Function of Ascorbic Acid in Plants*

Function of Ascorbic Acid in Plants* L. W. MAPSON Low Temperature Station for Research in Biochemistry and Biophysics, University of Cambridge, and Department of Scientifi and Industrial Research, Cambridge, England Page I. Introduction. . . . . . . . ..................................... 1 11. Role as Respiratory Carrier.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 2 2 1. Enzyme Systems Catalyzing the Oxidation of Ascorbic Acid.. . . . . . . . . . a. Polyphenolase and Laccase. . . . . . . . . ........... 3 b. Ascorbic Oxidase.. . . . . . . . . . . . . . . . . c. Cytochrome c-Cytochrome Oxidase System, . . . . . . . . . . . . . . . . . . . . . . 8 d. Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 2. Inhibition of the Enzymatic Oxidation of Ascorbic Acid by Suhstances Occurring in Plants.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9 3. Enzyme Systems Associated with the Reduction of Deh Acid (DHA). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Reducing Systems Linked with Coenzyme I . . . . . . . . . . . b. Dehydroascorbic Acid Reductase . . . . . . . . . . . . . . . . . . . . 16 c. Ascorbic Acid-Glutathione System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Evidence for the Action of Ascorbic Acid-Dehydroascorbic Acid as a 19 Respiratory Carrier in vivo.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 111. Ascorbic Acid as a Growth Factor.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 IV. Action of Ascorbic Acid on Enzymes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

I. INTRODUCTION The almost universal distribution of ascorbic acid in plant tissues, together with the fact that its occurrence there coincides quite generally with high metabolic activity, suggests that, as in animal tissues, it performs some essential function in cellular metabolism. Of these functions its action as a respiratory catalyst has received most attention because, no doubt, of the fact that the most characteristic property of the molecule is the ease with which it may be reversibly oxidized and reduced. Ascorbic acid occurs both in the reduced form (AA) and in the oxidized form, dehydroascorbic acid (DHA), in plant tissues, usually about 95 % of the total being present in the reduced form. On injury to the tissues in the presence of oxygen the reduced form is oxidized, generally with great rapidity, thus indicating the presence of oxidase enzymes. The demon*This paper was prepared as part of the program of The Food Investigation Ogaanization of The Department of Scientific and Industrial Research. 1

2

L. W. MAPSON

stration by Seent-Gyorgyi (1931), and since then by other workers, that there is in many plants an enzyme, ascorbic oxidase, capable of catalyeing a direct reaction between ascorbic acid and molecular oxygen, has added weight t o the idea that ascorbic acid may act like cytochrome as a respiratory catalyst. 11. ROLEAS RESPIRATORY CARRIER

A substance must exhibit the following characteristics before it can be considered t o behave as a respiratory carrier (Potter, 1940) : 1. The substance must be a natural component of tissues. 2. The substance must be capable of being reduced by tissues at a rate comparable with the rate of oxidation of substances whose oxidation it is presumed to catalyze. 3. The reduced compound must be capable of being oxidized by the tissues at an adequate rate. 4. The substance must be capable of stimulating the rate of hydrogen transfer in the system under investigation. 5. The compound must be directly reduced by one system and directly oxidized by a second system which is not identical with the first. To this might be added a sixth characteristic, namely, that it is essential t o show that the substance exhibits these properties in the presence of other systems in the intact cell. Ascorbic acid, as will be seen in the following review, satisfies many of these requirements, although in no one study have all these criteria been demonstrated. 1 . Enzyme Systems Catalyzing the Oxidation of Ascorbic Acid

The ease with which ascorbic acid may be oxidized in the presence of molecular oxygen in both enzymic and non-enzymic systems has been demonstrated by many workers over the last decade. Both copper and iron salts catalyze its oxidation, and the oxidative activity of many tissues may, in part, be attributed to them, despite the fact that the activity of these metals is reduced by other natural constituents such as glutathione, amino acids, or proteins. Many quinones which could be formed from a variety of compounds present in plant tissues are also capable of oxidizing ascorbic acid. Enzymes capable of oxidizing ascorbic acid are widely distributed in plants and are proteins containing either copper or iron. None of them can function in the absence of oxygen, or in the presence of cyanide; hence it follows that, as far as is known, any respiratory system in which ascorbic acid is acting as a catalyst will be inactivated in the presence of cyanide. There are at least five oxidases present in the tissue of higher plants that could be responsible for the entry of oxygen into the respiratory sys-

FUNCTION O F ASCORBIC ACID IN PLANTS

3

tem and that have been shown to catalyze the direct or indirect oxidation of ascorbic acid. These are ascorbic oxidase, polyphenol oxidase, cytochrome oxidase, laccase, and peroxidase, and only in the first is there a direct reaction between ascorbic acid and molecular oxygen. Of these enzymes, ascorbic oxidase, polyphenol oxidase (tyrosinase), and laccase, have been shown to be copper proteinates; they need molecular oxygen for their activity, and will bring about the oxidation of their substrates by means of methylene blue or similar dyes. Hydrogen peroxide is not formed in these reactions. It is the purpose of this review to deal in detail with the properties of these enzymes only in so far as they relate to ascorbic acid, but a few remarks on their general properties may be desirable. a. Polyphenolase and Laccase. These enzymes, as isolated from mushrooms or from potatoes (Kubowitz, 1937; Keilin and Mann, 1939) are characterized by their ability to catalyze the aerobic oxidation of both monohydric and o-dihydric phenols; they have no direct action on ascorbic acid. The observation that monophenolase activity is frequently lost during the preparation of the enzyme has suggested to many workers that two distinct enzymes are involved. Following Nelson and Dawson (1944), many authors (Mallette et al., 1948; Bordner and Nelson, 1939; Gregg and Nelson, 1940; Mallette and Dawson, 1949) support the view that mushroom polyphenolase is a single protein possessing both monophenolase and polyphenolase activity, and that the loss in monophenolase activity during preparation is due to the alteration in the enzyme protein rather than to the separation of two distinct enzymes. I n contrast to these conclusions, recent work by Kertesz (1952) suggests that polyphenolase catalyzes only the oxidation of o-dihydric phenols to quinone, and that the so-called monophenolase activity is due t o a reaction between the quinone and monohydric phenol which is catalyzed by nonprotein-bound metallic ions such as copper, cobalt, vanadium, or nickel. It would appear, therefore, that copper associated with certain specific proteins will account for the oxidase activity of plant tissues previously designated as polyphenolase, potato oxidase, or catechol oxidase. If the conclusions of Kertesz are confirmed, tyrosinase activity is t o be attributed to the action of a polyphenolase in conjunction with metallic ions such as copper. The important feature of the action of these enzymes from the point of view of the behavior of ascorbic acid is that the immediate oxidation products of the polyphenol substrate are o-quinone derivatives which are capable of oxidizing ascorbic acid very rapidly t o dehydroascorbic acid, with a consequent reduction of the quinone (Ludwig and Nelson, 1939; Miller and Dawson, 1941). The system then becomes a cyclic one until all the ascorbic acid has been oxidized. With monophenols the first stage is their conversion into an o-dihydric phenol, which is then oxidized in the same se-

4

L. W. MAPSON

quence of operations. It will be noted that such a system can function as a terminal oxidase only so long as reductants like ascorbic acid are present to regenerate the phenol from the o-quinone formed in the initial reaction. OI

\ /

Catechol

catalyzed by polyphenolase

H20

d

Lo-Benroquinone

\

f

uncatalyzed fast renction

/ \

Dehydroascorbic acid @HA) Ascorbicacid (AA)

Laccase resembles the other polyphenolases in its action on ascorbic acid. Crude preparations oxidize ascorbic acid (Keilin and Mann, 1939), but their activity decreases as the enzyme is purified; it can be restored by the addition of substrates such as o- or p-dihydric phenols. The enzyme is without action on monohydric phenols, and consequently these do not induce the oxidation of ascorbic acid. Bertrand (1945a,b) has claimed that purified laccase, like ascorbic oxidase, will oxidize ascorbic acid directly, although no confirmation of this finding has so far been reported. The polyphenolase group of enzymes therefore all act as oxidation catalysts for ascorbic acid, by virtue of their ability to form either o- or pquinones from their respective substrates. There is some evidence to suggest that phenolic derivatives of the flavonoid type may also be substrates for these enzymes: recent work with tea and potato polyphenolase has indicated that ascorbic acid is rapidly oxidized when certain flavones are added to the crude or purified enzyme (Roberts and Wood, 1951; Baruah and Swain, 1952). There is thus clear evidence that these enzymes can act in vitro as oxidation systems in which ascorbic acid is involved. What is less certain is whether such systems play any large role as terminal oxidases in vivo and thus whether ascorbic acid may be considered as the immediate hydrogen donator linking these oxidases with the dehydrogenase systems of the cell. The widespread occurrence of the polyphenolase in plants has, however, lpng made it seem likely that these oxidases play an important part in respiration. Onslow’s studies (1920-1924) emphasized the wide distribution in plant tissues of o-dihydric phenols such as caffeic and protocatechuic acids, and focused attention on the possibility that the enzymatic oxidation of such substances was an important part of the plant respiratory process. Since then many investigators have attempted to show that polyphenolase acts as a terminal oxidase. The addition of catechol and protocatechuic acids has been shown to increase the oxygen consumption of potato slices (Boswell and Whiting, 1938; Baker and Nelson, 1943). Boswell and Whiting succeeded in obtaining a substance soluble in alcohol and giving tests indicating a phenolic

5

FUNCTION O F ASCORBIC ACID IN PLANTS

character. When it was added to potato slices, this substance caused a sustained increase in oxygen uptake and carbon dioxide production; it was not identified. Robinson and Nelson (1944) isolated L-tyrosine from potato tissue and showed that this increased the oxygen consumption. The tyrosinase first catalyzed the oxidation of tyrosine to 3,4-dihydroxyphenylalanine (Dopa), and this was then oxidized to the quinone. In the presence of ascorbic acid the quinone was reduced to the phenolic compound. They showed that under these conditions only a trace of L-tyrosine was oxidized, despite the presence of polyphenolase. This state of affairs continued until all the ascorbic acid was oxidized, after which a rapid oxidation of tyrosine occurred. The affinity of Dopa for the enzyme is greater than that of tyrosine, and since Dopa is continually being regenerated by reduction of Dopa-quinone by ascorbic acid, no oxidation of the tyrosine occurred until all the ascorbic acid had been oxidized. The following cyclic series of reactions therefore serves as a potential means of transferring hydrogen from ascorbic acid to molecular oxygen. L-Tyrosine

\

I

+ O2

polyphenolase

3,4-Dihydroxyphenylalanine (Dopa)

DH)

IpolypixnY

Dopa-quinone

AA

-1

H 10

Melanin pigments

Ascorbic acid in this system is acting as an immediate hydrogen donator to the quinone and, in conjunction with the oxidase, confers the properties of a hydrogen carrier (reversible oxidation and reduction) on substances such as 3,4-dihydroxyphenylalanine. Tyrosine under such conditions serves as a reservoir for the supply of the dihydric phenolic derivative. Other evidence that polyphenolase functions as a terminal oxidase has been obtained in studies on tea (Sreerangachar, 1942; Roberts and Wood, 1951) and on spinach leaves (Bonner and Wildman, 1946), where it was found that 90 to 100 % of the respiration was inhibited by p-nitrophenol, an agent which inhibits polyphenolase but which is said to have no action on cytochrome. The idea that polyphenolase plays any significant role as a terminal oxidase in the potato has been criticized by Levy et al. (1948) on the grounds that the normal respiration of potato tissue is inhibited by CO and reversed by light-a characteristic of the iron oxidase systems, but not of

6

L. W. MAPSON

the polyphenolasea. Similar observations have also been made with other plant tissues (Allen and Goddard, 1938; Brown and Goddard, 1941). It is also true that the polyphenolase activity of homogenates from fresh potato tissue represents a hydrogen transfer some 100 times greater than the total respiration of the intact tuber (Mapson and Barker, 1951). It seems clear that in vivo the full potential effect of these enzymes is not realized, either because of some spatial separation of substrate and enzyme, or because substrates are in a form not acted upon by the enzyme. In this respect recent work (Baruah and Swain, 1952) on the flavonoids is of interest. It has been shown that, with a purified potato polyphenolase, the oxidation of ascorbic acid is rapidly catalyzed on the addition of the aglycone but not on the addition of the corresponding glycoside. Still more recent work by the same authors has suggested an alternative possibility. It has been generally assumed that L-ascorbic acid has no effect on the polyphenolase system other than its effect as a reducing agent for the o-quinone formed by the oxidation of the phenols. It has now been shown that ascorbic acid itself has an inhibitory action on the polyphenolase enzyme. When polyphenolase prepared from potato was treated with ascorbic acid under anaerobic conditions, and the ascorbic acid subsequently removed by dialysis, the activity of the enzyme was very considerably reduced. The enzyme after such treatment could not be reactivated by the addition of cupric salts and appeared to be irreversibly inactivated. It was also shown that neither dehydroascorbic acid nor the further oxidation products of dehydroascorbic acid were responsible for this result. There is a t present no explanation of the mechanism of this inhibitory action of ascorbic acid, but it is quite clear that, if these results are confirmed, other explanations are possible of why these enzymes do not exert their full potential effect i n vivo. b. Ascorbic Oxidase. It may be of some significance that those plants which possess no polyphenolase enzymes are usually found to possess the enzyme ascorbic oxidase. This enzyme is the only known enzyme to catalyze a direct reaction between ascorbic acid and oxygen. The primary product of the oxidation is dehydroascorbic acid: 1 gram atom of oxygen is absorbed per mole of ascorbic acid oxidized (Lovett-Janison and Nelson, 1940; Powers et al., 1944). It is a blue or greenish blue Cu proteinate containing approximately 0.25 7' Cu. Unlike the correbponding oxidation catalyzed by Cu++, no hydrogen peroxide is formed during the reaction (Steinman and Dawson, 1942; Hand and Greisen, 1942). Although ascorbic oxidase is a Cu protein containing nondialyzable Cu similar in amount t o that of the polyphenolase, it has no oxidative action on monoor polyhydric phenols (Tauber e2 al., 1935; Srinivasan, 1935; Johnson and Zilva, 1937). Artificial Cu proteinates have been prepared which exhibit

FUNCTION O F ASCORBIC ACID I N PLANTS

7

many of the properties of ascorbic oxidase. It would appear premature, however, to consider the natural enzyme as one in which the Cu ion was adsorbed on or associated with a nonspecific protein. Thus sedimentation constants and electrophoretic mobilities are reproducible in preparations of the enzyme containing very different amounts of inactive protein (Dunn and Dawson, 1951)) and recent studies have shown that the Cu in the purified enzyme does not exchange with radioactive Cus4, when the enzyme is allowed to remain in contact with the radioactive ionic copper for several hours (Joselow and Dawson, 1951; Dawson, 1950). These observations confirm the view that the copper-protein bond in the enzyme is of a nondissociable nature and are not in agreement with the work of Lampitt and Clayson (1945), who postulated that the catalytic activity of ascorbic acid oxidase was to be attributed to traces of ionic copper resulting from an ionization of the copper bound to nonspecific protein. Ascorbic oxidase oxidizes L-ascorbic acid, and certain analogs of ascorbic acid, D-araboascorbic acid, L-glucoascorbic acid, and L-galactoascorbic acid, are equally oxidized by the enzyme (Johnson and Zilva, 1937; Dodds, 1948). Compounds related t o L-ascorbic acid in which the oxygen bridge is on the right side of the carbon chain are directly oxidized by the enzyme a t a much higher rate than are their enantiomorphs. Of the latter the six-membered series (D-ascorbic) are oxidized more rapidly than the seven-membered series (D-ghcoascorbic acid and D-galactoascorbic acid). Other dienols (reductone and reductic acid) may also be oxidized, though at slower rates than the isomers of ascorbic acid (Snow and Zilva, 1938). The number of carbon atoms in the molecule and the stereochemical configuration of other asymmetric carbon atoms in the oxygen ring are important factors besides the dienol group in controlling the enzyme action. The configuration of the asymmetric carbon atoms outside the oxygen ring has, however, little influence. A noteworthy feature in the kinetics of the enzyme are the characteristic linear oxidation rates obtained which show no falling off in velocity until the substrate is virtually completely oxidized (Hopkins and Morgan, 1936). This implies a low Michaelis constant, which has been found t o be of the order of 2 X lo-" M (Crook, 1941). The enzyme is extremely sensiM will inhibit completely, and tive to cyanide; concentrations of M . Other chelating agents definite inhibitions may be observed with for copper such as diethyl thiocarbamate, 8-hydroxyquinoline1 sodium sulfide, and potassium ethyl xanthate all inhibit (Tauber and Kleiner, 1935; Stotz et al., 1937; Stotz, 1940; Giri and Seshagiri Rao, 1946). Carbon monoxide inhibits only slightly, and the inhibition is not affected by light (Matusukawa, 1940).

8

L. W. MAPSON

c, Cytochrome c-C'ytochrome Oxidase System. The works of Hill and Bhagvat (1939), and Bhagvat and Hill (1951) Goddard (1944), Goddard and Holden (1950), have shown the presence in plant tissues of cytochrome c and cytochrome oxidase. Cytochrome c will oxidize many mild reducing agents such as cysteine, p-phenylenediamine, hydroquinone, and other phenols. It is not surprising, therefore, that it will oxidize ascorbic acid (Schneider and Potter, 1943), and if, in addition, cytochrome oxidase is present, a cyclic oxidation system is established whereby ascorbic acid may be oxidized to completion (Keilin and Hartree, 1938). Cytochrome oxidase will not oxidize ascorbic acid in the absence of cytochrome c; the oxidative action of the enzyme is therefore an indirect one. To what extent ascorbic acid is oxidized in vivo by the oxidase system is not known. d. Peroxidase. One of the first enzymatic mechanisms to be described as catalyzing the oxidation of ascorbic acid was the peroxide-peroxidase system (Szent-Gyorgyi, 1928). The enzyme is widely distributed in plant tissues, but in purified form has no oxidative action on ascorbic acid in the presence of hydrogen peroxide. A rapid reaction ensues if phenolic compounds capable of being oxidized to quinones are added to the enzyme. The sequence of reactions may be expressed as

+

+ peroxidase

Phenolic compound HZOZ Quinone Quinone AA + DHA phenolic compound

+

The most efficient of these phenolic compounds are those of the benzopyran class (quercitrin, ereodictyol) (Huszak, 1937). The action of peroxidase therefore resembles that of the polyphenolase in that the oxidation of ascorbic acid results from the intermediate formation of quinone derivatives. It is clear from the foregoing that the oxidation of ascorbic acid may be catalyzed by all the well-known oxidase systems occurring in plants, but such biochemical studies furnish little evidence as to what extent these reactions proceed in vivo. All these oxidases carry oxidation only as far as the dehydroascorbic acid stage. The fact that ascorbic acid is always associated in fresh tissues with small but definite amounts of dehydroascorbic acid, combined with the probability that dehydroascorbic acid is continually being lost by irreversible conversion to 2,3-diketogulonic acid, makes it appear probable that there is a continuous oxidation of ascorbic acid in vivo. The low content of dehydroascorbic acid in most tissues is not inconsistent with this view, for the continuous oxidation may also be accompanied by an equally continuous reduction. The conflicting evidence as to whether the cytochrome, the polyphenolase, or the ascorbic oxidase is the main terminal oxidase does not concern us here except that, as far as ascorbic acid is concerned, all or any of these

FUNCTION OF ASCORBIC ACID IN PLANTS

9

oxidases would serve as a link with ascorbic acid and other hydrogentransporting systems in the cell. In so far as all the known oxidase systems oxidizing ascorbic acid are cyanide-sensitive, the participation of ascorbic acid as a respiratory catalyst cannot be greater than the extent to which the respiration of the tissue is reduced by cyanide. With some tissues the degree of inhibition by cyanide is high, whereas with others the effect of cyanide is slight (Marsh and Goddard, 1939). Evidence is accumulating that cyanide-insensitive respiratory systems exist in plants (Laties, 1950; James and Beevers, 1950) ;it seems unlikely that ascorbic acid can participate in these systems as a respiratory catalyst unless oxidase enzymes insensitive to cyanide and capable of oxidizing ascorbic acid are discovered. 2. Inhibition of the Enzymatic Oxidation of Ascorbic Acid by Substances

Occurring in Plants When the tissues of many fruits and vegetables are damaged by mechanical means, there is a rapid oxidation of ascorbic acid; with others this treatment has little or no effect (Zilva, 1934; Barron et al., 1936). Stone (1937) discussed the cause of the stability of ascorbic acid in some and its instability in others and concluded that differences between various plants were due to the presence or absence of oxidase enzymes. There are few references in the literature to the inhibition of oxidase enzymes by naturally occurring substances. The presence of a substance in many fruits and vegetables which effectively inhibits the oxidation of ascorbic acid, whether this is brought about by ascorbic oxidase, polyphenolase, peroxidase, or by inorganic Cu, has been reported by Somogyi (1944), but the nature of this substance or substances was not identified. Damodaran and Nair (1936) isolated a tannin from the Indian gooseberry (Phyllanthus emblica) which inhibited the oxidation of ascorbic acid in the press juice. Since the protective effect of this substance could be “overridden” by the addition of Cu, they concluded that its action depended on the suppression of metal catalysis. Kardo-Sysoeva and Nisenbaum (1938) reported a thermolabile stabilizer for ascorbic acid in the tomato but gave no indication of how it functioned. The same is true of the work of Giri and Krishnamurthy (1940), who separated from the juices of Cucumis sativus, Cucubite maxima, and Lu$a acutangula, ascorbic oxidase by fractionation with acetone from a substance, which prevented the oxidation of ascorbic acid even in the presence of Cu. The presence of volatile constituents which are alleged t o inhibit the enzymatic destruction of ascorbic acid in cabbage and other green vege-

10

L. W. MAPSON

tables has also been reported (Brand, 1949). Hooper and Ayres (1951) have found that black currants, a fruit in which ascorbic acid is remarkably stable, contain substances which inhibit the oxidation of ascorbic acid by the polyphenolase system of apples. The protective action was found to be associated with a red pigment (anthocyanin fraction) and with a yellow pigment (flavanone). Similar red pigments in beet roots also gave marked protection against the oxidase of apple juice. These substances from the black currant afforded no protection however against the enzymatic oxidation of ascorbic acid by ascorbic oxidase from cucumber juice; they were thus distinct from those of Somogyi. Extracts containing vitamin P were also tested but with negative results. The enzymatic oxidation of ascorbic acid is known to be inhibited by agents which possess the power to chelate with the copper of the oxidase. As in other enzymatic reactions, inhibition might also be expected to follow the addition of substances which by reason of their structure comPete with the natural substrates for the surface of the enzyme (competitive inhibition). If in addition these substances are not themselves oxidized, the resulting.inhibition may be high. The results of Somogyi and of Brand are more readily interpreted as a direct effect on the enzyme; for in Somogyi’s experiments, his substances inhibited the action of ascorbic oxidase and polyphenolase as well as that of inorganic copper, and in Brand’s, the probability seems high that his volatile constituents from cabbage contained sulfhydryl derivatives which have high chelating powers for copper. The experimental results of Hooper and Ayres, on the other hand, are more likely to be due to competitive inhibition, produced by the high concentration of glycosidic flavonoid substances present in their extracts, The protection they observed against polyphenolase but not against ascorbic oxidase would then be explicable. 3. Enzymic Systems Associated with the Reduction of Dehydroascorbic Acid ( D H A )

In his earlier experiments Szent-Gyorgyi (1928) found that ascorbic acid (AA) was rapidly oxidized on the addition of hydrogen peroxide but that, on standing, the ascorbic acid was regenerated. This latter observation could not be repeated if the juice was boiled. Since that date evidence has accumulated that AA and DHA are interconvertible in plant tissues. In some fruits, notably apples, the proportion of DHA to that of AA is high in the early stages of development but decreases as the fruit approaches maturity (Zilva et al., 1938). The direct formation of ascorbic acid from DHA was demonstrated in cabbage (Rubin et al., 1937) and in poplar leaves (Mapson and Barker, 1948)after DHA had been injected or fed to these tissues.

FUNCTION OF ASCORBIC ACID IN PLANTS

11

Since the concentration of DHA is usually very small in relation to that of AA in most plant tissues, it is reasonable to assume that the oxidative and reducing enzymic systems associated with AA are poised at such a level that normally most of the AA is maintained in the reduced form, This balance is normally maintained throughout the life of the cell. It may, however, be disturbed (1) by mechanical damage, (2) by the action of narcotics, or (3) by the action of certain enzymic poisons, e.g., iodoacetate, arsenite, or fluoride (Mapson and Barker, 1948). Under such conditions there is a rapid conversion of AA t o DHA. Various reasons have been advanced t o explain the phenomenon. On the one hand, there is the hypothesis that damage to the cell causes disorganization and allows the oxidases to come into more effective contact with their substrates, resulting in a greatly increased rate of oxidation. A second explanation is that the enzymic systems concerned with the reduction of AA are impaired. A third explanation that on mechanical damage the oxygen tension in the tissues is raised seems improbable in view of experiments which show that raising the oxygen tension from that of air to that of pure oxygen has no immediate effect on the balance of AA-DHA in the intact cell (Mapson and Barker, 1948; Barker and Mapson, 1952a). Whichever explanation is correct, it seems certain that reducing systems are present in plant tissues which maintain AA in the reduced form. Although the oxidation of ascorbic acid by enzymic systems in vitro is relatively easy t o demonstrate, the proof that enzymic systems capable of reducing DHA exist has presented a more difficult problem. The failure to discover such reducing systems has, in the past, been the chief obstacle to acceptance of the view that ascorbic acid may act as a respiratory catalyst. In the past too much attention may have been paid to the fact that ascorbic acid occurs in the plant in the reduced form and t o its effect as a yydrogen donator. The fact that AA accumulates in the cell in the reduced form may simply mean that the systems which reduce it are more active in vivo than are those which oxidize it. What indeed may be of greater significance is the concentration of DHA, which by reason of its ability t o accept hydrogen may determine the level at which the ascorbic acid system acts as a respiratory catalyst (cf. Thomas, 1947). a. Reducing Systems Linked with Coenzyme I . Evidence for the participation of coenzyme I in certain metabolic reactions which may be linked with the reduction of dehydroascorbic acid has been advanced by a number of workers. James and Cragg (1943) were the first to suggest a link between certain dehydrogenase systems and ascorbic acid. Having demonstrated the existence of an ascorbic oxidase in barley seedlings, they found that three acids, viz., glycolic, lactic, and tartaric, increased the oxygen consumption in the presence of ascorbic acid but not in its absence; other

12

L. W. MAPSON

acids, including citric, ascorbic, malic, acetic, pyruvic, and succinic, were without effect. The increased oxygen consumption with the former group of acids was due ultimately to their oxidation and not to the oxidation of ascorbic acid, since little or no loss of ascorbic acid occurred. I n the case of lactic acid the formation of pyruvic acid was shown. The presence of a lactic dehydrogenase was claimed on the evidence that the rate of reduction of indophenol dye under anaerobic conditions was increased in the presence of added lactate; similar experiments with methylene blue, however, gave negative results. On the basis of these experiments James and Cragg proposed the following reactions, in which the increased oxygen consumption observed by them was to be attributed to the Participation of ascorbic acid as a respiratory carrier. R*CHOH.COOH+ DHA AA

+

$02

dehydrogenase

*

ascorbic oxidase

R.CO.COOH DHA

+ AA

+ HzO

Clagett et al. (1949) in a study of the oxidation of a-hydroxy acid by enzymes obtained from the leaves of soybean, tomato, and potato plants found the presence of an enzyme capable of oxidizing both lactic and glycolic acids. This enzyme was not activated by ascorbic acid; in fact, with glycolic acid the oxidation was somewhat inhibited. In further experiments (James et al., 1944) it was shown that, with barley saps to which hexose diphosphate and ascorbic acid were added, an increased oxygen uptake occurred in excess of that caused by the addition of ascorbic acid alone. This oxygen uptake could be still further increased by the addition of coenzyme I. The breakdown of hexose diphosphate to phosphoglyceric acid which was observed was stimulated by the addition of ascorbic acid. The course of hydrogen transport was therefore believed to be triose phosphate + coenzyme 1 4 ascorbic acid + 02. Whether coenzyme I was concerned in the earlier experiments was not determined and has not since been determined. The evidence from these studies was suggestive of the participation of ascorbic acid as a respiratory carrier. It was, however, only suggestive and not conclusive, for the authors did not demonstrate either with their lactate dehydrogenase or hexose diphosphate systems the direct reduction of dehydroascorbic acid to ascorbic acid. Results of a similar character were obtained by Davison (1949) in pea seeds and pea seedlings. These tissues contain an active formic dehydrogenase which will reduce dyes such as Nile blue in the presence of coenzyme I ; the reduced dye could be reoxidized by dehydroascorbic acid. As with the work of James et al., the oxygen consumption of such tissue was increased when formate was added and still further increased on the

FUNCTION OF ASCORBIC ACID IN PLANTS

13

addition of ascorbic acid, but no direct demonstration of the ability of such tissue to reduce dehydroascorbic acid was attempted. Further evidence of like character in support of the participation of coenzyme I in reactions associated with the reduction of dehydroascorbic acid has been advanced by Waygood (1950). Cell-free extracts of wheat seedlings were found to contain a malic dehydrogenase enzyme, reducing coenzyme I, as well as ascorbic oxidase and peroxidase enzymes. When to such extracts malic acid, coenzyme I, and ascorbic acid were added, together with a fixative for the oxalacetate formed in the reaction, the system absorbed oxygen in excess of that required for the complete oxidation of ascorbic acid. In this system methylene blue could replace ascorbic acid. Other substrates such as fumarate, alcohol, or hexose diphosphate were also found to give similar results. The increased oxygen consumption in most of these experiments was greater than 1 mole of oxygen per mole of ascorbic acid added, i.e., greater than that required for the complete oxidation of ascorbic acid to oxalic and threonic acid. This fact, combined with the finding of small amounts of dehydroascorbic acid at the end of the experiment (13% of the original amount of AA added), is taken to indicate that the oxygen consumption observed was due to formation of dihydrocoenzyme I, which reduced dehydroascorbic acid and is itself reduced in the presence of the dehydrogenase enzyme and substrate. I n an incomplete system (no coenzyme I added), neither ascorbic nor dehydroascorbic acid could be detected. This evidence like that of James and Cragg and Davison rests mainly upon the observation of increased oxygen consumption above that required for the oxidation of the ascorbic acid added, and no direct observations were made to ascertain whether the system could reduce added dehydroascorbic acid. I n Waygood’s experiments one might have expected to find both ascorbic and dehydroascorbic acids in his complete system if these were involved simply as carriers, for there was no evidence that the substrate providing the hydrogen was exhausted at that stage in his experiments when determinations of these substances were made. In fact, he found only small amounts of dehydroascorbic acid, and the test used for its presence (titration with indophenol after reduction with hydrogen sulfide) is known to be rather unspecific. More direct observations of reactions linking coenzyme I with ascorbic acid have been published by Mathews (1951). Extracts of soaked peas were found to catalyze the oxidation of reduced coenzyme I (followed spectrophotometrically) by oxygen in the presence of either ascorbic acid or methylene blue. These reactions were not inhibited by cyanide. The enzyme preparation also catalyzed the oxidation of reduced coenzyme I1

14

L. W. MAPSON

wit,h methylene blue but not with ascorbic acid; it thus contained a diaphorase I and I1 similar to those reported by Davison (1950). The most interesting feature of these experiments was the observation that dehydroascorbic acid prepared chemically (oxidation by iodine) could not act as hydrogen acceptor in the reactions. On purely speculative grounds Mathews makes the suggestion that the hydrogen acceptor is not dehydroascorbic acid but a semiquinone intermediate. If this suggestion is correct, it may be the reason why the present writer was unable to demonstrate (unpublished experiments) the reduction of dehydroascorbic acid (prepared by oxidation with bromine or activated charcoal) by either coenzyme I specific formate or ethanol dehydrogenase preparations from peas containing both coenzyme I and substrates. An observation was made by Waygood (1950) which is of interest in this connection. He found that in extracts in which ascorbic acid appeared to be acting as a carrier, a pigment was formed in solution a t a rate directly proportional to the oxygen consumption. It was formed only when ascorbic acid was being oxidized and could not be produced by other oxidants such as oxygen or hydrogen peroxide. On the available evidence the suggestion was made that the pigment is part of an oxido-reduction system and exists colored (reddish-violet) in the oxidized state and colorless when reduced. Since both oxygen and dehydroascorbic acid were necessary for its appearance, this substance must be positioned as a carrier between coenzyme I arid ascorbic acid: if it were a terminal oxidase, oxygen, but not dehydroascorbic acid, would be required for its development. Waygood proposed the following scheme for the system in wheat: Substrate (malate, alcohol, hexasediphosphate)

-

Leucopigmeot

+

I1

Pigment

diaphorasa

CO I HZ

Flavine Flavine HI

- I1 -4 DHA AA AA

ascorbic oxidase

H 00

The failure to detect the pigment in a system containing ascorbic acid but no coenzyme I is explained by the author as being due to the failure t o retain dehydroascorbic acid sufficientlylong to enable the pigment to be formed. This implies that the postulated reaction between leucopigment and dehydroascorbic acid is relatively slow. These observations were all made on extracts a t a pH of 7.5, a pH a t which the conversion of dehydroascorbic to diketogulonic acid is rapid. A repetition of these experiments at a lower pH would seem worth while, together with experiments de-

FUNCTION O F ASCORBIC ACID IN P L A N T S

15

signed to study the effect of adding DHA on the formation of the pigment. Before the above interpretation of Waygood’s observations can be accepted, such experiments would appear to be essential. A scheme such as this may, however, serve as a working hypothesis for future work; it is attractive in that it postulates the need for an additional carrier between coenzyme I and dehydroascorbic acid. The failure to demonstrate the direct reduction of dehydroascorbic acid in tissue extracts in the presence of coenzyme I might therefore be explained either by the absence of the pigment described by Waygood or on the lines of the hypothesis put forward by Mathews. It is a t present, however, impossible to decide between these and other possibilities, and, until there is further clarification of these points, the link between coenzyme I and the ascorbic acid system remains uncertain. b. Dehydroascorbic Acid Reductase. The close association between glutathione and ascorbic acid in plant tissues is well established. In germinating seeds and potato tubers ascorbic acid and glutathione appear at the same time (Pett, 1936; Hopkins and Morgan, 1943). The increase in ascorbic acid that occurs on the surface of potato tubers during the process of wound healing is accompanied by increases in the concentration of glutathione. Similar results are also observed when potato tubers are treated with ethylene chlorohydrin (Guthrie, 1937). The protective effect of glutathione (GSH) on the oxidation of ascorbic acid has been observed by several workers, who have, however, explained it in different ways. The fact that GSH combines readily with copper was believed by some workers to be the explanation. That this is not the sole reason is shown by the fact that GSH will reduce dehydroascorbic acid in solution above pH 6.5 without added catalysts (Borsook et al., 1937; Bukin, 1943; Yamaguchi and Joslyn, 1951). Following Szent-Gyorgyi, many authors have ascribed the power of tissues or tissue extracts to reduce DHA to their content of GSH (De Caro and Giani, 1934; Mawson, 1935; Barron et al., 1936; Borsook et al., 1937; Rubin et al., 1937). The rate of the uncatalyzed reaction is, however, too slow to be of much consequence, the half-time period for the reduction of dehydroascorbic acid by GSH at physiological temperatures and a t pH values and in coiicentrations usually found in vivo being of the order of 15 minutes, whereas under the same conditions the conversion of DHA to 2,3-diketogulonic acid has a half life of only 2 minutes (Ball, 1937). Pfankuch (1934) was the first to describe enzymatic reduction of DHA by sulfhydryl compounds. He found an enzyme in potato juice which catalyzed the reduction of DHA by cysteine. This work was extended by Hopkins and his collaborators (Hopkins and Morgan, 1936; Crook and Hopkins, 1938; Crook, 1941), who showed that an enzyme, dehydroascor-

16

L. W. MAPSON

bic acid reductase, catalyzed the reduction of DHA b y GSH in accordance with the following equation : PGSH 3-

DHA + GSSG

+ AA

The enzyme was prepared from cauliflower juice and separated from ascorbic oxidase. It had the properties of an enzyme in that it was thermolabile and was precipitated by (NHSzS04 and in that its activity was affected by p H in a manner characteristic of enzymes. Cysteine and thiolactic acid could replace GSH, although GSH was twice as effective as cysteine and four times as effective as thiolactic acid. The distribution of the enzyme was investigated, and it was found to be present in 22 of the 30 species of plants examined, the most active sources being broad beans and cauliflowers. Other workers, notably Kertesz (1938), have been unable t o repeat these observations, for reasons which are obscure. Dehydroascorbic acid reductase has been found, however, in leguminous seeds b y Kohman and Sanborn (1937), while Yamaguchi and Joslyn (1951) have also found it in peas, particularly in the meristematic regions where the respiration rate is high. The possibility that this enzyme is one stage in a hydrogen transfer system was suggested by Crook (1941), who visualized the following action : H

plant substrates

+ -%S-

dehydroascorbic reductaae

DHA -+ Atmosphere 02

This suggestion was crit.icized by Barron (1939), on the grounds th a t there is little GSH in plants and that the concentrations of GSH and AA used in the work of Hopkins and his collaborators were unphysiological. These objections have been answered by Crook (1941), who has pointed out that the protection of AA from oxidation has been shown to hold for low concentrations of GSH, and th at the concentrations of reactants used in his experiments were well within the physiological range occurring in some plants. c. Ascorbic Acid4lutathione System. Szent-Gyorgyi (1937) suggested on the basis of the known behavior of ascorbic acid that it might, in conjunction with glutathione, act as a respiratory carrier. The following hypothetical reactions were visualized as occurring :

+ + + + + HzO + aacorbia oxidase + 4. Flavone + H202 Flavone oxide + H20 peroxidaae 6. Flavone oxide + AA -+ Flavone + DHA 1. DHA GSH- AA GSSG 2. GSSG glucose phosphate4 GSH CO1 3. AA 02DHA Hz02

-

FUNCTION O F ASCORBIC ACID IN PLANTS

17

Since the discovery of the existence of dehydroascorbic acid reductase, the chief difficulty in accepting glutathione as a link in the respiratory chain with the ascorbic acid system was the lack of evidence connecting the former substance with any of the natural substrates of respiration. Evidence has been accumulating that both plant and animal tissue possess compounds t o SH compounds. The work of the power t o reduce -S-SKohman and Sanborn (1937) and Ganapathy (1938) indicated that GSSG may be reduced to GSH by plant juices. Firket and Comhaire (1929) and Vivario and Lecloux (1930) observed that sulfhydril compounds rapidly appear after hydration of seeds. In 1943, Hopkins and Morgan established with pea seeds that the sulfhydril compound produced was mainly GSH, and furthermore they were able to show that if GSSG was added t o extracts from dried pea seeds under anaerobic conditions the GSSG was rapidly reduced. Bukin (1943) claimed that GSSG may be reduced by dihydrocoenzyme I in a simple nonenzymatic reaction, but this claim that dihydrocoenzyme I will reduce GSSG either in a chemical reaction or in a reaction catalyzed by enzymes in plant tissues has not been substantiated (Mapson and Goddard, 1951; Conn and Vennesland, 1951a,b). It seems probable, therefore, as will be seen later, that coenzyme I1 may have been present as an impurity in the preparation of coenzyme I used, or that it was formed from coenzyme I in Bukin’s experiments. The observation of Meldrum and Tarr (1935) that GSSG was reduced by enzyme extracts from blood or yeast in the presence of hexose monophosphate and by extracts containing coenzyme I1 has now been substantiated for plant tissue. Mapson and Goddard (1951) and Conn and Vennesland (1951a,b), working with peas and wheat, respectively, have shown the presence of an enzyme, glutathione reductase, in these tissues. This enzyme catalyzes the reduction of GSSG by dihydrocoeiizyme 11, and the equilibrium point of the reaction is all in favor of the formation of GSH. The enzyme appears to be highly specific for GSSG, for it will not catalyze the reduction of cystine, homocystine, a-glutamyl cystine, or aspartathione. It is also specific for coenzyme 11;there is no reaction with coenzyme I. The possibility of a hydrogen transfer system involving enzyme 11, glutathione, and ascorbic acid is a t once apparent. Such a hydrogen transfer has in fact been demonstrated in pea seed extracts (Mapson and Goddard, 1951). Extracts from this tissue contain both malate and isocitrate dehydrogenase enzymes which reduce coenzyme 11, together with glutathione and dehydroascorbic acid reductase, and it was shown that, as a result, hydrogen from isocitrate or malate may be transferred to dehydroascorbic acid in accordance with the following reactions.

18

L. W. MAPSON

gg'tte+ Co I1 +

Co I1 Ht

+ Oxalosuccinate Oxalacetate

Oxalosuccinate Mn and decarboxylase Oxalacetate * ' COa

a-Ketoglutarate

+ Pyruvate glutathione reductme COI1 He + GSSG . PGSH + Co I1 GSH

+ DHA dehydroascorbicreductase' AA + GSSG

Hydrogen from substrates of dehydrogenase enzymes linked with coenzyme I1 may therefore be transferred in the presence of this system to dehydroascorbic acid and, by means of the ascorbic acid so formed and an appropriate oxidase, to molecular oxygen. In this respect the system provides a pathway alternative to that of coenzyme II-diaphorasecytochrome. Direct evidence that the system is active in one plant tissue in vivo is provided by the work of Barker and Mapson (1952a) who found that when potato tubers are placed in pure oxygen there is, after a latent period which may be as long as 50 days, first a fall in the GSH content, followed closely by a fall in ascorbic acid and by a corresponding rise in DHA. In later work (1952b) they found that under these conditions the fall in GSH is accompanied by a rise in GSSG, and the rate of change in the GSH/GSSG ratio is reflected in corresponding changes in that of AA/DHA. The maintenance of the ascorbic acid in its reduced form thus appears to be dependent on the maintenance of GSH. The results of these experiments support the view that the dehydroascorbic acid reductase which can be demonstrated in vitro in potato extracts is also operative in vivo. During the phase in which ascorbic acid was disappearing there was no evidence for any loss of activity of dehydroascorbic reductase; the increase of DHA, as the fall in GSH and the rise in GSSG indicate, appeared to be associated with an impairment of the system responsible for the reduction of GSSG to GSH. We have as yet no information as to the importance of such a system in the general respiratory activity of plant tissues. Its importance as a means of maintaining GSH, and hence of activating and conserving the activity of the so-called SH enzymes (enzymes which are dependent on the maintenance of certain SH groups in their molecules), is self-evident but does not concern us here. The participation of ascorbic acid in a respiratory chain of reactions will be prevented in the presence of cyanide, for the last stage, the enzymatic oxidation of ascorbic acid, will be inhibited. The extent to which hydrogen is transferred in the system will depend inter alaa on the concentration of GSH and dehydroascorbic acid and on the

FUNCTION O F ASCORBIC ACID IN P L A N T S

19

relative affinities of glutathione reductase and other diaphorase enzymea reacting with dihydrocoenzyme 11. That this cannot be the only mechanism reducing DHA in plant tissues seems evident from other considerations. Crook and Morgan (1943) found no evidence of the presence of a dehydroascorbic acid reductase in 8 out of 30 species of plants examined. Some plants which have been shown to reduce DHA readily, e.g., hydrangea (Rubin et al., 1937), do not possess an active dehydroascorbic acid reductase.

4. Evidence for the Action of Ascorbic Acid-Dehydroascorbic Acid as a Respiratory Carrier in vivo

The biochemical evidence reviewed here suggests that the ascorbic system may act as a carrier positioned between either coenzyme I or I1 and the terminal oxidase. It emphasizes the role of dehydroascorbic acid as hydrogen acceptor, the concentration of which, rather than the concentration of ascorbic acid, might be expected to determine the respiratory changes through this channel. There has, to the present date, been no comprehensive attempt to determine whether there is any correlation between the concentration of DHA and the level of respiration in plant tissues. Some attempts have been made to correlate the level of respiration of certain plant tissues with their ascorbic acid content. Rubin et al. (1946) found that with the fruit of two forms of the dog rose, R. cinnamanea and R. spinosissima, both the level of respiration and the ascorbic acid were higher in the former than in the latter species. An increase of ascorbic acid of the fruit, induced by the infiltration of glucose, increased the respiration. However, this correlation was not observed during ripening, for here the level of respiration rose but the concentration of ascorbic acid decreased. The failure to observe a complete correlation under all conditions was explained by the hypothesis that only a part of the total ascorbic acid was participating. This argument gains some support from the fact that the concentration of ascorbic acid in some plant tissues may be very high, which makes it difficult to believe that it is all functioning as a respiratory catalyst. The level of ascorbic acid may in fact be altered in the plant cell without having any significant effect on the respiratory level. For instance, it was observed (Chen and Mapson, 1951) that cress seedlings, grown under conditions where the amount of ascorbic acid synthesized varied greatly, did not show any difference in their general level of respiratory activity. We must conclude that such studies have so far given little positive evidence either for or against ascorbic acid functioning as a respiratory catalyst.

20

L. W. MAPSON

If the DHA-AA system is acting as a respiratory carrier in vivo, then one would expect that the subjection of plant tissues to anaerobic conditions would lead to a fall in concentration, if not to the complete disappearance of DHA. An analogous phenomenon is certainly observed with cytochrome in portions of intact potato tissue. The reduction of cytochrome under anaerobic conditions and its reoxidation on admittance of air may be observed spectroscopically by visual examination of the cytochrome spectrum of the tubers in vivo (Hill and Scarisbrick, 1951). Moreover, these changes are produced quite rapidly within 60 to 90 minutes of the alteration of the atmosphere around the tubers (Hill and Barker, 1951). I n similar experiments with ascorbic acid there was no immediate change either in the concentration of DHA or in the ratio of DHA/AA. Only when the potato tubers were left 18 to 22 hours under water was there any significant fall in the concentration of DHA (Mapson and Barker, 1952). Such evidence would suggest that the bulk of the hydrogen transport is via cytochrome rather than via ascorbic acid. It is, however, difficult to conclude positively th at this is so for the following reasons. In the first place the technique of observing the changes in AA and DHA is not so simple as that employed for cytochrome, which may be followed without subjection of the tissue to chemical manipulation. I n the second place, in the later stages of storage in nitrogen a rise of DHA and a fall of AA were observed. Thus, even under completely anaerobic conditions, substances capable of oxidizing AA appear to be produced, and, if these were present in the early stage of anaerobiosis, production of DHA due t o these may have complicated the picture. One such oxidant is known, viz., nitrous acid, which is formed from nitrite during the acid extraction of tissue prior t o the estimation of ascorbic acid, and which oxidizes the latter and gives misleading values for DHA (Mapson and Ingram, 1951). Nitrite is formed during the storage of potatoes in nitrogen, and while its interference during extraction was prevented in the experiments quoted, other substances acting likewise may have been produced. Investigation of the rate of loss of ascorbic acid from plant tissue under anaerobic condition, though it suggests that DHA is certainly being formed in vivo, throws very little additional light on this problem. The conversion of DHA to diketogulonic acid is the only known cause of the irreversible loss of ascorbic acid from tissues. This reaction, which is the first stage in a series of disruptive reactions (Herbert et al., 1933; Mills et al., 1949) is not affected by the presence or absence of oxygen (Penney and Zilva, 1943). One would, therefore, expect that the rate of loss would depend on the concentration of DHA in the tissue. If DHA was not continually being formed from ascorbic acid by oxidation in tissues in

FUNCTION OF ASCORBIC ACID IN PLANTS

21

air, the rate of loss of ascorbic acid should be no greater in air than in nitrogen. Studies on this subject have shown, however, that with detached leaves the complete exclusion of oxygen has been found to retard, and sometimes to prevent entirely, a decrease in the ascorbic acid (Wood et al., 1944; Mapson and Barker, 1948). Lower concentrations of oxygen than those in air have been found to retard the loss of ascorbic acid in vegetables (Platenius and Jones, 1944), and, conversely, pure oxygen increases the loss from both leaves and potato tubers (Guthrie, 1937; Barker and Mapson, 1952a). Although the biochemical evidence would suggest therefore that ascorbic acid may act as a respiratory carrier, the previous remarks have served to emphasize that, as yet, we have no clear evidence that it is so acting in vivo.

111. ASCORBIC ACIDAS

A

GROWTHFACTOR

Observations showing that ascorbic acid is most highly concentrated in the more actively growing regions of plant tissues have led to experiments designed to determine whether it acts as a growth factor. Kogl and Haagen-Smit (1936) were unable to detect any effect of ascorbic acid on the growth of young pea seedlings when it was added to the culture solution. On the other hand, Havas (1935), Davies et al. (1937), and Dennison (1940) among others have found that ascorbic acid may promote the growth of certain plants. Reid (1937) studied the relation of the content of ascorbic acid to volume, area, and dry weight of individual cells in the root tips of cow pea seedlings. The ascorbic acid continued to increase in concentration in the cell until elongation ceased and maturation began. There appeared to be a connection between the relative surface area of cells a t different stages of development and the concentration of ascorbic acid. The author believes that the accumulation of ascorbic acid precedes and possibly conditions cell expansion. Bonner and Bonner (1938) found that ascorbic acid added in culture solution to the excised embryos of certain varieties of peas (Perfection, Alaska) stimulated the growth, whereas with other varieties (Wrinkled Winner) it had no effect. Correlated with these observations were some which showed that those varieties giving negative results were able to synthesize the vitamin to a much greater extent than those in which positive effects were observed. The fact that some varieties did not respond to feeding with ascorbic acid with an increase in growth may simply mean that they are able to synthesize sufficient of the substance for their needs; consequently, negative results do not necessarily indicate that the substance is not a growth factor. Assimilation of Nitrate. Von Hausen (1936) observed that if the cotyledons of pea seedlings were removed after a period of 5 to 9 days

22

L. W. MAPSON

after the start of germination and the cotyledonless seedlings transferred to a sterile nutrient solution, growth, which was a t first feeble, soon ceased. If ascorbic acid in a concentration of 30 to 40 mg. per liter of nutrient solution was added, the plants grew until the flowering stage. In a continuation of the work Virtanen and von Hausen (1949) found that it was necessary to add three times this level of ascorbic acid to promote growth equal to that observed in normal plants. All these experiments were carried out in nutrient media in which nitrate was used as a source of nitrogen. When similar experiments were carried out with ammonium sulfate as the source of nitrogen, in both wheat and pea seedlings, normal growth was observed in the cotyledonless seedling even in the absence of ascorbic acid: only when nitrate was used as the supplier of nitrogen was ascorbic acid found to be necessary. Later experiments showed that other reducing substances, e.g., glutathione, cysteine, and reductone could also promote growth under these conditions. The authors suggest that this effect of ascorbic acid is due to its reducing properties, and indicate that the regulation of the redox potential is apparently an important function of the vitamin in normal growth of the plant. The fact that this effect of ascorbic acid is closely associated with the presence of nitrate and not with ammonium salts might also indicate that the reduction and utilization of nitrate is dependent on the presence of ascorbic acid or other reductants. The suggestion of Virtanen and von Hausen (1949) that the sulfhydryl compounds act by conserving the small amounts of ascorbic acid present in cotyledonless seedlings by regenerating AA from DHA by reduction would, if true, suggest a higher degree of specificity for ascorbic acid than the experimental data indicated. The observation that reductone can function like ascorbic acid would still remain to be explained. The evidence from these studies implies that ascorbic acid is not necessary as a general growth factor but only as a possible participant in reactions enabling the plant to reduce and thus utilize nitrate for cell expansion and growth. This suggestion that ascorbic acid may be essential for the assimilation and utilization of nitrate nitrogen by the plant receives some support from the work of Hewitt and his collaborators. I n molybdenum deficiency there was found to be a significant reduction of the ascorbic acid concentration (25 to 30%) in the foliage of cauliflower, cabbage, kale, sprouts, tomato, beet, and other crops (Hewitt et al., 1950). It has also been shown that nitrate accumulates in the foliage of molybdenum-deficient plants (Wilson and Waring, 1948; Hewitt, 1950). Injection of microgram quantities of molybdenum produced a marked increase in the ascorbic acid level within a period of 3 to 5 days. Moreover Hewitt found that homogenized extracts from the foliage of molybdenum-deficient plants had a lowered

FUNCTION OF ASCORBIC ACID IN PLANTS

23

ability to reduce nitrite in vitro a t 37" C. compared with extracts from normal plants. The concentrations of ascorbic acid found in normal and molybdenum-deficient foliage are consistent with the relative ability of homogenized foliage of plants grown with or without molybdenum to reduce added nitrite (Hewitt, 1951). Further investigation may reveal more definitely whether ascorbic acid plays a part in the reduction of nitrate or nitrite to nitrogenous derivatives which are essential for the growth of the plant. Raadts and Soding (1948) found that dehydroascorbic acid stimulates the growth of Avena coleoptiles. They suggest that this was due to the greater formation of indoleacetic acid from some precursor. However, they also observed that methylene blue and, in some cases, hydrogen peroxide had similar effects. Wetmore and Morel (1949) let auxin diffuse from Equisetum tissue to agar blocks and found that the addition of ascorbic acid to the receiving blocks greatly increased the curvatures obtained in the Avena test. Further work along these lines is needed to determine the mechanism of this action of ascorbic acid or its derivatives.

IV. ACTION OF ASCORBIC ACID ON ENZYMES The activities of several enzymes appear to be markedly influenced by ascorbic acid. In some cases the effect observed is one of activation, in others of inhibition. Cathepsin is activated by ascorbic acid and the effect is increased in the presence of iron salts (Euler et al., 1934). Activation of arginase by ascorbic acid has been reported by Edlbacher and Leuthardt (1933). Papain is inhibited by ascorbic acid alone but activated in the presence of ferrous salts (Maschmann and Helmert, 1934). Inhibition of enzymic activity by ascorbic acid has also been reported for urease (Edlbacher and Leuthardt, 1933; Elson, 1943) and for 0-amylase (Purr, 1935; Hanes, 1935). In the case of liver esterase the claim was made that the enzyme consists of a protein (apoenzyme) combined with ascorbic acid (coenzyme) when i t was found that liver esterase loses its activity on dialysis with dilute hydrochloric acid but can be reactivated by the addition of ascorbic acid (Pantschenko-Jurewicz and Kraut, 1936). These results have not, however, been confirmed by later workers (Kertesz, 1938; Strachitskii and Meerzon, 1939). Many enzymes have been shown to depend for their activity on the integrity of an -SH group in the molecule (Hellerman, 1937), and the activating effect of ascorbic acid has been suggested as being due to the protection of such -SH groups from oxidation (Harrer and King, 1941).

24

L. W. MAPSON

However, it has been shown that the activity of one such enzyme, urease, is inhibited by ascorbic acid, although this inhibition was eliminated in the presence of cysteine (Elson, 1943). A suggestion that dehydroascorbic acid was the agent responsible for reacting with the -SH group of the enzyme and that the protective effect of thiol compounds was due to the reduction of dehydroascorbic acid (Quastel, 1943) was found to be untenable when it was shown that dehydroascorbic acid did not inactivate urease (Giri and Seshagiri Rao, 1944). In further studies (Mapson, 1946) it was found that ascorbic acid itself does not inhibit urease activity. In the presence of Cutt-, however, it does so by effecting the reduction of Cu++ .+ Cu+, which latter ions have a much higher affinity for -SH groups than have the former. With mercury salts the reverse effect of ascorbic acid was observed, namely an activation of urease activity. This was correlated with the fact that enzymic activity was reduced more by Hg++ than by Hg+ salts. The action of ascorbic acid on urease could therefore be explained in terms of its reducing action on metallic ions present in solution. The inhibiting action of ascorbic acid on plant P-amylase is probably explicable on a similar basis, since it has been shown that the inhibition was increased in the presence of small amounts of copper salts (Hanes, 1935). It is of interest to note that the activating effect of ascorbic acid on papain occurs only if ferrous ions are present; otherwise the effect of the vitamin is depressant (Maschmann and Helmert, 1934). There is evidence suggesting that the ascorbic acid-iron complex activates by first reducing dithiol compounds associated with the enzyme, and that these thiol compounds in turn activate the enzyme (Purr, 1935). Similar reactions may be involved in the activation of arginase by the ascorbic acid-iron complex (Purr, 1933). The action of ascorbic acid on many enzymes appears to be conditioned by other substances, notably metallic ions, present in reaction mixtures; certainly where its action has been critically examined, this has been found to be so. Whether ascorbic acid in vivio has any regulatory influence on these enzymes is uncertain. Still more improbable is the view that this action of ascorbic acid constitutes one of its essential roles in the living cell, since it has been shown both for P-amylase and urease that other dienols (reductone, dehydroxymaleic acid, reductic acid, hydroxytetronic acid) which are biologically inactive react similarly (Hanes, 1935; Mapson, 1946). There is also no evidence at present t o suggest that ascorbic acid promotes the activity of enzymes of plant tissues in a manner comparable to its effect on the phosphatase activity in animal tissues.

FUNCTION O F ASCORBIC ACID I N PLANTS

25

REFERENCES Allen, P. J., and Goddard, D. R. 1938. Am. J. Botany 26, 613-621. Baker, D. L., and Nelson, J. M. 1943. J . Gen. Physiol. 26, 269-276. Ball, E. G. 1937. J. Biol. Chem. 118, 219-239. Barker, J., and Mapson, L. W. 1952a. New Phytologist 61, 90-115. Barker, J., and Mapson, L. W. 1952b. Unpublished data. Barron, E. S. G. 1939. Cold Spring Harbor Symposia Quant. Biol. 7, 145-147. Barron, E. S. G., Barron, A. G., and Klemperer, F. 1936. J. Biol. Chem. 116,563-573. Baruah, P., and Swain, T. 1952. Unpublished data. Bertrand, D. 1945a. Bull. SOC. chim. biol. 27, 396-398. Bertrand, D. 194513. Compt. rend. 221, 35-36. Bhagvat, K., and Hill, R. 1951. New Phytologist 60, 112-120. Bonner, J., and Bonner, D. 1938. Proc. Natl. Acad. Sci. U . S. 24, 70-75. Bonner, J., and Wildman, S. G. 1946. Arch. Biochem. 10, 497-518. Rordner, C. A., and Nelson, J. M. 1939. J. Am. Chem. SOC.61, 1507-1513. Borsook, H., Davenport, H. W., Jeffrys, C., and Warner, R. C. 1937. J. Biol. Chem. 117, 237-279. Boswell, J. G., and Whiting, G. C. 1938. Ann. Botany (London) 2, 847-863. Brand, A. K. 1949. Deut. Lebensm.-Rundschau 46, 372-376. Brown, A. H., and Goddard, D. R. 1941. Am. J. Botany 28, 319-324. Bukin, V. N. 1934. Biokhimiya 8, 60-76. Chen, T., and Mapson, L. W. 1951. Unpublished data. Claggett, C. O., Tolbert, N. E., and Burris, R. H. 1949. J. Biol. Chem. 178, 977-987. Conn, E. E., and Vennesland, B. 1951a. Nature 167, 976. Conn, E. E., and Vennesland, B. 1951b. J. Biol. Chem. 192, 17-28. Crook, E. M. 1941. Biochem. J. (London) 36, 226-236. Crook, E. M., and Hopkins, F. G. 1938. Biochem. J. (London) 32, 1356-1763. Crook, E. M., and Morgan, J. 1943. Biochem. J. (London) 38, 10-15. Damodaran, M., and Nair, K. 1936. Biochem. J. (London) 30, 1014-1020. Davies, W., Atkins, G. A., and Hudson, P. C. 1937. Ann. Botany (London) 7,329-351. Davison, D. C. 1949. Proc. Linnear SOC.N . S . Wales 74, 37-56. Davison, D. C. 1950. Nature 166, 265. Dawson, C. R. 1950. Copper Metabolism, Johns Hopkins Press, Baltimore. Dennison, R. A. 1940. Science 92, 17. De Caro, L., and Giani, M. 1934. Hoppe-Seyler’s 2. physiol. Chemie 228, 13-24. Dodds, M. L. 1948. Arch. Biochem. 18, 51-58. Dunn, F. J., and Dawson, C. R. 1951. J. Biol. Chem. 891, 485-497. Edlbacher, S., and Leuthardt, F. 1933. Klin. Wochschr. 12, 1843. Elson, L. A. 1943. Nature 162, 49. Euler, H. von, Karrer, P., and Zehender, F. 1934. Helv. Chim. Acta 17, 157-162. Firket, M. J., and Comhaire, M. 1929. Bull. acad. TOY.m8d. Belg. 9, 93-122. Ganapathy, C. V. 1938. Current Sci. (India) 6, 451-452. Giri, K. V., and Krishnamurthy, S. 1940. Nature 146, 99. Giri, K. V., and Seshagiri Rao, P. 1946. Proc. Indian Acad. Sci. 24B, 264-278. Giri, K. V., and Seshagiri Rao, P. 1944. Nature 163, 253-254. Goddard, D. R. 1944. Am. J. Botany 31, 270-276. Goddard, D. R., and Holden, C. 1950. Arch. Biochem. 27, 41-47. Gregg, D. C., and Nelson, J. M. 1940. J. Am. Chem. SOC.62, 2500-2505.

26

L. W. MAPSON

Guthrie, J. D. 1937. Contrib. Boyce Thompson Znst. 9, 17-39. Hand, D.B.,and Greisen, E. C. 1942. J. Am. Cham. SOC.64,358-361. Hanea, C . 8. 1935. Biochem. J . (London) 29, 2588-2603. Harrer, C. J., and King, C. G. 1941. J. Biol. Chem. 138, 111-121. Hausen, S.von. 1936. Ann. Acad. Sci. Fennicae 46A,No. 3, 134 pp. Havaa, L. 1935.Nature 136, 435. Hellerman, L. 1937.Physiol. Revs. 17, 454-484. Herbert, R. W.,Hirst, E. L., Percival, E. G., Reynolds, R. J., and Smith, F. 1933. J . Chem. SOC.,pp. 1270-1290. Hewitt, E. J. 1951. Ann. Rev. Plant Physiol. 2, 25-52. Hewitt, E.J. 1950. Rep. No. 11669 on Sand Culture. Agricultural Research Council. Mineral Deficiencies Conference 1950. Hewitt, E. J., Agaryala, 9. C., and Jones, E. W. 1950. Nature 166, 1119-1120. Hill, R.,and Bhagvat, K. 1939.Nature 143,726. Hill, R.,and Barker, J. 1951. Unpublished data. Hill, R., and Scarisbrick, R. 1951. New Phytologist 60, 98-111. Hooper, F. C., and Ayres, A. D. 1950. J. Sci. Food Agr. 1,5-8. Hopkins, F. G., and Morgan, E. J. 1943.Nature 162, 288-290. Hopkins, F. G., and Morgan, E. J. 1936. Biochem. J. (London) SO, 1446-1461. Husz&k,S. 1937. Hoppe-Seyler's Z.physiol. Chem. 247, 239-247. James, W.O.,and Beevers, H. 1950. New Phytologist 49, 353-374. James, W.O.,and Cragg, J. M. 1943. New Phytologist 42,28-44. James, W.O.,Heard, C. R. C., and James, G. M. 1944. New Phytologist 49, 62-74. Johnson, S. W.,and Zilva, S. 6. 1937. Biochem. J . (London) 91,438-453, 1366-1374. Joselow, M., and Dawson, C. R. 1951. J. Biol. Chem. 191, 11-20. Kardo-Sysoeva, E. K., and Nisenbaum, R. F. 1938. Biokhimiya 3, 348-354. Keilin, D., and Hartree, E. F. 1938.Proc. Roy. SOC.(London) 126B, 171-186. Keilin, D., and Mann, T. 1939. Nature 143,23-24. Kertesz, Z.I. 1938.Arkiv, Kemi, Mineral. Geol. l2B, No. 57, 4 pp. Kertesz, Z. I. 1938.Biochem. J . (London) 32, 621-625. Kertesz, D. 1952. Biochim. et Biophys. Acta 9, 170-179. Kogl, F.,and Haagen-Smit, A. J. 1936.Hoppe-Seyler's Z. physiol. Chem. 243,209-226. Kohman, E. F.,and Sanborn, N. H. 1937. Znd. Eng. Chem. 29, 189, 1195-1199. Kubowitz, F. 1937.Biochem. Z.292,221-229. Lampitt, L. H., and Clayson, D. H. 1945. Biochem. J . (London) 39,p. xv. Laties, G. G. 1950. Arch. Biochem. 27, 404-409. Levy, H., Schade, A. L., Bergmann, L., and Harris, S. 1948. Arch. Biochern. 19, 273286. LovethTanison, P. L., and Nelson, J. M. 1940. J . Am. Chem. SOC.62, 1409-1412. Ludwig, B.J., and Nelson, J. M. 1939. J. Am. Chem. SOC.61,2601-2606. Mallette, M. F.,Lewis, S., Ames, S. R., Nelson, J. M., and Dawson, C. R. 1948. Arch. Biochem. 16,283-289. Mallette, M. F., and Dawson, C. R. 1949. Arch. Biochem. 29, 29-44. Mapson, L. W.1946.Biochem. J . (London) 40, 240-247. Mapson, L. W.,and Barker, J. 1952. Unpublished data. Mapson, L. W., and Barker, J. 1951. Unpublished data. Mapson, L. W., and Barker, J. 1948. Unpublished data. Mapson, L. W., and Ingram, M. 1951. Biochem. J. (London) 48, 551-559. Mapson, L. W.,and Goddard, D. R. 1951. Nature 167, 975. Mapson, L. W.,and Goddard, D. R. 1951. Biochem. J . (London) 49, 592-601.

FUNCTION O F ASCORBIC ACID I N PLANTS

27

Marsh, P. B., and Goddard, D. R. ,1939. Am. J . Botany 26, 724-728. Maschmann, E., and Helmert, E. 1934. Hoppe-Seyler's 2.physiol. Chem. 223, 127-135. Mathews, M. B. 1951. J. Biol. Chem. 189, 695-704. Matusukawa, D. 1940. J. Biochem. (Japan) 32, 257-267. Mawson, C. A. 1935. Biochem. J. (London) 29, 569-579. Meldrum, N. U., and Tarr, H. L. A. 1935. Biochem. J . (London) 29, 108-115. Merry, J., and Goddard, D. R. 1941. PTOC. Rochester Acad. Sci. 8, 28-44. Miller, W. H., and Dawson, C. R. 1941. J . Am. Chem. SOC.63,3368-3374,3375-3382. Mills, M . B., Damron, C. M., and Roe, J. H. 1949. Anal. Chem. 21, 707-709. Nelson, J. M., and Dawson, C. R. 1944. Advances in Enzymol. 4, 99-152. Onslow, M. W. 1920-24. Biochem. J. (London) 1920, 14, 535-540, 541-547; 1921, 16, 107-112, 113-117; 1923, 17, 216-219; 1924, 18, 549. Pantschenko-Jurewicz, W. V., and Kraut, H. 1936. Biochem. Z. 286, 407-419. Penney, J. R., and Zilva, S. S. 1943. Biochem. J . (London) 37, 39-44. Pett, L. B. 1936. Biochem. J. (London) 30, 1228-1232. Pfankuch, E. 1934. Naturwissenschaften 22, 821-837. Platenius, H., and Jones, J. B. 1944. Food Research 9, 378-385. Potter, V. R. 1940. Medicine 19, 441-474. Powers, W. H., Lewis, S., and Dawson, C. R . 1944. J . Gen. Physiol. 27, 167-180. Purr, A. 1933. Biochem. J. (London) 27, 1703-1705. Purr, A. 1935. Biochem. J . (London) 29, 5-12, 13-20. Quastel, J. H. 1943. Nature 162, 215. Raadts, E., and Soding, H. 1948. Naturwiseenschaften'34, 344. Reid, M. E. 1937. Am. J. Botany 24,445-447. Robinson, E. S., and Nelson, J. M. 1944. Arch. Biochem. 4, 111-117. Roberts, E. A. H., and Wood, D. J. 1951. Nature 167, 608. Rubin, B. A., Sisakian, N. M., and Lutikova, 0. T. 1937. Bull. acad. sci. U.S.S.R. 16, 495-498. Rubin, B. A., Arkikhovskaya, E. V., and Roskwinikova, T. A. 1946. Biokhimiya 11, 349-358. Schneider, W. C., and Potter, V. R. 1943. J. Biol. Chem. 149, 217-227. Snow, G. A., and Zilva, S. S. 1938. Biochem. J . (London) 32, 1926-1937. Somogyi, J. C. 1944. Helv. Physiol. et Pharmacol. Acta 2, 269-274. Srinivasan, M. 1935. Current Sci. (India) 4, 407-408. Steinman, H. G., and Dawson, C. R. 1942. J . Am. Chem. SOC.64, 1212-1219. Stone, W. 1937. Biochem. J . (London) 31, 508-512. Stotz, E. 1940. J. Biol. Chem. 133, Proc. p . 100. Stotz, E., Harrer, C. J., and King, C. G. 1937. J. Biol. Chem. 119, 511-522. StrachitskiI, K. I., and Meerzon, T. I. 1939. Biokhimiya 4, 60-67. Sreerangacher, H. B. 1943. Biochem. J . (London) 37, 653-655. Szent-Gyorgyi, A. 1928. Biochem. J. 22, 1387-1409. Szent-Gyorgyi, A. 1931. J . Biol. Chem. 90, 385-393. Szent-Gyorgyi, A. 1937. Studies on Biological Oxidation, Leipzig, 1937. Tauber, H., Kleiner, I. S., and Mishhind, D. 1935. J. Biol. Chem. 110, 211-218. Tauber, H., and Kleiner, I. S. 1935. Proe. SOC.Exptl. Biol. Med. 33, 391-392. Thomas, M. 1947. Plant Physiology, London. Virtanen, A. I., and Von Hausen, S. 1949. Nature 163, 482-483. Vivario, R., and Lecloux, J. 1930. Arch. intern. physiol. 32, 1-14. Waygood, E. R. 1950. Can. J . Research 28C, 7-62. Wetmore, R. H., and Morel, G. 1949. Am. J. Botany 36, 830-831.

28

L. W. MAPSON

Wilson, R. D., and Waring, E. J. 1948. J . Australian Inst. Agr. Sci. 14, 141-145. Wood, J. G., Mercer, F. V., and Pedlow, C. 1944. Australian J . Exptl. Biol. Med. Sci. aa, 37-43. Yamaguchi, M., and Joslyn, M. A. 1951. Plant Physiol. 26, 757-771. Zilva, S. S. 1934. Biochem. J . (London) 28, 663-666. Zilva, S. S., Kidd, F., and West, C. 1938. New Phytologist 37, 345-357.