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Peroxide genesis in plant tissues and its relation to indoleacetic acid destruction
Peroxide Genesis in Plant Tissues and Its Relation to Indoleacetic Acid Destruction S. M. Siegel and A. W. G&ton1 From the Ke~ckhoff Laboratories of Biology, California Technology, Pasadena, California Received
Institute
of
May 7, 1954
INTRODUCTION Peroxides have been traditionally viewed as toxic waste products of metabolism, but investigations of recent years indicate that they may be of great importance in the biochemical activities of the higher plant. For example, peroxides are now known to be involved in the destruction of the plant growth hormone, 3-indoleacetic acid (1, 2), in the peroxidative condensation of hydroxylated phenylpropanes into ligninlike polymers (3, 4), and possibly also in photosynthesis (5). These peroxides are generally presumed to arise from certain of the flavin enzymes known to occur in the higher plant (6, 7), but .other possible pathways for the generation of Hz02 and organic peroxides (8) may exist. Our present experiments stem from previous work on the enzyme system which destroys indoleacetic acid (IAA). This system, which may be inhibited by catalase and by heavy-metal poisons (1, 2), has been characterized as consisting of a peroxide-generating component coupled through HzOz to a peroxidase which includes IAA among its substrates (2). The activity of the oxidase system as a whole is increased markedly by visible radiation (9), the manganous ion (lo), and certain substituted phenols, particularly 2 ,Pdichlorophenol (DCP) (11). Previous evidence (12, 13) suggested that these agents operate on the peroxide-generating system rather than in the peroxidative step. The present paper will pro1 Aided by financial support provided by the National Science Foundation under Grant NSF G-329. We wish to thank Dr. H. R. Highkin, of the Earhart Plant Research Laboratory for supplying the green pea stem tissue used in certain of the experiments, and Dr. A. J. Haagen-Smit of these laboratories for the dihydrophenolphthalein reagent for peroxides. 102
PEROXIDE GENESIS IN PLANT TISSUES
103
vide a direct demonstration that in the IAA-oxidase system, all three factors speed the reaction by enhancing the generation of peroxides. MATERIALS
AND METHODS
Alaska pea (Pisum satiuum) was used as the experimental plant. Excised root tips were obtained from seedlings grown in darkness for 2 or 3 days at 28”C., and terminal buds and epicotyls from 9-11-day-old seedlings. Additional experiments were carried out using green pea stem tissue obtained from a-week-old peas grown in light either at 14 or 26°C. Tissues were harvested and handled only in weak green light phototropically and photomorphogenically inactive. Tissue samples were randomized and weighed on a Roller-Smith torsion balance of 500 mg. eapacity; groups of stem sections of equal weight and approximately equal number were used in each experiment. Measurement of peroxide formation was accomplished by shaking tissues in 10 ml. of 0.05 M KHzPOl containing 100 Nmoles of pyrogallol. Under experimental conditions, oxidation of the phenol by endogenous peroxidase is known to be limited by the peroxide-generating capacity of the tissue, as addition of known amounts of Hz02 could increase the rate of oxidation of the pyrogallol by more than two orders of magnitude. In no case was more than 2.5% of the total pyrogall01 oxidized. The peroxidation product of pyrogallol, purpurogallin (14), was identified by determination of its absorption spectrum using the Beckman model DU spectrophotometer. For routine determination of purpurogallin, the following procedure was employed: The aqueous reaction mixture was decanted and its color measured using the Klett-Summerson photoelectric calorimeter with a blue (No. 42) filter. Tissues were then placed in 10 ml. of 1.2 N HCl in acetone and allowed to stand at least 4 hr. for extraction of purpurogallin in the tissue. The optical density of the acetone-HCl extract was then determined and the amount of purpurogallin ascertained from the combined readings. Each mole of purpurogallin represents two mole equivalents of Hs02 . Controls in each experiment consisted of nonenzymatic blanks containing pyrogallol, but no tissue, and turbidity blanks containing tissue only. Monochromatic light was obtained, as previously described (15), by passing radiation from an incandescent bulb through appropriate combinations of Corning glass filters and Bausch and Lomb interference filters. The energy could be varied at will by adjusting a variable rheostat in series with each light source to give some predetermined reading on a calibrated phototube radiation meter. This latter instrument, specially constructed by Dr. G. H. Bowen, has as its sensitive element a type 926 vacuum phototube with a type S-3 spectral sensitivity, which is fairly uniform over the visible range. Current measurement is accomplished by means of a high gain d.c. amplifier. Accurate readings can be obtained over the range 3 X lo-” to 3 X lo+ amp. RESULTS
1. Peroxide Generation from Endogenous Substrates
In the course of 2-4 hr. of shaking, a standard reaction mixture (10 ml. of 0.05 M KHzPOd , 100 pmoles of pyrogallol) containing 100 mg.
104
S. M.
SIEGEL
AND
A.
W.
GALSTON
of pea root tissue formed a considerable amount of yellow-brown pigment, in the tissues and in the medium. The reaction mixture was acidified with 0.1 N HCI and the pigment extracted with ethyl ether. The absorption spectrum of the pigment was then compared with that of a known sample of purpurogallin, the expected oxidation product. The absorption spectra of the products were essentially identical with that recently presented by Tauber (16) ; hence purpurogallin may be taken to be the product of pyrogallol oxidation by the pea tissue. Pyrogallol, although the most sensitive substrate used, was not the only phenol suitable for measurement of peroxide generation. Guaiacol and thymol, both of low activity as substrates for tyrosinase (17), were oxidized when incubated with the pea tissue under usual conditions. All parts of the pea seedling tested, and both green and etiolated stem tissue, form peroxides (Table I). Older, green stem tissue has a lower peroxide-generating capacity than younger, etiolated material; and plants grown at 26*C. produce more peroxide than those grown at 14°C. Efforts were made to determine whether peroxides accumulated in the tissue or whether they are decomposed as quickly as formed. For this purpose, tissues were incubated for several hours with buffer alone, and peroxide levels were measured by subsequent additions of either pyrogall01 or an alkaline solution of dihydrophenolphthalein containing CU++ (19). With both methods of measurement, no more than traces of peroxides were detected. It may therefore be concluded that no significant accumulation of peroxides occurs in pea tissues. Brei preparations had only about 10% of the peroxigenic activity expected of a comparable amount of intact tissue. Structural integrity of the cell or of one of its components would therefore seem to be essential for this process. TABLE Generation Tissue
I by Pea Tissue
of Peroxides
used
Basal oxide
rate of pergeneratmn
DCP;tJance-
??ZpdtS/lOO
mg.lhr.
3-day-old root 11-day-old epicotyl (etiolated) .ll-day-old terminal buds (etiolated) a-week old green stem grown at 14°C. grown at 26°C.
124 126 40 35 64
DCP
concn.
%
M
61 226 87
10-d 5 x 10-d 10-d
-
-
PEROXIDE
GENESIS
IN
TABLE Effect of DCP
Concentration
DCP Y
Pormation
formation
m~wmles/100
126 126 160
10-d
200
105
TISSUES
II
on Peroxide
Peroxide
10-G 10-s
0
PLANT
mg./hr.
2. Ej’ects of 2,4-Dichlorophenol
DCP
by Pea Roots
enhancement %
0 27 75
(DCP)
When DCP was added to a standard reaction mixtdre, marked increases in the quantity of peroxide formed were observed, the magnitude of the increase depending on the type of tissue and the DCP concentration (Tables I and II). The formation of purpurogallin began promptly on addition of pyrogallol, and mainOained an approximately linear course for the entire period of measurement, 10 hr. (Fig. 1). Addition of 1c4 M DCP nearly doubled the rate of peroxide formation, this enhanced activity also exhibiting a linear course throughout the experiment.
3.0 -
FIG.
1.
REACTION TIME - HRS. The effect of 2,4dichlorophenol on the course of peroxide genesis.
106
S. M.
SIEGEL
AND
A.
W.
GALSTON
3. E$ect of Visible Light Thus far, we have described only the formation of peroxide in tissue maintained in the dark. In certain experiments parallel groups of reaction mixtures were prepared containing root tissue and varying concentrations of DCP. One set of vessels was enclosed in aluminum foil, and both uncovered and shielded vessels were shaken together under a fluorescent light source of 450 ft.-candles intensity. In darkness, NY6 and 1O-4 M DCP enhanced peroxide formation; in light, the effect of DCP was apparent at 1O-6 M as well (Fig. 2). The results indicate an over-all nonadditive enhancement of peroxide formation by light and the phenol. Further experimentation revealed that the effect of light could also be inductive, that is, that tissues irradiated without pyrogallol would exhibit a subsequent increase in peroxide-generating capacity when supplied with pyrogallol in darkness. Entire, dark-grown seedlings were exposed for 2 hr. to a ruby-red tungsten filament lamp delivering 50 ergs/sq. cm./sec. at bud level. Terminal buds excised from irradiated plants produced 57 % more peroxide than dark controls when incubated subsequently with pyrogallol in darkness. Incubation of such preirradiated buds under the fluorescent source produced no further increase in peroxide formation. In contrast, controls not receiving the red radia-
NlOLARlTY OF DCP FIG.
2. The interaction
of 2,bdichlorophenol peroxide genesis.
and light
in enhancing
PEROXIDE
GENESIS
IN
PLANT
107
TISSUES
tion previously were enhanced by light given during incubation with pyrogallol. An inductive treatment with red light as brief as 5 min. effected a 30 % increase in subsequent dark-production of peroxide and a concomitant reduction in the response to light applied during the incubation with pyrogallol. An attempt was made to determine an action spectrum for the inductive response in the hope of elucidating the nature of the photoreceptor. Although all wavelengths tested were effective, the complicated doseresponse relations of the peroxide-forming system (Fig. 3) did not permit ready determination of an action spectrum. 4. E$ects of the Manganous Ion The peroxide-generating capacity of the pea tissue was increased markedly by Mn++ (Fig. 4). The cobaltous ion was entirely without effect over the same range of concentrations, but caused a 25 % inhibition of peroxide formation at 1O-4 M. When Mn++ and DCP were added in combination, the response was roughly additive, the DCP eliciting a greater response than the manganese. 5. Peroxigenic Substrates A survey of possible peroxide-generating systems was carried out by adding to various tissues the substrates for known flavoproteins and then
460
740
-0
20
40
60
80
mp
mp
100
KILOERGSlCM* 3. Peroxide formation as affected by increasing doses of monochromatic light of three different wavelengths. All light administered inductively, prior to addition of pyrogallol. FIG.
108
S. M.
SIEGEL
AND
A.
W.
GALSTON
MOLARITY OF Mn++ FIG.
4. The effect
of manganous
ion on peroxide
AGED
FRESHLY
0
.I
1.0
EXCISED
genesis.
BUD
BUD
10.0
BUTANAL- JAMOLES Fro.
5. The differential
peroxide
effect of a presumed flavoprotein substrate genesis in freshly excised and aged tissue.
(butanal)
on
PEROXIDE
GENESIS
TABLE The Effect of Various
Presumed
Root Epicotyl Bud
PLANT
109
TISSUES
III
Flavoprotein Substrates Aged Pea Tissue
Substrate
Tissue
IN
on Peroxide
Formation
Amount added
Increment H20a
pmoles
pmoles
1.0 0.1 0.1 0.5 2.0
0.802 0.110 0.088 0.05 0.290
Glycolic acid Xanthine Butanal Glycolic acid DL-Alanine
in
Per cent utilization
40 110 85 5 29
incubating the tissues in darkness with pyrogallol. Xanthine, nn-alanine, butanal, and glycolic acid were used. In no case was enhancement of peroxide formation observed; rather, moderately low levels of these substances were markedly inhibitory. On the other hand, when excised tissues were first aged lo-13 days at 2’C. on moist filter paper, all subsequently added substrates were found to enhance oxidation of pyrogallol. Both the inhibition observed with fresh tissues and the enhanced peroxide formation in aged tissues are given in Fig. 5 for butanal. When the utilization of various compounds was computed on the basis of the maximum amount of available hydrogen for HZ02 formation, a considerable percentage of the substrate provided was found to have been consumed (Table III). It was now possible to learn whether the effects of DCP and manganese were associated with the production of Hz02 from flavoprotein substrates or whether they represented independent pathways of peroxide generation. For this purpose, aged epicotyl was incubated with glycolic acid, Mn++, and DCP in various combinations. Peroxide generation was enhanced to the same absolute degree by glycolic acid irrespeetive of the presence of Mn* or the phenol (Table IV). It may therefore TABLE The Effect of Manganous
IV
Ion and DCP on Peroxide Acid
Formation
from Glycolic
Micromoles peroxide formed Addition
No glycolic acid
None 1OW M Mn++ lo-” M DCP Bot.h
1.25
1.95
1.55 1.40 1.65
2.25 2.15 2.40
With glycolic acid Increment due to glycolic acid
0.70 0.70 0.75 0.75
110
S. M. SIEQEL
AND A. W. GALSTON
MxIO-~ IAA FIG. 6. The effect
of indoleacetic
acid on peroxide
genesis
be inferred that the peroxigenic effects of Mn++ and of DCP are mediated by some system other than the flavoprotein acting on glycolic acid. The aged tissues tested thus far had lost their sensitivity to light, hence the effect of light on utilization of flavoprotein substrates could not be determined. It has previously been conjectured (19) that IAA itself might serve as a source of peroxide for its own peroxidatic destruction. Proof of the validity of this conjecture was obtained by supplying IAA to pea epicotyl tissue aged for 18 days at l-2°C. (Fig. 6). IAA similarly supplied to fresh tissue produced only an inhibition of peroxide genesis. DISCUSSION
In the present work, it has been demonstrated that the peroxide required in the inactivation of IAA can be derived endogenously from manganese- and phenol-sensitive nonflavoprotein sources, as well as through the participation of conventional flavoprotein substrates. Presumably, it is among the indicated flavoproteins or others not yet demonstrated that the blue-light sensitive (21) component of IAA-oxidase will be found.
PEROXIDE
GENESIS
IN
PLANT
TISSUES
111
The finding that IAA is itself peroxigenic, foreshadowed by the experiments of Andreae (22) on the peroxidation of scopoletin, indicated that the auxin molecule may be attacked in two ways, first by a dehydrogenation pesumably involved in peroxide formation, and secondly, by peroxidation. It may be assumed that the peroxidase-catalyzed synthesis of lignins (3, 4), and transformations of aromatic substances in general, can be supported by these peroxigenic systems. The recent work of Kenten and Mann (13) has expanded the possible functions of HzOz-peroxidase, implicating peroxides in the degradation of aliphatic organic acids. Siegel and Weintraub (23) showed that Hz02 and ether peroxides could reversibly inhibit growth response of the Arena coleoptile to applied IAA. The inhibition due to Hz02 could be completely removed by catalase, hence destruction of IAA was not involved, but rather, a prevention of its action. In view of the fact that auxins are believed to attach to some receptor via -SH linkages, and that peroxides are known to convert -SH groups to the -S-Sform (24), it is possible that interference with auxin action results from such an attack on -SH groups. It also raises the possibility that the in vivo formation of peroxides may antagonize auxin action. Any of three enzymes may be involved in the direct oxidation of phenolic substances. These are peroxidase (17) and catalase (16, 25), which consume oxygen as peroxide, and the phenolases (17), which consume molecular oxygen directly. We believe phenolases to be inoperative in our system for the following reasons: (a) If phenolases were involved, 90 % of the activity would not disappear as a consequence of homogenization of the tissue; (5) phenols such as thymol and guaiacol, which are not active tyrosinase substrates, may be substituted for pyrogall01 in our experiments; (c) the increased oxidation of pyrogallol accomplished by adding flavoprotein substrates to aged tissues is consistent with the presence in peas of a considerable peroxide-generating capacity. The marked inhibitions conferred by all flavoprotein substrates when supplied to fresh tissue suggest saturating substrate levels may be present in this material. In the action of xanthine oxidase, the reaction velocity is known to be reduced when the substrate level exceeds an optimal concentration (26). Organic peroxides, as well as hydrogen peroxide, may contribute to the oxidation of pyrogallol by peroxidase, and evidence for the existence of these compounds in plants has appeared in recent years. Kolesnikov has reported the isolation of peroxyformic acid from green tissue by
112
S. M. SIEGEL
AND A. W. GALSTON
direct chemical procedures (8). The conversion of hydroxylamine to hydroxamic acid by unstable substances in leaves has also been adduced as evidence for the presence of peroxides in plants (5). The peroxigenic effect of the Mn* ion demonstrated here offers a possible explanation for the well-known mutagenic effects of MnCh on .Escherichia coli (27). In view of the known mutagenicity of Hz& and other peroxides (28), it seems reasonable to postulate that Mn* applied to the bacteria gives rise to peroxides, which are then effective in producing mutations. In support of this suggestion, preliminary experiments of the present authors in conjunction with Dr. A. M. M. Berrie, indicate that 1t3 M Mn++ is peroxigenic in E. coli as it is in pea tissue. SUMMARY
I. All tissues of etiolated pea plants and the stems of green pea plants produce peroxides from endogenous substrates. Such peroxides are not accumulated; in the absence of exogenous peroxidizable substrate, they decay, and in the presence of exogenous pyrogallol, they result in the appearance of purpurogallin in the tissue and in the medium. 2. The rate of peroxide formation is increased by visible light, 2,4dichlorophenol, and manganous ion. Thus, the enhancement of IAA oxidase activity by these three agents is believed due to their effect on peroxide genesis. 3. Peroxide formation in suitably starved tissue may be increased by the addition of typical flavoprotein substrates, such as glycolic acid, xanthine, butanal, and alanine, or by indoleacetic acid itself. Peroxide generation from such substrates is unaffected by Mn++ or dichlorophenol. These peroxigenic materials must therefore be presumed to operate via other peroxide-generating pathways. 4. Some implications of the metabolic role of peroxides are discussed. REFERENCES 1. GOLDACRE, P. L., Australian J. Sci. Resewrch, Ser. B 4, 293 (1951). 2. GALSTON, A. W., BONNER, J., AND BAKER, R. S., Arch. Biochem. and Biophys.
42, 456 (1953). 3. FREUDENBERG, K., REZNIK, H., BOESENBERG, H., AND RASENACK, D., Chem. Ber. 86, 641 (1952). 4. SIEGEL, S. M., Physiol. Plantarum 7, 41 (1954). 5. KUZIN, A. M., AND SHKOLNJK, P. J., Doklady Akad. Nauk U.S.S.R. 66, 719 (1952). 6. LOCKHART, E. E., Biochem. J. 33,613 (1939). 7. OKUNUKI, K., Acta Phytochim. (Japan) 11, 249 (1940).
PEROXIDE
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
GENESIS
IN
PLANT
TISSUES
113
KOLESNIKOV, P. A., Doklady Akad. Nauk U.S.S.R. 64,117 (1949). GALSTON, A. W., AND BAKER, R. S., Am. J. Botany 38, 190 (1951). WAGENKNECHT, A. C., AND BURRIS, R. H., Arch. Biochem. 26, 30 (1950). GOLDACRE, P. L., GALATON, A. W., AND WEINTRAUB, R. L., Arch. Biochem. and Biophys. 43, 358 (1953). GALSTON, A. W., AND BAKER, R. S., Abstracts, Botan. Sot. Amer. Meeting, Columbus, Ohio, 1950. KENTEN, R. H., AND MANN, P. J. G., Biochem. J. 62, 125 (1952). MAEHLY, A. C., 2. Vitamin-, Hormon- u. Fermentforsch. 3, 115 (1949). TODD, G. W., AND GALSTON, A. W., Plant Physiol. 29, 311 (1954). TAUBER, H., J. Biol. Chem. 206, 395 (1953). SUMNER, J. B., AND SOMERS, G. F., “Chemistry and Methods of Enzymes.” Academic Press, New York, 1947. HAAGEN-SMIT, A. J., BRADLEY, C. E., AND Fox, M. M., Ind. Eng. Chem. 46, 2086 (1953). GALSTON, A. W., Science 111, 619 (1950). TANG, Y. W., AND BONNER, J., Arch. Biochem. 13, 11 (1947). GALSTON, A. W., AND BAKER, R. S., Anz. J. Botany 36, 773 (1949). ANDREAE, W. A., AND ANDREAE, S., Can. J. Botany 31, 426 (1953). SIEGEL, S. M., AND WEINTRAUB, R. L., Physiol. Plantarum 6, 241 (1952). BARRON, E. S. G., J. Gen. Physiol. 33, 229 (1950). KEILIN, D., AND HARTREE, E. F., Proc. Roy. Sot. (London) B119, 141 (1936). WILSON, P. W., in Respiratory Enzymes (Lardy, H. A., ed.). Burgess Publ. Co., Minneapolis, 1949. DENEREC, M., AND HANSON, J., (‘old Spring Harbor Symposia Quant. Biol. 16, 215 (1951). DEXEREC, M., BERTANI, G., AND FLINT, J., Am. Naturalist 66, 119 (1951).
Institute
of
May 7, 1954
INTRODUCTION Peroxides have been traditionally viewed as toxic waste products of metabolism, but investigations of recent years indicate that they may be of great importance in the biochemical activities of the higher plant. For example, peroxides are now known to be involved in the destruction of the plant growth hormone, 3-indoleacetic acid (1, 2), in the peroxidative condensation of hydroxylated phenylpropanes into ligninlike polymers (3, 4), and possibly also in photosynthesis (5). These peroxides are generally presumed to arise from certain of the flavin enzymes known to occur in the higher plant (6, 7), but .other possible pathways for the generation of Hz02 and organic peroxides (8) may exist. Our present experiments stem from previous work on the enzyme system which destroys indoleacetic acid (IAA). This system, which may be inhibited by catalase and by heavy-metal poisons (1, 2), has been characterized as consisting of a peroxide-generating component coupled through HzOz to a peroxidase which includes IAA among its substrates (2). The activity of the oxidase system as a whole is increased markedly by visible radiation (9), the manganous ion (lo), and certain substituted phenols, particularly 2 ,Pdichlorophenol (DCP) (11). Previous evidence (12, 13) suggested that these agents operate on the peroxide-generating system rather than in the peroxidative step. The present paper will pro1 Aided by financial support provided by the National Science Foundation under Grant NSF G-329. We wish to thank Dr. H. R. Highkin, of the Earhart Plant Research Laboratory for supplying the green pea stem tissue used in certain of the experiments, and Dr. A. J. Haagen-Smit of these laboratories for the dihydrophenolphthalein reagent for peroxides. 102
PEROXIDE GENESIS IN PLANT TISSUES
103
vide a direct demonstration that in the IAA-oxidase system, all three factors speed the reaction by enhancing the generation of peroxides. MATERIALS
AND METHODS
Alaska pea (Pisum satiuum) was used as the experimental plant. Excised root tips were obtained from seedlings grown in darkness for 2 or 3 days at 28”C., and terminal buds and epicotyls from 9-11-day-old seedlings. Additional experiments were carried out using green pea stem tissue obtained from a-week-old peas grown in light either at 14 or 26°C. Tissues were harvested and handled only in weak green light phototropically and photomorphogenically inactive. Tissue samples were randomized and weighed on a Roller-Smith torsion balance of 500 mg. eapacity; groups of stem sections of equal weight and approximately equal number were used in each experiment. Measurement of peroxide formation was accomplished by shaking tissues in 10 ml. of 0.05 M KHzPOl containing 100 Nmoles of pyrogallol. Under experimental conditions, oxidation of the phenol by endogenous peroxidase is known to be limited by the peroxide-generating capacity of the tissue, as addition of known amounts of Hz02 could increase the rate of oxidation of the pyrogallol by more than two orders of magnitude. In no case was more than 2.5% of the total pyrogall01 oxidized. The peroxidation product of pyrogallol, purpurogallin (14), was identified by determination of its absorption spectrum using the Beckman model DU spectrophotometer. For routine determination of purpurogallin, the following procedure was employed: The aqueous reaction mixture was decanted and its color measured using the Klett-Summerson photoelectric calorimeter with a blue (No. 42) filter. Tissues were then placed in 10 ml. of 1.2 N HCl in acetone and allowed to stand at least 4 hr. for extraction of purpurogallin in the tissue. The optical density of the acetone-HCl extract was then determined and the amount of purpurogallin ascertained from the combined readings. Each mole of purpurogallin represents two mole equivalents of Hs02 . Controls in each experiment consisted of nonenzymatic blanks containing pyrogallol, but no tissue, and turbidity blanks containing tissue only. Monochromatic light was obtained, as previously described (15), by passing radiation from an incandescent bulb through appropriate combinations of Corning glass filters and Bausch and Lomb interference filters. The energy could be varied at will by adjusting a variable rheostat in series with each light source to give some predetermined reading on a calibrated phototube radiation meter. This latter instrument, specially constructed by Dr. G. H. Bowen, has as its sensitive element a type 926 vacuum phototube with a type S-3 spectral sensitivity, which is fairly uniform over the visible range. Current measurement is accomplished by means of a high gain d.c. amplifier. Accurate readings can be obtained over the range 3 X lo-” to 3 X lo+ amp. RESULTS
1. Peroxide Generation from Endogenous Substrates
In the course of 2-4 hr. of shaking, a standard reaction mixture (10 ml. of 0.05 M KHzPOd , 100 pmoles of pyrogallol) containing 100 mg.
104
S. M.
SIEGEL
AND
A.
W.
GALSTON
of pea root tissue formed a considerable amount of yellow-brown pigment, in the tissues and in the medium. The reaction mixture was acidified with 0.1 N HCI and the pigment extracted with ethyl ether. The absorption spectrum of the pigment was then compared with that of a known sample of purpurogallin, the expected oxidation product. The absorption spectra of the products were essentially identical with that recently presented by Tauber (16) ; hence purpurogallin may be taken to be the product of pyrogallol oxidation by the pea tissue. Pyrogallol, although the most sensitive substrate used, was not the only phenol suitable for measurement of peroxide generation. Guaiacol and thymol, both of low activity as substrates for tyrosinase (17), were oxidized when incubated with the pea tissue under usual conditions. All parts of the pea seedling tested, and both green and etiolated stem tissue, form peroxides (Table I). Older, green stem tissue has a lower peroxide-generating capacity than younger, etiolated material; and plants grown at 26*C. produce more peroxide than those grown at 14°C. Efforts were made to determine whether peroxides accumulated in the tissue or whether they are decomposed as quickly as formed. For this purpose, tissues were incubated for several hours with buffer alone, and peroxide levels were measured by subsequent additions of either pyrogall01 or an alkaline solution of dihydrophenolphthalein containing CU++ (19). With both methods of measurement, no more than traces of peroxides were detected. It may therefore be concluded that no significant accumulation of peroxides occurs in pea tissues. Brei preparations had only about 10% of the peroxigenic activity expected of a comparable amount of intact tissue. Structural integrity of the cell or of one of its components would therefore seem to be essential for this process. TABLE Generation Tissue
I by Pea Tissue
of Peroxides
used
Basal oxide
rate of pergeneratmn
DCP;tJance-
??ZpdtS/lOO
mg.lhr.
3-day-old root 11-day-old epicotyl (etiolated) .ll-day-old terminal buds (etiolated) a-week old green stem grown at 14°C. grown at 26°C.
124 126 40 35 64
DCP
concn.
%
M
61 226 87
10-d 5 x 10-d 10-d
-
-
PEROXIDE
GENESIS
IN
TABLE Effect of DCP
Concentration
DCP Y
Pormation
formation
m~wmles/100
126 126 160
10-d
200
105
TISSUES
II
on Peroxide
Peroxide
10-G 10-s
0
PLANT
mg./hr.
2. Ej’ects of 2,4-Dichlorophenol
DCP
by Pea Roots
enhancement %
0 27 75
(DCP)
When DCP was added to a standard reaction mixtdre, marked increases in the quantity of peroxide formed were observed, the magnitude of the increase depending on the type of tissue and the DCP concentration (Tables I and II). The formation of purpurogallin began promptly on addition of pyrogallol, and mainOained an approximately linear course for the entire period of measurement, 10 hr. (Fig. 1). Addition of 1c4 M DCP nearly doubled the rate of peroxide formation, this enhanced activity also exhibiting a linear course throughout the experiment.
3.0 -
FIG.
1.
REACTION TIME - HRS. The effect of 2,4dichlorophenol on the course of peroxide genesis.
106
S. M.
SIEGEL
AND
A.
W.
GALSTON
3. E$ect of Visible Light Thus far, we have described only the formation of peroxide in tissue maintained in the dark. In certain experiments parallel groups of reaction mixtures were prepared containing root tissue and varying concentrations of DCP. One set of vessels was enclosed in aluminum foil, and both uncovered and shielded vessels were shaken together under a fluorescent light source of 450 ft.-candles intensity. In darkness, NY6 and 1O-4 M DCP enhanced peroxide formation; in light, the effect of DCP was apparent at 1O-6 M as well (Fig. 2). The results indicate an over-all nonadditive enhancement of peroxide formation by light and the phenol. Further experimentation revealed that the effect of light could also be inductive, that is, that tissues irradiated without pyrogallol would exhibit a subsequent increase in peroxide-generating capacity when supplied with pyrogallol in darkness. Entire, dark-grown seedlings were exposed for 2 hr. to a ruby-red tungsten filament lamp delivering 50 ergs/sq. cm./sec. at bud level. Terminal buds excised from irradiated plants produced 57 % more peroxide than dark controls when incubated subsequently with pyrogallol in darkness. Incubation of such preirradiated buds under the fluorescent source produced no further increase in peroxide formation. In contrast, controls not receiving the red radia-
NlOLARlTY OF DCP FIG.
2. The interaction
of 2,bdichlorophenol peroxide genesis.
and light
in enhancing
PEROXIDE
GENESIS
IN
PLANT
107
TISSUES
tion previously were enhanced by light given during incubation with pyrogallol. An inductive treatment with red light as brief as 5 min. effected a 30 % increase in subsequent dark-production of peroxide and a concomitant reduction in the response to light applied during the incubation with pyrogallol. An attempt was made to determine an action spectrum for the inductive response in the hope of elucidating the nature of the photoreceptor. Although all wavelengths tested were effective, the complicated doseresponse relations of the peroxide-forming system (Fig. 3) did not permit ready determination of an action spectrum. 4. E$ects of the Manganous Ion The peroxide-generating capacity of the pea tissue was increased markedly by Mn++ (Fig. 4). The cobaltous ion was entirely without effect over the same range of concentrations, but caused a 25 % inhibition of peroxide formation at 1O-4 M. When Mn++ and DCP were added in combination, the response was roughly additive, the DCP eliciting a greater response than the manganese. 5. Peroxigenic Substrates A survey of possible peroxide-generating systems was carried out by adding to various tissues the substrates for known flavoproteins and then
460
740
-0
20
40
60
80
mp
mp
100
KILOERGSlCM* 3. Peroxide formation as affected by increasing doses of monochromatic light of three different wavelengths. All light administered inductively, prior to addition of pyrogallol. FIG.
108
S. M.
SIEGEL
AND
A.
W.
GALSTON
MOLARITY OF Mn++ FIG.
4. The effect
of manganous
ion on peroxide
AGED
FRESHLY
0
.I
1.0
EXCISED
genesis.
BUD
BUD
10.0
BUTANAL- JAMOLES Fro.
5. The differential
peroxide
effect of a presumed flavoprotein substrate genesis in freshly excised and aged tissue.
(butanal)
on
PEROXIDE
GENESIS
TABLE The Effect of Various
Presumed
Root Epicotyl Bud
PLANT
109
TISSUES
III
Flavoprotein Substrates Aged Pea Tissue
Substrate
Tissue
IN
on Peroxide
Formation
Amount added
Increment H20a
pmoles
pmoles
1.0 0.1 0.1 0.5 2.0
0.802 0.110 0.088 0.05 0.290
Glycolic acid Xanthine Butanal Glycolic acid DL-Alanine
in
Per cent utilization
40 110 85 5 29
incubating the tissues in darkness with pyrogallol. Xanthine, nn-alanine, butanal, and glycolic acid were used. In no case was enhancement of peroxide formation observed; rather, moderately low levels of these substances were markedly inhibitory. On the other hand, when excised tissues were first aged lo-13 days at 2’C. on moist filter paper, all subsequently added substrates were found to enhance oxidation of pyrogallol. Both the inhibition observed with fresh tissues and the enhanced peroxide formation in aged tissues are given in Fig. 5 for butanal. When the utilization of various compounds was computed on the basis of the maximum amount of available hydrogen for HZ02 formation, a considerable percentage of the substrate provided was found to have been consumed (Table III). It was now possible to learn whether the effects of DCP and manganese were associated with the production of Hz02 from flavoprotein substrates or whether they represented independent pathways of peroxide generation. For this purpose, aged epicotyl was incubated with glycolic acid, Mn++, and DCP in various combinations. Peroxide generation was enhanced to the same absolute degree by glycolic acid irrespeetive of the presence of Mn* or the phenol (Table IV). It may therefore TABLE The Effect of Manganous
IV
Ion and DCP on Peroxide Acid
Formation
from Glycolic
Micromoles peroxide formed Addition
No glycolic acid
None 1OW M Mn++ lo-” M DCP Bot.h
1.25
1.95
1.55 1.40 1.65
2.25 2.15 2.40
With glycolic acid Increment due to glycolic acid
0.70 0.70 0.75 0.75
110
S. M. SIEQEL
AND A. W. GALSTON
MxIO-~ IAA FIG. 6. The effect
of indoleacetic
acid on peroxide
genesis
be inferred that the peroxigenic effects of Mn++ and of DCP are mediated by some system other than the flavoprotein acting on glycolic acid. The aged tissues tested thus far had lost their sensitivity to light, hence the effect of light on utilization of flavoprotein substrates could not be determined. It has previously been conjectured (19) that IAA itself might serve as a source of peroxide for its own peroxidatic destruction. Proof of the validity of this conjecture was obtained by supplying IAA to pea epicotyl tissue aged for 18 days at l-2°C. (Fig. 6). IAA similarly supplied to fresh tissue produced only an inhibition of peroxide genesis. DISCUSSION
In the present work, it has been demonstrated that the peroxide required in the inactivation of IAA can be derived endogenously from manganese- and phenol-sensitive nonflavoprotein sources, as well as through the participation of conventional flavoprotein substrates. Presumably, it is among the indicated flavoproteins or others not yet demonstrated that the blue-light sensitive (21) component of IAA-oxidase will be found.
PEROXIDE
GENESIS
IN
PLANT
TISSUES
111
The finding that IAA is itself peroxigenic, foreshadowed by the experiments of Andreae (22) on the peroxidation of scopoletin, indicated that the auxin molecule may be attacked in two ways, first by a dehydrogenation pesumably involved in peroxide formation, and secondly, by peroxidation. It may be assumed that the peroxidase-catalyzed synthesis of lignins (3, 4), and transformations of aromatic substances in general, can be supported by these peroxigenic systems. The recent work of Kenten and Mann (13) has expanded the possible functions of HzOz-peroxidase, implicating peroxides in the degradation of aliphatic organic acids. Siegel and Weintraub (23) showed that Hz02 and ether peroxides could reversibly inhibit growth response of the Arena coleoptile to applied IAA. The inhibition due to Hz02 could be completely removed by catalase, hence destruction of IAA was not involved, but rather, a prevention of its action. In view of the fact that auxins are believed to attach to some receptor via -SH linkages, and that peroxides are known to convert -SH groups to the -S-Sform (24), it is possible that interference with auxin action results from such an attack on -SH groups. It also raises the possibility that the in vivo formation of peroxides may antagonize auxin action. Any of three enzymes may be involved in the direct oxidation of phenolic substances. These are peroxidase (17) and catalase (16, 25), which consume oxygen as peroxide, and the phenolases (17), which consume molecular oxygen directly. We believe phenolases to be inoperative in our system for the following reasons: (a) If phenolases were involved, 90 % of the activity would not disappear as a consequence of homogenization of the tissue; (5) phenols such as thymol and guaiacol, which are not active tyrosinase substrates, may be substituted for pyrogall01 in our experiments; (c) the increased oxidation of pyrogallol accomplished by adding flavoprotein substrates to aged tissues is consistent with the presence in peas of a considerable peroxide-generating capacity. The marked inhibitions conferred by all flavoprotein substrates when supplied to fresh tissue suggest saturating substrate levels may be present in this material. In the action of xanthine oxidase, the reaction velocity is known to be reduced when the substrate level exceeds an optimal concentration (26). Organic peroxides, as well as hydrogen peroxide, may contribute to the oxidation of pyrogallol by peroxidase, and evidence for the existence of these compounds in plants has appeared in recent years. Kolesnikov has reported the isolation of peroxyformic acid from green tissue by
112
S. M. SIEGEL
AND A. W. GALSTON
direct chemical procedures (8). The conversion of hydroxylamine to hydroxamic acid by unstable substances in leaves has also been adduced as evidence for the presence of peroxides in plants (5). The peroxigenic effect of the Mn* ion demonstrated here offers a possible explanation for the well-known mutagenic effects of MnCh on .Escherichia coli (27). In view of the known mutagenicity of Hz& and other peroxides (28), it seems reasonable to postulate that Mn* applied to the bacteria gives rise to peroxides, which are then effective in producing mutations. In support of this suggestion, preliminary experiments of the present authors in conjunction with Dr. A. M. M. Berrie, indicate that 1t3 M Mn++ is peroxigenic in E. coli as it is in pea tissue. SUMMARY
I. All tissues of etiolated pea plants and the stems of green pea plants produce peroxides from endogenous substrates. Such peroxides are not accumulated; in the absence of exogenous peroxidizable substrate, they decay, and in the presence of exogenous pyrogallol, they result in the appearance of purpurogallin in the tissue and in the medium. 2. The rate of peroxide formation is increased by visible light, 2,4dichlorophenol, and manganous ion. Thus, the enhancement of IAA oxidase activity by these three agents is believed due to their effect on peroxide genesis. 3. Peroxide formation in suitably starved tissue may be increased by the addition of typical flavoprotein substrates, such as glycolic acid, xanthine, butanal, and alanine, or by indoleacetic acid itself. Peroxide generation from such substrates is unaffected by Mn++ or dichlorophenol. These peroxigenic materials must therefore be presumed to operate via other peroxide-generating pathways. 4. Some implications of the metabolic role of peroxides are discussed. REFERENCES 1. GOLDACRE, P. L., Australian J. Sci. Resewrch, Ser. B 4, 293 (1951). 2. GALSTON, A. W., BONNER, J., AND BAKER, R. S., Arch. Biochem. and Biophys.
42, 456 (1953). 3. FREUDENBERG, K., REZNIK, H., BOESENBERG, H., AND RASENACK, D., Chem. Ber. 86, 641 (1952). 4. SIEGEL, S. M., Physiol. Plantarum 7, 41 (1954). 5. KUZIN, A. M., AND SHKOLNJK, P. J., Doklady Akad. Nauk U.S.S.R. 66, 719 (1952). 6. LOCKHART, E. E., Biochem. J. 33,613 (1939). 7. OKUNUKI, K., Acta Phytochim. (Japan) 11, 249 (1940).
PEROXIDE
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
GENESIS
IN
PLANT
TISSUES
113
KOLESNIKOV, P. A., Doklady Akad. Nauk U.S.S.R. 64,117 (1949). GALSTON, A. W., AND BAKER, R. S., Am. J. Botany 38, 190 (1951). WAGENKNECHT, A. C., AND BURRIS, R. H., Arch. Biochem. 26, 30 (1950). GOLDACRE, P. L., GALATON, A. W., AND WEINTRAUB, R. L., Arch. Biochem. and Biophys. 43, 358 (1953). GALSTON, A. W., AND BAKER, R. S., Abstracts, Botan. Sot. Amer. Meeting, Columbus, Ohio, 1950. KENTEN, R. H., AND MANN, P. J. G., Biochem. J. 62, 125 (1952). MAEHLY, A. C., 2. Vitamin-, Hormon- u. Fermentforsch. 3, 115 (1949). TODD, G. W., AND GALSTON, A. W., Plant Physiol. 29, 311 (1954). TAUBER, H., J. Biol. Chem. 206, 395 (1953). SUMNER, J. B., AND SOMERS, G. F., “Chemistry and Methods of Enzymes.” Academic Press, New York, 1947. HAAGEN-SMIT, A. J., BRADLEY, C. E., AND Fox, M. M., Ind. Eng. Chem. 46, 2086 (1953). GALSTON, A. W., Science 111, 619 (1950). TANG, Y. W., AND BONNER, J., Arch. Biochem. 13, 11 (1947). GALSTON, A. W., AND BAKER, R. S., Anz. J. Botany 36, 773 (1949). ANDREAE, W. A., AND ANDREAE, S., Can. J. Botany 31, 426 (1953). SIEGEL, S. M., AND WEINTRAUB, R. L., Physiol. Plantarum 6, 241 (1952). BARRON, E. S. G., J. Gen. Physiol. 33, 229 (1950). KEILIN, D., AND HARTREE, E. F., Proc. Roy. Sot. (London) B119, 141 (1936). WILSON, P. W., in Respiratory Enzymes (Lardy, H. A., ed.). Burgess Publ. Co., Minneapolis, 1949. DENEREC, M., AND HANSON, J., (‘old Spring Harbor Symposia Quant. Biol. 16, 215 (1951). DEXEREC, M., BERTANI, G., AND FLINT, J., Am. Naturalist 66, 119 (1951).