola are consistent with these observations.
2.0 ? 1.1 0.9 ? 0.3 0.6 ? 0.1
DISCUSSION Flavonoids as Electron Donors to Peroxidase
The present study has demonstrated that flavonol glycosides in leaves of S. arboricola have the potential to act as reducing agents in a manner similar to ascorbate (Figs. 3 and 4). The concept of antioxidative function of flavonoids is not novel, as seen in the “vitamin P” concept proposed 60 years ago (Bors et al., 1990), but has been largely bypassed in the physiological research of plants. The chemical basis of the antioxidative potential of flavonoids has been ascribed to the hydroxy groups present in their structures. Bors et al. (1990) have suggested three structural features that are important determinants for the radical-scavenging potential of flavonoids: (a) the odihydroxy (catechol) structure in the B ring; (b) the 2,3double bond in conjunction with 4-0x0 function; and (c) the presence of 3- and 5-OH groups (see Fig. 2). Similar to nonenzymatic radical-scavenging efficiency, the electrondonating activity of flavonoids to peroxidases also requires these structures (Takahama, 1986; Takahama and Egashira, 1991). Among them, the 3-OH group is the most’significant determinant of electron-donating activity (Takahama and Egashira, 1991); aglycones are oxidized much faster than 3-glycosides (Fig. 3). However, it is unlikely that aglycones act as substrates for peroxidase in vivo because they are localized in the nonaqueous phase. Thus, the catechol structure in the B ring, rather than 3-OH, may be actually more important in determining the efficiency in vivo. In this context, quercetin glycosides, which dominated the flavonoid profile of young leaves (Table III), are postulated to be superior electron donors than kaempferol glycosides in vivo. Participation of Vacuolar Peroxidase in the H,O,-lnduced Oxidation of Flavonoids
The inhibitor experiments (Table 11) suggest that flavonoids are oxidized to a greater extent by GuPX than APX in leaf extracts. Although GuPXs are known to be localized in vacuoles and apoplasts, the former dominate the total activity of leaf extracts (Takahama and Egashira, 1991). Results in Table 11, therefore, can be largely explained by the enzymatic activity of vacuolar GuPXs in S. arboricola. Takahama and Egashira (1991) have isolated a basic peroxidase from vacuoles of broad bean and demonstrated that flavonols are good electron donors to vacuolar peroxidase. Because the concentrations of flavonols in vacuoles
A Flavonoid Redox Cycle as the H,O,-Scavenging
Mechanism
When quercetin is oxidized in vitro by the peroxidaseH,O, system, dimerized and trimerized quercetin are produced as the major oxidized products (Schreier and Miller, 1985). Ascorbate can reduce the primary oxidized product of flavonols (probably a flavonoid radical) and consequently inhibits the subsequent formation of degraded products (Takahama, 1986; Jan et al., 1991). A scheme that can account for these reactions is: 2 FlavOH + H202+ 2 FlavO .f 2 H20
(1)
2 FlavO + 2 ASA -+ 2 FlavOH + 2 MDA
(2)
MDA + MDA + ASA + DHA
(3)
H202+ ASA
(4)
+
2 H 2 0+ DHA
where FlavOH is a flavonoid containing a free hydroxyl group, FlavO. is a flavonoid phenoxyl radical, MDA is the monodehydroascorbic acid radical, ASA is ascorbic acid, and DHA is dehydroascorbic acid. Reaction 1 is catalyzed by peroxidase, whereas reactions 2 and 3 proceed nonenzymically. If ascorbate is absent, polymerization products of flavonoids, similar to the case of tannin formation, may be irreversibly generated. The results shown in Figure 4 can be accounted for by the sum of reactions 1, 2, and 3, namely, no apparent oxidation of flavonoids, as shown in reaction 4. If ascorbate regeneration by the cytosolic DHA reductase and glutathione reductase system is coupled with reaction 4, it is possible that the vacuolar flavonoidperoxidase system could function as an H,O,-scavenging mechanism, as previously proposed in broad bean (Takahama, 1992). A Role of Flavonoids in U V Tolerance
With respect to their proposed functions in leaves, flavonoids are thought to be primarily involved in the protection against UV light. It has long been proposed that flavonols act as interna1 UV-screening molecules for protecting photosynthetic tissues (Koes et al., 1994; Shirley, 1996). Analyses of mutants defective in flavonoid biosynthesis have indicated the importance of flavonoids for UV tolerance (Lois and Buchanan, 1994; Shirley, 1996). Recently, however, Landry et al. (1995) have demonstrated that a mutant of Arabidopsis thaliana defective in ferulic acid hydroxylase Vah 1 ) is more susceptible to UV damage than a chalcone isomerase-deficient mutant (tt 5). They suggest that hydroxycinnamate derivatives are more important than flavonoids for UV tolerance (Landry et al., 1995). Like flavonols, hydroxycinnamate derivatives also have antioxidative properties (Castelluccio et al., 1995) and act as electron donors to GuPX (Takahama, 1988a). UV light induces
1411
H,O,-Scavenging Function of Flavonoids
A
---- ASA
m
H20
c
B
hv
V
Figure 6. A proposed diagram for the protective function of flavonoids during stress and growth. A, Scheme of the H,O,-scavenging mechanism by flavonoids. vPX, Vacuolar peroxidase; F, flavonoid; F., flavonoid radical; ASA, ascorbic acid; DHA, dehydroascorbic acid; hv, light energy; and cDHAR, cytosolic dehydroascorbic acid reductase. The diffusive nature of H,O, enables vPX to scavenge it in vacuoles, even if the generating site is other than a vacuole (6).This concept can be expanded to the cell-cell interaction. The photoproduced H,O, may leak out from mesophyll cells and be scavenged in epidermal cells that have a high flavonoid content (C).
oxidative stress and activates the production of active oxygen species, including free radicals (Mount, 1996). Therefore, it is likely that polyphenolic "sunscreen" pigments protect cells from UV damage by indirect means such as H,Oz scavenging in addition to their absorption properties. Infuh and tt mutants UV irradiation increases lipid peroxidation and APX activity (Landry et al., 1995).This strongly suggests that active oxygens participate in the mechanism of UV-B-induced injury (Foyer et al., 1994). Flavonoids as Stress Protectants
Flavonoids are known to be induced not only by exposure to UV-B but also by various type of stresses (Dixon and Paiva, 1995; Shirley, 1996). They often accumulate in response to wounding, pathogen infection, high light, chilling, ozone, or nutrient deficiency (Dixon and Paiva, 1995). Also, they are often abundant during senescence or shoot growth even under favorable conditions. These conditions are prone to produce H,Oz in cells. Recently, we have demonstrated that anthocyanins (cyanidin-3-sophoroside) can scavenge excess H,O, in conjunction with peroxidase (Yamasaki, 1997). These results suggest that flavonoids may contribute to the overall mechanism for protecting cells from oxidative damage in addition to their action as optical filters (Gould et al., 1995). Figure 6 is a schematic diagram of a proposed function of flavonoids in the H,O,-scavenging system in cells. In most plants vacuoles dominate the cell volume, and peroxidases may be localized in the inner surface of tonoplast mem-
branes (Thomas and Jen, 1980). This spatial distribution readily enables vacuolar peroxidase to scavenge H,O, leaked out from other organelles (Fig. 6B). The concept of delocalized scavenging of H,O, by vacuoles can be applied not only to organelle-organelle interactions but also to the cell-cell interaction. The epidermal cells usually contain much higher concentrations of flavonoids than mesophyll cells (Hrazdina et al., 1982). The H,O, leaked out from mesophyll cells under light stress can, according to this scheme, be scavenged by the flavonoid-peroxidase system in epidermal cells (Fig. 6C). Consistent with this idea, blackening of the epidermis after severe light stress is frequently observed in many species in the field, a phenomenon that has been ascribed to the polymerization of vacuolar phenolics as the result of the penetration of H,O, into epidermal cells. In Pinaceae species hydrophilic flavono1 glycosides are found in the cell wall (Strack et al., 1988), where GuPXs are also present (Takahama, 1993). The possibility that the apoplastic flavonoid-GuPX may participate in H,O, scavenging (Takahama and Oniki, 1992) cannot be excluded from Figure 6. CONCLUDING REMARKS
Because the generation and scavenging of active oxygen is usually a localized event, the mechanism proposed here (Fig. 6) is unlikely to function as a primary detoxification system. However, it will be important when cellular H,O, levels are increased under conditions of high stress or rapid growth, or when ascorbate availability is limited, such as in
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juvenile leaves (Fig. 5) or in ascorbate-deficient mutants (Yamasaki et al., 1995a, 1995b; Conklin et al., 1996). It is plausible that flavonoids support the primary detoxification system as a backup defense mechanism of vascular plants. The negative correlation between foliar flavonoid and ascorbate content (Fig. 5) may reflect a decrease of the requirement for the flavonoid antioxidant system during development. This functional dispensability might allow chemical modifications that would produce a wide variety of structures and new specific functions. It should be emphasized that the antioxidative function is not a specific feature of flavonoids, but is a general feature of plant phenolics (Takahama, 1988a; Castelluccio et al., 1995).This view has been largely overlooked in plant stress research. Although further evidence is required to confirm the flavonoids’ role in vivo, it is clear that the antioxidative function must be taken into consideration to assess the physiological roles of those molecules. ACKNOWLEDCMENTS
We gratefully acknowledge Dr. Umeo Takahama (Kyushu Dental College, Kitakyushu, Japan) for his helpful suggestions and comments. We also thank Dr. Stephen Grace (The Australian National University, Canberra) for his critica1 reading of the manuscript. Received May 12, 1997; accepted August 28, 1997. Copyright Clearance Center: 0032-0889/97/115/1405/08. LITERATURE CITED
Amako K, Chen G-X, Asada K (1994) Separate assays specific for ascorbate peroxidase and guaiacol peroxidase and for the chloroplastic and cytosolic isozymes of ascorbate peroxidase in plants. Plant Cell Physiol 3 5 497-504 Asada K (1992) Ascorbate peroxidase: a hydrogen peroxidescavenging enzyme in plants. Physiol Plant 8 5 235-241 Bors W, Heller W, Michel C, Saran M (1990) Flavonoids as antioxidants: determination of radical-scavenging efficiencies. Methods Enzymol 1 8 6 343-355 Bors W, Michel C, Saran M (1994) Flavonoid antioxidants: rate constants for reactions with oxygen radicals. Methods Enzymol 234: 420-429 Castelluccio C, Paganga G , Melikian N, Bolwell GP, Pridham J, Sampson J, Rice-Evans C (1995) Antioxidant potential of intermediates in phenylpropanoid metabolism in higher plants. FEBS Lett 368 188-192 Charriere-Ladreix Y, Tissut M (1981) Foliar flavonoid distribution during Spinacia chloroplast isolation. Planta 151: 309-313 Conklin PL, Williams EH, Last R (1996) Environmental stress sensitivity of an ascorbic acid-deficient Arabidopsis mutant. Proc Natl Acad Sci USA 93: 9970-9974 Dixon RA, Paiva N (1995) Stress-induced phenylpropanoid metabolism. Plant Cell 7: 1085-1097 Foyer CH (1993) Ascorbic acid. In RG Alscher, JL Hess, eds, Antioxidants in Higher Plants. CRC Press, Boca Raton, FL, pp 31-58 Foyer CH, Lelandais M, Kunert KJ (1994) Photooxidative stress in plants. Physiol Plant 92: 696-717 Gould KS, Kuhn DN, Lee DW, Oberbauer ST (1995) Why leaves are sometimes red. Nature 378: 241-242 Grace SC, Logan BA (1996) Acclimation of foliar antioxidant systems to growth irradiance in three broad-leaved evergreen species. Plant Physiol112 1631-1640 Hrazdina G, Marx GA, Hoch HC (1982) Distribution of secondary plant metabolites and their biosynthetic enzymes in pea (Pisum sativum L.) leaves. Plant Physiol 70: 745-748
Plant Physiol. Vol. 11 5, 1 9 9 7
Jan CY, Takahama U, Kimura M (1991) Inhibition of photooxidation of a-tocopherol by quercetin in human blood cell membranes in the presence of hematoporphyrin as a photosensitizer. Biochim Biophys Acta 1086 7-14 Koes RE, Quattrocchio F, Mo1 JNM (1994) The flavonoid biosynthetic pathway in plants: function and evolution. BioEssays 1 6 123-132 Landry LG, Chapple CCS, Last R (1995) Arabidopsis mutant lacking phenolic sunscreens exhibits cnhanced ultraviolet-B injury and oxidative damage. Plant Physiol 109: 1159-1166 Lois R, Buchanan BB (1994) Severe sensitivity to ultraviolet radiation in an Arubidopsis mutant deficient in flavonoid accumulation 11. Mechanisms of UV resistance in Arabidopsis. Planta 194: 504-509 Markham KR (1993) Flavones, flavonols and their glycosides. In J Harborne, ed, Methods in Plant Biochemistry, Vol 1. Academic Press, San Diego, CAPpp 197-235 Mehlhorn H, Lelandais M, Korth HG, Foyer CH (1996) Ascorbate is the natural substrate for plant peroxidase. FEBS Lett 378 203-206 Miller E, Schreier P (1985) Studies on flavonol degradation by peroxidase (donor: H,O,-oxidoreductase, EC 1.11.1.7). Part 1: kaempferol. Food Chem 17: 143-154 Mount DW (1996) Reprogramming transcription. Nature 383: 763-764 Schreier P, Miller E (1985) Studies on flavonol degradation by peroxidase (donor: H,O,-oxidoreductase, EC 1.11.1.7). Part 2: quercetin. Food Chem 18: 301-317 Shirley BW (1996) Flavonoid biosynthesis: ’new’ functions for an ’old’ pathway. Trends Plant Sci 1: 377-382 Stafford HA (1994) Anthocyanins and betalains: evolution of mutually exclusive pathways. Plant Sci 101: 91-98 Strack D, Heilemann J, Momken M, Wray V (1988) Cell wallconjugated phenolics from Coniferae leaves. Phytochemistry 27: 3517-3521 Takahama U (1986) Spectrophotometric study on the oxidation of rutin by horseradish peroxidase and characteristics of the oxi* dized products. Biochim Biophys Acta 882 445451 Takahama U (1988a) Hydrogen peroxide-dependent oxidation of flavonoids and hydroxycinnamic acid derivatives in epidermal and guard cells of Tradescuntia virginiana L. Plant Cell Physiol29: 475481 Takahama U (198813)Oxidation of flavonols by hydrogen peroxide in epidermal and guard cells of Vicia fuba L. Plant Cell Physiol 29: 433-438 Takahama U (1989) A role of hydrogen peroxide in the metabolism of phenolics in mesophyll cells of Vicia fubu L. Plant Cell Physiol 30: 295-301 Takahama U (1992) Hydrogen peroxide scavenging system in vacuoles of mesophyll cells of Vicia fuba. Phytochemistry 31: 1127-1133 Takahama U (1993) Regulation of peroxidase-dependent oxidation of phenolics by ascorbic acid: different effects of ascorbic acid on the oxidation of coniferyl alcohol by the apoplastic soluble and cell wall-bound peroxidases from epicotyls of V i g n u uizguluris. Plant Cell Physiol. 34: 809-817 Takahama U, Egashira T (1991) Peroxidase in vacuoles of Vicia faba leaves. Phytochemistry 30: 73-77 Takahama U, Oniki T (1992) Regulation of peroxidase-dependent oxidation of phenolics in the apoplast of spinach leaves by ascorbate. Plant Cell Physiol 33: 379-387 Thomas RL, Jen JJ (1980) The cytochemical localization of peroxidase in tomato fruit cells. J Food Biochem 4: 247-259 Yamasaki H (1997) A function of colour. Trends Plant Sci 2: 7-8 Yamasaki H, Heshiki R, Ikehara N (1995a) Leaf-goldening induced by high light in Ficus microcurpu L. f., a tropical fig. J Plant Res 108: 171-180 Yamasaki H, Heshiki R, Yamasu T, Sakihama Y, Ikehara N (199513) Physiological significance of the ascorbate regenerating system for the high-light tolerance of chloroplasts. In P Mathis, ed, Photosynthesis: From Light to Biosphere, Vol IV. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 291-294 Yamasaki H, Uefuji H, Sakihama Y (1996) Bleaching of the red anthocyanin induced by superoxide radical. Arch Biochem Biophys 332: 183-186
