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The effect of gibberellic acid and ethephon on enzymatic browning of redhaven peaches Paulson, Allan Thomas 1978

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THE EFFECT OF GIBBERELLIC ACID AND ETHEPHON ON ENZYMATIC BROWNING OF REDHAVEN PEACHES ALLAN THOMAS PAULSON B.Sc.(Agr.), University of British Columbia, 1973 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Department of Food Science We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA by March, 1978 Allan Thomas Paulson, 1978. In presenting this thesis in partial fulfilment of the requirements f o r an advanced degree at the University of British Columbia, I agree t h a t the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department o r by his representatives. It is understood that copying o r publication o f this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date MARCH W78 6 - i i -ABSTRACT Redhaven peaches treated with gibberellic acid (GA, 100 ppm) and ethephon (75 and 150 ppm) 21 and 46 days after f u l l bloom were evaluated for enzymatic browning in the ripe f r u i t . Treated f r u i t had less browning than untreated f r u i t , and f r u i t treated 46 days after bloom had less browning than f r u i t treated 21 days after bloom. Fruit pH and fresh weight were affected by treatment, but o-diphenol content and polyphenoloxidase (PPO) activity were not. Forward stepwise multiple regression on browning showed that 81% of the variation i n browning was explained by differences i n treatment, treatment application date, o-diphenol content, PPO activity, and fresh weight. Twenty-one polyphenolic compounds from Redhaven peaches were separated by two-dimentional thin layer chromatography. Eight were oxidized by PPO, and were tentatively identified as four chlorogenic acid isomers, three leucoanthocyanidins, and catechin. No differences in qualitative distributions of phenolic compounds were observed i n peaches receiving the different treatments. Polyacrylamide disc gel electrophoresis of peach PPO preparations showed the presence of up to eleven isozymes with activity toward catechol. The isozymes had different substrate s p e c i f i c i t i e s and were present in different amounts. PPO from peaches treated 21 days after bloom appeared to have a catechol reactive isozyme not present in untreated peaches or peaches treated 46 days after bloom. One PPO isozyme from peaches treated 46 days after bloom with 150 ppm - i i i -ethephon appeared to have decreased substrate spec i f i c i t y toward pyrogallol. Crude PPO preparations from untreated f r u i t and f r u i t receiving the 46-day treatments oxidized o-dihydroxyphenolic compounds only. The relative a c t i v i t i e s of the PPO preparations with these compounds varied with treatment. The same PPO preparations exhibited two pH optima; pH 4.4 and 6.2 for untreated and GA treated peaches (46-day treatment), and pH 4.4 and 6.6 for peaches treated with ethephon (75 or 150 ppm, 46-day treatment). PPO from the treated peaches had a lower proportion of total activity at pH 4.4 than PPO from untreated peaches. The Michaelis constant for PPO from untreated peaches was 9.1 x 10~3M. - iv -TABLE OF CONTENTS Page ABSTRACT . • • i i TABLE OF CONTENTS • • • iv LIST OF TABLES . . . . v i LIST OF FIGURES • v i i ACKNOWLEDGEMENTS v i i i INTRODUCTION 1 LITERATURE REVIEW 3 Enzymatic Browning Reaction 3 Polyphenoloxidase 4 Browning Substrates . 7 Gibberellic Acid and Ethephon .9 EXPERIMENTAL 12 Application of Growth Regulators . . . . . . . . . . . . . . .12 Enzymatic Browning 13 Polyphenoloxidase Extraction and Assay 13 Extraction and Assay of o-Diphenolic Compounds 14 St a t i s t i c a l Methods 15 Thin Layer Chromatography of Peach Phenolic Compounds . . . . 16 Electrophoresis 19 PPO Substrate Specificity 24 PPO pH Optima 24 Effect of Substrate Concentration. . 25 - V RESULTS AND DISCUSSION 2 6 Enzymatic Browning 26 Thin Layer Chromatography 35 Polyphenoloxidase Isozymes 49 PPO Substrate S p e c i f i c i t y 5 5 PPO pH Optima • .57 Effect of Substrate Concentration • 57 SUMMARY AND CONCLUSIONS 6 1 LITERATURE CITED 6 4 - v i -Table LIST OF TABLES Page I Treatment Contrasts 17 II Orthogonal Multipliers for Treatment Contrasts . . . 17 III Enzymatic Browning, o-Diphenol Content, PPO Activity, Fresh Weight, and pH of Redhaven Peaches Receiving Growth Regulator Treatments . . . .21 IV Analysis of Variance of Browning of Redhaven Peaches 27 V Individual Degrees of Freedom for the Effects of Treatments on Browning 27 VI Analysis of Variance of pH of Redhaven Peaches . . . 29 VII Individual Degrees of Freedom for the Effects of Treatments on pH . 29 VIII Analysis of Variance of Fresh Weight of Redhaven Peaches 31 IX Individual Degrees of Freedom for the Effects of Treatments on Fresh Weight .31 X Stepwise Multiple Regression on Browning . . . . . . 33 XI Color Characteristics and Rf Values of Polyphenolic Compounds Extracted from Redhaven Peaches 37 XII Values of Authentic Polyphenolic Compounds. . . . 45 XIII Relative Activity of PPO from Treated and Untreated Redhaven Peaches with Phenolic Compounds at pH 6.3. .56 - v i i -LIST OF FIGURES Figure Page 1 Effect of Ethephori Concentration and Application Date on Fresh Weight of Redhaven Peaches 32 2 Peach Polyphenols Visualized with Folin-Cioucalteau Reagent . 38 3 Peach Polyphenols Visualized with Diazotized p-Nitroaniline Reagent 40 4 Peach Polyphenols Visualized with Vanillin-HCl Reagent 41 5 Peach Polyphenols Visualized with Sodium Molybdate Reagent 42 6 Peach Polyphenols Visualized with Polyphenoloxidase. 43 7 Reactions of Polyphenoloxidase Isozymes with o-Diphenolic Substrates . . . 51 8 Effect of pH on Polyphenoloxidase Activity from Untreated and Treated (46-day treatment) Redhaven Peaches 58 9 Double Reciprocal Plot of Crude Polyphenoloxidase from Untreated Redhaven Peaches . . . . . 60 - v i i i -ACKNOWLEDGEMENTS The author wishes to express his gratitude to Dr. J . Vanderstoep, Research Supervisor, for his help and encouragement during this study, to Dr. S.W. P o r r i t t for assistance i n the f i e l d work, and the Canada Agriculture Research Station, Summerland, B.C., for making available the Redhaven peach trees used i n t h i s study. Special thanks go to Dr. G.W. Eaton for his advice and assistance i n the s t a t i s t i c a l analyses. - 1 -INTRODUCTION The enzymatic browning of peaches is a result of the enzyme catalyzed oxidation of polyphenolic compounds to colored pigments. This reaction occurs when the fruit tissue has been disrupted during handling, packaging, or processing and may result in deleterious changes in color, odor, flavor, and nutritional value. The control of enzymatic browning has been the subject of much research, as the rejection of badly bruised or browned fruit products and the time and labor involved in browning control represent a large cost to the food industry. Traditional methods of controlling enzymatic browning are based on controlling some aspects of its enzyme : oxygen : substrate system. These methods include heat denaturation of the browning enzyme, exclusion of oxygen by vacuum packing, addition of reducing agents such as ascorbic acid and sulfur dioxide, adjustment of pH, and freezing (Ponting, 1960; Mathew andParbia, 1971). The use of plant growth.regulators to control browning has been of more recent interest. In 1969 i t was reported that "Early Amber" peaches sprayed two weeks after f u l l bloom with the growth regulators gibberellic acid (GA) and ethephon (both at 50 ppm) had less enzymatic browning at harvest than untreated fruit (Buchanan et al., 1969). This was found to be due to a decrease in polyphenoloxidase, the browning enzyme, of over 90% (Knapp et al., 1970). Paulson (1973) found that "Redhaven" peaches sprayed with 100 ppm GA four weeks after - 2 -f u l l bloom had decreased enzymatic browning at harvest and attributed this to a decrease i n polyphenolic substrate. GA and ethephon at 50 ppm had no effect and "Fairhaven" peaches receiving the same treatment displayed no change in browning. Porritt (1974) however, found that "Redhaven" peaches receiving 75 ppm ethephon sprays 46 days after f u l l bloom had decreased enzymatic browning at maturity. Italian prunes sprayed four weeks before harvest with GA (Proebsting and M i l l s , 1966) and with ethephon combined with GA (Proebsting and M i l l s , 1969) had lowered internal browning, and applies sprayed with ethephon ten days before harvest had lower levels of polyphenoloxidase and were more resistant to browning on cutting than untreated f r u i t (Sal'kova et a l . , 1977). In view of these varying reports, the present study was undertaken to determine the effect of GA (100 ppm) or ethephon (75 or 150 ppm), applied at either of two application dates, on enzymatic browning of Redhaven peaches. - 3 -LITERATURE REVIEW Enzymatic Browning Reaction The fundamental step in the enzymatic browning of peaches i s the oxidation of ortho-dihydroxyphenolic compounds (o-diphenols) to o-quinones catalyzed by the enzyme polyphenoloxidase (PPO) (Luh and Phithakpol, 1972). The reaction involves two substrates with o-diphenols serving as hydrogen donors and oxygen as the hydrogen acceptor. The order of binding of the substrates isn't known with certainty. Data from different sources have indicated a sequential mechanism with oxygen binding f i r s t , a sequential mechanism with oxygen binding second, or a random mechanism (Rivas and Whitaker, 1973; Lerner and Mayer, 1976). Studies of the mode of action of PPO's have suggested that they possess separate binding sites for oxygen and the phenolic substrate (Walker and Wilson, 1975). The o-quinones produced by the oxidation reaction are themselves colored red to reddish-brown, but they are highly reactive and take part i n non-enzymatic secondary reactions leading to the formation of more intensely colored secondary products. Such secondary reactions include 1) coupled oxidations of compounds that aren't PPO substrates or are oxidized with d i f f i c u l t y , 2) complexing with amino acids and proteins, and 3) condensation and polymerization with polyphenols to higher molecular weight more intensely colored compounds (Mathew and Parbia, 1971). In most food products the intense color of enzymatic browning occurs only after such complexing. PPO undergoes - 4 -"reaction inactivation" due to the formation of a covalent linkage between the enzyme molecule and a quinone at or near the active site (Whitaker, 1972). It i s assumed that browning doesn't take place i n the intact c e l l because PPO and the polyphenolic substrates are spacially separated (Ponting, 1960). There has been l i t t l e concrete evidence demonstrating the sub-cellular location of PPO and phenolic compounds however (Anderson, 1968). After the ce l l s are damaged such as by impact or cutting, the enzyme and substrate are free to mix and in the presence of oxygen the browning reaction can proceed. The function of the browning complex i s uncertain. It has been suggested that PPO functions as a terminal oxidase i n respiration (Boswell, 1963) but i t has been found to compete poorly with the respiratory chain at low pa r t i a l pressures of oxygen (Anderson, 1968). It has also been implicated in disease resistance of plants. Oxidized polyphenols are more potent anti-fungal agents than the unoxidized precursors (Walker, 1975). The rate of browning i n various foods has been related to PPO level, substrate level, and a combination of both factors (Kahn, 1975), but browning of peaches was most closely related to levels of polyphenols (Guadagni et a l . , 1949; Nakabayashi et a l . , 1963). Polyphenoloxidase Polyphenoloxidase (o-diphenol: oxygen oxidoreductase EC also known as catechol oxidase, phenolase, diphenolase) contains copper - 5 -as i t s active prosthetic group. Lanzarini et a l . (1972) demonstrated that the active enzymatic form i s associated with Cu + ions, with ++ + very l i t t l e in the Cu form. They suggested that a Cu -C^ interaction would activate molecular oxygen and the reaction would occur according to the scheme of Mason (1957). It also appears to involve an active site with a high a f f i n i t y for the aromatic ring and a basic group which promotes phenol to phenolate ionization (Bright et a l . , 1963; Duckworth and Coleman, 1970). It i s assumed, based on studies of the effects of inhibitors and ring substitution on reaction velocity that oxidation occurs via an electrophilic attack (Lanzarini et a l . , 1972). PPO's from different sources usually have differences in such properties as substrate s p e c i f i c i t i e s , pH optima, and reaction kinetics. They also usually exist as isozymes which can be separated by electrophoretic and chromatographic means. Peach PPO has been shown to oxidize o-diphenols almost exclusively, with negligible activity with monophenols (Luh and Phithakpol, 1972; Reyes and Luh, 1960). Slight activity with the p-diphenol quinol has been reported and varies with maturity (Reyes and Luh, 1960; Harel et a l . , 1970). PPO from some sources has the a b i l i t y to catalyze both the hydroxylation of monophenols to o-diphenols and their subsequent oxidation to o-quinones (Mason, 1957). Constantinides and Bedford (1967) resolved mushroom PPO into nine isozymes, three of which had a c t i v i t y with monophenols. Taneja and Sarkar (1974) reported that the monophenolase and diphenolase a c t i v i t i e s of wheat were separable and reside in different enzymes. - 6 -The pH optimum of peach PPO ranges from 5.9 to 6.3 depending on the type of buffer used (Luh and Phithakpol, 1972; Reyes and Luh, 1960). Jen and Kahler (1974) found that as "Redhaven" peaches matured the PPO pH optimum changed from a single optimum at pH 6.2 to double optima at pH 6.0 and 6.5, suggesting the synthesis of new isozymes with maturity. Wong et a l . (1971a) separated PPO from "Cortez" peaches into four isozymes which differed from each other in pH optima, substrate spec i f i c i t y , rate constants, and susceptability to inhibitors and heat. Harel and Mayer (1970) electrophoretically separated PPO from "Salvey" peaches into five bands, one of which was active with quinol, a characteristic of the enzyme laccase, not normally found i n peaches. The sub-cellular location of PPO i s poorly understood. Sections of mature peach f r u i t stained for PPO with catechol showed the enzyme to be present in localized patches of parenchyma ce l l s (Reeve, 1959). Harel et a l . (1970) found peach PPO to be present i n both the particulate and soluble form, which were present in differing amounts at different -stages of maturity. The insoluble form of apple PPO has been found to be associated with both chloroplasts and mitochondria (Harel et a l . , 1965). Such particulate PPO can often be solubilized by treatment with various detergents, which i s often accompanied by PPO activation (Sato and Hasegawa, 1976). Soluble PPO sometimes also exists in a latent form that can be activated by storage, temperature change, detergent treatment, or denaturing agents, contributing additional PPO activity (Kahn, 1977). - 7 -Due to the complexity of PPO i t i s d i f f i c u l t to ascribe any single role in cellular metabolism. Conn (1964) suggested a role for PPO in biosynthesis of phenolic compounds, but any explanation of function must take into account the demonstrated multiplicity of forms. Browning Substrates The ortho-dihydroxyphenolic configuration i s essential for PPO activity, but not a l l compounds with such a configuration are oxidized, and those that are exhibit different rates of oxidation. Substituents on the benzene ring have been shown to influence reaction rate depending on position and electron donating or attracting character (Lanzarini et a l . , 1972). Natural browning substrates found in food are usually cinnamic acid derivatives which arise from the shikimic acid pathway, and flavonoid compounds of which the "A" ring i s derived from the acetate-malonate pathway and the "B" ring from cinnamic acid derivatives (Hess, 1975). Of the cinnamic acid derivatives the most important in enzymatic browning i s chlorogenic acid (3-caffeoylquinic acid) a 3-depside of quinic acid with caffeic acid. Chlorogenic acid content has been related to browning of several varieties of applies (Walker, 1962). Flavonoids involved i n browning include catechins, leucoanthocyanidins, anthocyanins, and flavonols. Anthocyanins are not primarily significant - 8 -as PPO substrates but have been shown to be involved in secondary reactions (Mathew and Parbia, 1971). The main polyphenols involved in enzymatic browning of peaches were found to be leucoanthocyanidins, chlorogenic acid isomers, and catechins (Craft, 1961; Fel'dman and Kostinskaya, 1970; Luh et a l . , 1967). Craft (1961) observed no qualitative change in polyphenolic pattern with ripening and the relative proportions remained constant. Phenolic compounds were seen to increase in concentration during the early stages of f r u i t growth, reach a maximum at pit-hardening, and then slowly decline u n t i l harvest (Craft, 1961; Harel et a l . , 1970). The amount of phenols on a whole f r u i t basis was seen to increase however (Craft, 1961). L i et a l . (1972) found that both flavor and color of peaches were negatively correlated with total phenols. Astringency of f r u i t s has been associated with polyphenolic concentration, particularly catechins and leucoanthocyanidins (Goldstein and Swain, 1963; Craft, 1961). Peaches show a decrease in astringency during ripening (Craft, 1961), but the reasons for this are unknown. The functions of plant phenols are obscure perhaps because they are secondary products and play no role i n metabolism. Anthocyanins, flavones, and flavonols, due to their coloration, probably play a role in attracting insects (Salisbury and Ross, 1969). Hydroxylated cinnamic acids are believed to be important precursors of lignin (Van Buren, 1970). In peach, the highest total phenolic concentration i s at pit-hardening. Phenolic compounds have also been suggested to - 9 -control auxin concentrations in some plants through their effects on the enzyme indole acetic acid (IAA) oxidase. Monophenols have been shown to enhance IAA oxidase activity while o-diphenols inhibit the enzyme (Nitsch, 1970). Gibberellic Acid and Ethephon Gibberellic acid (abbreviated GAorGA^), best known for i t s stimulating effects on plant growth, i s one of many different structural variations of the plant growth hormones known as gibberellins. As well as stimulating growth, gibberellins have been found to have regulatory effects on plant development. Plants have been found to have selectivity of response to the different forms. The term "gibberellin" i s often used rather loosely in the literature as being synonymous with gibberellic acid (Stuart and Cathey, 1961). Recent reviews of the gibberellins have been made by Lang (1970) and Jones (1973). Ethephon (2-chloroethylphosphonic acid) breaks down in the plant releasing the plant hormone ethylene (Yang, 1969). Ethylene i s best known for i t s effect i n triggering ripening of climacteric f r u i t but has also been found to be important in regulation of plant development. The physiology of ethylene has recently been reviewed by Abeles (1972). GA and ethylene often have similar effects (e.g. breaking of seed dormancy) as well as opposing effects (e.g. ethylene promotes but GA delays ripening and senescence in many types of fruit) (Leopold and Kriedmann, 1975). - 10 -Peach f r u i t growth Is characterized by two stages of rapid growth (Stages I and III) separated by a period of slow growth (Stage II) during which l i g n i f i c a t i o n of the stone occurs (pit-hardening) . Both fresh weight and dry weight growth follow similar double-sigmoid growth patterns (Chalmers and van den Ende, 1975). Growing f r u i t act as physiological sinks, attracting nutrients at the expense of the remainder of the tree. Both GA and ethylene are thought to be involved i n this effect (Nitsch, 1970; Chalmers, et a l . , 1976). Jackson (1968) found gibberellin in the seeds of peaches immediately after f u l l bloom, and later in the mesocarp and endocarp. Gibberellin was found to be closely associated with the rate of c e l l expansion i n each tissue but not with c e l l division. Ogawa (1965) reported that gibberellins i n peach seeds began to increase 35 days post-bloom, reached their maximum amount by 50 days post-bloom, and decreased rapidly thereafter. Looney et a l . (1974) found an association between ethylene level and growth rate i n Stage I of peach growth. Applied GA and ethephon have varying effects on peach growth. GA has been shown to induce parthenocarpy (seedlessness), stimulate vegetative growth (Jerie and Taylor, 1971), achieve a thinning effect at flowering (Edgerton, 1966), and delay maturation and ripening (Leopold and Kriedmann, 1975; Paulson, 1973). Rom and Scott (1971) found that ethephon applied during the f i n a l swell of peach growth accellerated maturation. Byers and Emerson (1973) found that ethephon applied during Stage II of peach growth Induced early onset of Stage III. Looney et a l . (1974), however, found no effect of ethephon application - l i -on length of Stage II. Other ethephon induced effects include f r u i t thinning and more uniform maturation (Stembridge and Raff, 1973; Rom and Scott, 1971). The modes of action of GA and ethephon are unknown. Both have been seen to regulate enzyme formation, possibly by control of RNA directed protein synthesis, and both have been linked to alterations in cellular membranes (Leopold and Kriedmann, 1975). The manner in which a single early season application of a growth regulator can induce a response months later i s presently unknown. Byers et a l . (1969) attributed the belated effects of ethephon to a higher rate of endogenous ethylene production, rather than ethephon degradation. Stembridge and Raff (1973) suggested that ethephon induces a lingering increase in ethylene level in immature peach f r u i t which persists u n t i l the f r u i t develop sufficiently to respond. Lavee and Martin (1974) however, showed that penetration of ethephon through peach exocarp into the mesocarp was very low; most of the ethephon accumulated on the exocarp and most of this could be removed by washing. They later found, however, that ethephon binds rapidly to sugars i n the peach mesocarp and suggested that the formation of stable sugar-ethephon complexes may be involved in long-term responses, rather than release of ethylene at the f r u i t surface (Lavee and Martin, 1975). This i s similar to the binding of GA as glycosides, which may be a storage form of this hormone (Leopold and Kriedmann, 1975). - 12 -EXPERIMENTAL ' Application of Growth Regulators Nine-year old "Redhaven" peach trees (Prunus persica) on Prunus tomentosa seedling roots were used in this study. Twenty-eight trees at the Canada Agriculture Research Station at Summerland, B.C. were randomly divided into seven groups of four trees each in the spring of 1975. Full bloom date was May 13, 1975. On June 3, twenty-one days after f u l l bloom, gibberellic acid (as activol GA, Chipman Chemicals) at 100 ppm, and ethephon (as Ethrel, Anchem Products) at 75 ppm and 15 ppm were applied by hand sprayer to runoff (2.5 - 3 liters per tree) to three of the seven groups. 0.1% Rhodes R-ll spreader-activator was used as a wetting agent. Hand thinning of a l l trees to a desirable crop load was accomplished on June 24. On June 29, forty-six days after f u l l bloom, the treatments were repeated on three of the remaining groups. The seventh group of trees served as the control. Three picks were made as the fruit attained commercial picking maturity; Aug. 16, 22 and 28. Fruits from the second pick (Aug.22) were used for a l l analyses, and tree identity was maintained throughout. Fifteen fruits from each tree were weighed and the average weight per fruit recorded. - 13 -The f r u i t s were placed in cold storage (0°C) immediately after picking. Five peaches from each tree were removed from cold storage and allowed to ripen at 21°C for determination of enzymatic browning and pH. The remainder of the fruits were transported to Vancouver, ripened at 21°C, halved, pitted, vacuum packaged with a nitrogen backflush in aluminum f o i l pouches, then placed in frozen storage (-35°C) u n t i l used. Enzymatic Browning One-third sectors of five ripe peaches from each tree were pureed for 20 seconds at half speed in a Waring blendor at 21°C. The degree of browning was measured with a Hunterlab D-25 Color Difference Meter as the difference in Rd (lightness) between readings taken at one minute and thirty minutes after blending. A yellow t i l e (Rd =68.7, a =-3.8, b = 25.2) was used for standardizing the instrument. The pH's of the purees were measured by a Fisher glass-electrode pH meter. Polyphenoloxidase Extraction and Assay The methods used were modifications of those of Wong et al.(1971a). Unless otherwise indicated a l l extraction procedures were carried out at 4°C. Three frozen peach halves per tree were allowed to thaw at 4°C for 4 hours before extraction. Twenty-five grams from each of the 3 halves were blended with 150 ml cold acetone (-35°C) for 25 seconds at half speed plus an additional 5 seconds at f u l l speed in a stainless steel Sorvall Omnimixer jar. The resulting slurry was allowed to stand - 14 -for 5 minutes in an ice bath and then suction f i l t e r e d through Whatman no. 4 f i l t e r paper. The f i l t e r cake was washed with 500 ml cold acetone (-35°C) to remove pigments. The f i l t e r cake was suspended in 75 ml of 0.1 M Na phosphate buffer (pH 6.3) and shaken for 4 hr on a rotary shaker at 4°C, then centrifuged at 33,000 x G for 15 min at 0°C in a Sorvall RC-2 refrigerated centrifuge. The supernatant was collected as the crude enzyme solution for PPO assay and the pellet was discarded. The spectrophotometric method of Wong et a l . (1971a) was used for assay of PPO a c t i v i t i e s . The standard reaction mixture consisted of 0.1 ml crude PPO plus 2.9 ml 0.01 M catechol i n 0.1 M Na phosphate buffer (pH 6.3). Preliminary investigation had found this to be the optimum pH of "Redhaven" peach PPO i n Na phosphate buffer. To insure that oxygen was not limiting, the catechol solution was aerated by bubbling oxygen through i t for 5 min. Reaction temperature was maintained at 25°C. The enzymatic formation of benzoquinone was followed at 420 nm (Ponting and Joslyn, 1948) with a Unicam SP-800 spectrophotometer with expanded scale recorder and a temperature controlled cuvette holder. One cm cuvettes were used. The i n i t i a l increase in absorbance was used as a measure of PPO activity, and was recorded as A Abs^QO. 1ml PPO * min The reaction was performed in duplicate with PPO from each tree and the results averaged. Extraction and Assay of o-Diphenolic Compounds Twenty-five grams from each of 3 frozen peach halves per tree were macerated in 300 ml boiling 80% ethanol for 3 minutes at half speed - 15 -in an Osterizer blender under a nitrogen atmosphere. The slurry was suction f i l t e r e d through Whatman no. 1 f i l t e r paper using "Hyflo Supercel" (Fisher) as a f i l t e r aid. The f i l t e r cake was washed with 2 aliquots of 300 ml boiling 80% ethanol then discarded. The f i l t r a t e was allowed to cool to room temperature, made up to 1 l i t e r with 80% ethanol, then f i l t e r e d through Whatman no. 4 f i l t e r paper to remove haze formed during cooling. o-Dihydroxy and vicinal-trihydroxy phenolic compounds in the ethanol extract were determined by a modification of the method of Mapson et a l . (1963). This method measures the yellow color resulting from a complex formed between 2 polyphenol molecules and one molybdate ion (Haight and Paragamian, 1960). One ml of 5% Na molybdate i n 0.1% Na phosphate buffer (pH 7.0) was mixed with 3 ml of the same buffer. To this was added 1 ml of the ethanolic extract followed by immediate mixing. After 45 min at room temperature Abs^y^ was measured by a Beckman DB spectrophotometer using a blank consisting of 4.0 ml phosphate buffer plus 1 ml of the ethanolic extract. Preliminary experimentation had determined 375 nm to be the optimum wavelength for measuring this reaction. The reaction was performed in duplicate and the values averaged. The concentration of o-dihydroxy and vicinal-trihydroxy phenolics was determined from a standard curve prepared using 0.02-0.2 mg catechol/ml. The results were expressed as mg catechol/g peach tissue. S t a t i s t i c a l Methods Data were analyzed by analysis of variance. To obtain information regarding specific treatment effects, the treatment sums of squares - 16 -and degrees of freedom were partitioned using the individual degree of freedom technique (Li, 1964a). The six treatments and the control were arranged as six treatment contrasts (Table I) and an orthogonal set multipliers obtained (Table II). The f a c i l i t i e s of the U.B.C. Computing Center Triangular Regression Package (Le and Tenisci, 1977) were used for multiple regression analyses. Thin Layer Chromatography of Peach Phenolic Compounds Qualitative analyses of peach phenolic compounds were accomplished by 2-dimentional thin layer chromatography (TLC). Peaches from one replicate of each treatment and control were used. To improve the po s s i b i l i t y of detecting treatment effects, the replicate from each treatment was chosen on the basis of low browning and/or low o-diphenol content, and the untreated replicate was chosen on the basis of high browning and high o-diphenol content. The peaches used were from the second pick (Aug. 22, 1975) as were those analyzed for browning. Twenty grams from each of 5 frozen peach halves were blended with 300 ml boiling 95% ethanol for 3 min under a nitrogen atmosphere in an Osterizer blender. The slurry was suction f i l t e r e d through Whatman no. 54 f i l t e r paper using "Hyflo Supercel" as a f i l t e r aid. The f i l t e r cake was washed with an additional 300 ml boiling 95% ethanol. The yellow f i l t r a t e was evaporated on a flash evaporator at 32° - 35° C u n t i l an aqueous solution remained. This was extracted twice with an equal volume of hexane to remove carotenoids (Schaller and von Elbe, 1970), saturated with NaCl, then f i l t e r e d through Whatman no. 4 f i l t e r - 17 -TABLE I TREATMENT CONTRASTS Symbol Definition a. C/Tr b. E/L c. GA/Eth d. Lo/Hi e. B x C f. B x D Control vs. Treated Early Treatment (21 days) vs. Late Treatment (46 days) Treatment with GA vs. Treatment with Ethephon Low Ethephon Treatment (75 ppm) vs. High Ethephon Treatment (150 ppm) E/L x GA/Eth E/L x Lo/Hi TABLE II ORTHOGONAL MULTIPLIERS FOR TREATMENT CONTRASTS Treatment Application Treatment Contrast Time (days) C/Tr E/L GA/Eth Lo/Hi BxC BxD Control — Ethephon (75ppm) 21 Ethephon (150ppm) 21 Gibberellic Acid 21 Ethephon (75ppm) 46 Ethephon (150ppm) 46 Gibberellic Acid 46 -6 1 1 1 1 1 1 0 -1 -1 -1 1 1 1 0 1 1 -2 1 1 -2 0 -1 1 0 -1 1 0 0 -1 -1 2 1 1 -2 0 1 -1 0 -1 1 0 - 18 -paper. The filtrate was extracted three times with equal volumes of ethyl acetate, then dried by stirring over anhydrous sodium sulfate. The ethyl acetate extract was concentrated to 3 - 4 ml by vacuum evaporation at 32°C, centrifuged at 1000 x G for 1 min to remove insoluble material, then stored in the dark under a nitrogen atmosphere at 4°C until used. Qualitative separation of peach phenolic compounds was carried out by ascending two-dimentional TLC on 20cm x 20cm plastic plates of 0.1-mm thick MN300 cellulose (M. Nagel and Co.). Three microliters of the concentrated phenol extract wereapplied 2 cm from the lower left corner of 5 thin layer plates. The chromatography tanks (27cm x 27cm x 7cm i.d.) were allowed to equilibrate with the developing solvent before each run. Development took place at room temperature. The chromatograms were developed in the first direction with butanol: acetic acid:water 12:3:5 v/v/v (BAW 12:3:5) until the solvent front was 1 cm from the top of the plates. The plates were removed from the tanks, air dried, then developed in the second direction with 5% acetic acid (5% HOAc) until the solvent front was 1 cm from the top of the plate. The air dried chromatograms were observed under ultra-violet light before and after fuming with cone, ammonia. Phenolic compounds were visualized on one chromatogram with a spray of Folin-Ciocalteau reagent (diluted 3 times with water) followed by a spray of 10% aq. sodium carbonate (Krebs et al., 1969). The phenolic compounds were detected as blue spots on a light blue background. The intensity of the spot was proportional to the concentration of the phenol. - 19 -A second chromatogram was sprayed with diazotized p-nitroaniline (DPNA) reagent, prepared by mixing in an ice bath 0.5% p-nitroaniline In 2N HCI, 5% NaN02> and 20% sodium acetate (w/v) i n a ratio of 1:10:30 (v:v:v) (Luh et a l . , 1967). This reagent gives characteristic colors with phenolics. A third chromatogram was sprayed with freshly prepared v a n i l l i n reagent made by mixing a 10% ethanolic solution of v a n i l l i n with an equal volume of cone. HCI. This reagent gives pink to orange colors with leucoanthocyanidins and catechins (Swain and H i l l i s , 1959). A fourth chromatogram was sprayed with a freshly prepared solution of 5% Na molybdate i n 0.1 M Na phosphate buffer (pH 7.0). Phenolic compounds containing an o-dihydroxy or vicinal-trihydroxy configuration form yellow complexes with this reagent (Haight and Paragamian, 1960) and were visualized as yellow spots on the chromatogram. The f i f t h chromatogram was sprayed with a crude preparation of PPO from control f r u i t (see Electrophoresis) and incubated in a humid chamber for 2 hours. PPO substrates showed up as yellow to brown spots (Siegelman, 1955). values of a l l spots were calculated and recorded. Authentic samples of chlorogenic acid, caffeic acid, and 1-epicatechin ( a l l 5 mg/ml ethanol) were also chromatographed as described above and R^  values were determined. Electrophoresis Polyacrylamide gel electrophoresis of peach PPO preparation was - 20 -performed according to Davis (1964). Peach PPO has been shown to exist as isozymes separable by electrophoresis (Wong et a l . , 1971; Harel and Mayer, 1970). To determine the effect of growth regulator treatment on PPO isozymes, one tree from each treatment was selected on the basis of low browning and/or low PPO activity for electrophoresis, (Table III). The control replicate was chosen on the basis of high browning and high PPO activity. For preparation of PPO for electrophoresis, 10 g of frozen peach tissue from each of 5 peach halves were blended with 100 ml cold acetone (-35°C) for 3 bursts of 10 seconds each at high speed in an Osterizer blender and allowed to stand at 4°C for 5 min. The slurry was suction f i l t e r e d through Whatman no. 4 f i l t e r paper and then washed with 500 ml cold acetone (-35°C). The f i l t e r cake was suspended in 100 ml of 0.1 M Na phosphate buffer (pH 7.0) by s t i r r i n g for 1.5 hr at 4°C. The suspension was centrifuged at 33,000 x G for 15 min in a Sorvall RC2 refrigerated centrifuge at 0°C. The supernatant (crude PPO preparation) was collected and the pellet discarded. To precipitate pectins, 0.5 M CaC^ was added to the supernatant to a f i n a l concentration of 0.05 M. The solution was adjusted to pH 6.8 by addition of 0.1 M NaOH and the precipitate was removed by centrifugation at 15,000 x G and 0°C for 10 min in a Sorvall RC-2 centrifuge. Twenty ml of the supernatant was diluted to 50 ml with 0.1 M Na phosphate buffer (pH 6.8) and then concentrated to 6.5 ml by u l t r a f i l t r a t i o n (Diaflo PM-10 membrane, Amicon Corp.) under a pressure of 60 p . s . i . of nitrogen at 4°C. Protein concentration was determined - 21 -by the method of Lowry et a l . (1951) as modified by Potty (1969) using crystalline bovine serum albumin as standard. This modification allows for the estimation of protein in the presence of phenolic compounds. For electrophoresis, 150-200 u l of enzyme solution (containing approx. 65 ug of protein) was mixed with 1 drop of 40% sucrose and 1 drop of 0.001% bromophenol blue tracking dye and applied to the top of 5 geltubes containing 7% acrylamide running gel (5.0 cm long) and 1.25% stacking gel (1.0 cm long). A l l gels were run i n a Pharmacia Model GE-4 electrophoresis apparatus. The starting pH was 8.3 and the running pH was 9.5. A current of 3.5 mA per tube was employed u n t i l the tracking dye migrated close to the end of the tube. The electro-phoresis chamber was kept cool with circulating cold tap water. The position of the tracking dye was marked on the gel with a needle containing India ink. One gel was stained for protein by immersion for 1.5 hr in 0.25% Coomassie blue dissolved i n a mixture of 7% methanol and 5% acetic acid. The gel was destained by soaking i n several changes of 7% methanol-5% acetic acid solution u n t i l a clear background was obtained. The position and intensity of the bands were recorded visually and then by densitometry using a Gelman Gelscan densitometer. Four other gels were soaked for 30 min in a solution of 0.1% m-phenylenediamine (MPD) i n 0.1 M Na phosphate buffer (pH 6.3) and then placed i n a solution of catechol (0.03 M), pyrogallol (0.03 M), caffeic acid (0.03 M), or chlorogenic acid (0.01 M) a l l in Na phosphate buffer - 22 -(pH 6.3). The gels were vigorously aerated by bubbling oxygen through the solutions for 5 min. MPD is a coupling agent which reacts with the quinones produced at the site of substrate oxidation by PPO (van Loon, 1971; Harel et a l . , 1965). The color of the bands varied depending on the substrate used. Band development was complete in 1.5 hours and their position and intensity recorded. values of the bands of a l l gels were calculated as distance of band migration  distance of tracking dye migration and averaged. PPO Substrate Specificity Several compounds with polyphenolic or monophenolic configurations were used for this study. 2.8 ml of catechol (lOmM), pyrogallol (lOmM), p-cresol (lOmM), quinol (lOmM), or chlorogenic acid (4.5mM) in 0.1M Na phosphate buffer (pH 6.3) was aerated and then rapidly mixed with 0.2 ml of a crude PPO preparation (see Electrophoresis). The temperature of the substrate solution was 25 C. The reaction was followed at 420 nm with a Beckman DB spectrophotometer with Photovolt recorder and the data for each substrate, with each PPO preparation,was recorded as activity relative to activity with catechol. PPO pH Optima Activity of the crude PPO preparations relative to pH was determined - 23 -as above by rapidly mixing 0.2 ml of crude PPO preparation with 2.8 ml of 10 mM catechol in 0.1 M citrate-0.2 M Na phosphate buffer over a pH range of 4.0 to 7.4. Activity at each pH was plotted as per cent of maximum activity attained. Effect of Substrate Concentration 0.2 ml of crude PPO preparation from control peaches (see Electrophoresis) was rapidly mixed with 2.8 ml of catechol in 0.1 M Na phosphate buffer (pH 6.3) to f i n a l concentrations in the reaction mixture ranging from 9.3 mM to 28 mM, and PPO activity determined as above. The data were plotted as 1/substrate concentration (M) versus 1 / i n i t i a l velocity (v ). _ 24 -RESULTS AND DISCUSSION Enzymatic Browning Treated f r u i t had less browning at harvest than untreated f r u i t (Tables IV and V). Fruit treated 46 days after bloom had less browning than f r u i t treated 21 days after bloom (P ^  .01) regardless of the type of treatment. Nakabayashi et a l . (1963) correlated browning of peaches with polyphenol content, Grice et a l . (1952) showed that the rate of browning of frozen peaches was influenced by both polyphenol and PPO contents of the f r u i t , while Guadagni et a l . (1949) found i n i t i a l browning tendency of peaches to be governed by original PPO activity but total amount of browning depended on polyphenol content. The failure of "Early Amber" peaches to undergo enzymatic browning after early season applications of GA and ethephon was due to a reduction in PPO activity by over 90% in the treated f r u i t , with .slight reductions i n o-diphenol content (Knapp et a l . , 1970). Paulson (1973) attributed the reduction in browning of "Redhaven" peaches after a post-bloom application of GA to a reduction in available substrate. Sal'kova et a l . (1977) found that apples treated with ethephon had lower levels of PPO, peroxidase (PRO), and ascorbic acid oxidase, and were more resistant to browning on cutting. GA applied to West Indian cherries was seen to cause a marked reduction in PPO and ascorbic acid oxidase a c t i v i t i e s (Srinivasan et a l . , 1973). TABLE III ENZYMATIC BROWNING, O-DIPHENOL CONTENT, PPO ACTIVITY, FRESH WEIGHT, AND pH OF "REDHAVEN" PEACHES RECEIVING GROWTH REGULATOR TREATMENTS Application Tree Browning o-diphenols PPO Activity Fresh Weight Treatment Time(days) No. ARd(29min) mg catechol/g tissue AAbs^g 0.1 m l - l min~l g/fruit pH Control - 1 36.6 .1.027 0.163 170.3 3.70 2 33.6 1.253 0.160 • 148.4 3.80 3 32.2 1.067 0.163 181.6 3.75 4«b 36.2 1.840 0.183 177.6 3.70 Ethephon(75ppm) 21 1 33.6 1.507 0.135 164.6 3.85 2 b 32.1 1.053 0.280 201.7 3.70 3 a 31.0 1.347 0.145 191.4 3.80 4 35.9 1.693 0.190 177.7 3.80 Ethephon(150ppm) 21 1 34.4 1.013 0.170 178.0 3.85 2 33.7 1.600 0.145 150.3 3.80 3 a 34.4 1.000 0.150 151.3 3.85 4b 32.8 0.840 0.230 154.2 3.80 Gibberellic Acid 21 1 30.9 1.027 0.170 185.5 3.85 (100 ppm) 2 34.5 1.507 0.178 189.2 3.75 • 3 b 32.6 0.987 0.175 198.5 3.85 4 a 35.3 1.533 0.155 162.1 3.60 Continued TABLE III (Continued) Application Tree Browning o-diphenols PPO Activity Fresh Weight Treatment Time(days) No. ARd(29min) mg catechol/g tissue A A D S 4 2 0 0 * 1  Tal~ 1 min - 1 g/fruit pH Ethephon(75ppm) 46 1 31.6 1.640 0.165 141.9 3. 75 2ab 21.4 0.653 0.148 193.2 3. 85 3 27.4 0.747 0.125 158.9 3. 90 4 29.3 1.280 0.170 123.2 3. 75 Ethephon(150ppm) 46 1 28.9 1.333 0.188 168.0 3. 85 2ab 22.1 0.840 0.138 232.4 3. 80 3 26.8 0.880 0.135 208.3 3. 85 4 29.4 1.667 0.133 185.2 3. 75 Gibberellic Acid 46 1 32.9 1.280 0.200 147.6 3. 80 (100 ppm) 33.4 1.467 0.140 133.5 3. 65 3b 26.3 1.080 0.170 174.6 3. 75 4 36.2 1.600 0.255 160.2 3. 70 aTrees chosen for PPO isozyme electrophoresis Trees chosen for TLC TABLE IV ANALYSIS OF VARIANCE OF BROWNING OF REDHAVEN PEACHES Source of Variation Degrees of Freedom Mean Square F-ratio Treatment 6 40.30 ** Error 21 8.74 Total 27 ** Denotes significance at P<.01 TABLE V INDIVIDUAL DEGREES OF FREEDOM FOR THE EFFECTS OF TREATMENTS ON BROWNING Contrast '. Degrees of Freedom Q 2 F-ratio C/Tr 1 • 42.70 * E/L 1 128.34 ** GA/Eth 1 32.34 n.s. Lo/Hi 1 0.00 n.s. B x C 1 36.75 n.s. B x D 1 1.69 n.s. Treatment 6 241.82 Denotes significance at P<.01 * Denotes significance at P^.05 Note: Q 2 = (MiT! + M 2T 2 + . . . + M kT k) 2 = (X MT) 2 n(Mi 2+ M 2 2 . . . + Mfc2) n l M 2 where M = Multipliers (from Table II) Q 2 = An independent component of the treatment sum of squares T = Treatment total n = Number of observations per treatment - 28 -Analysis of variance of PPO and o-diphenol data from the present study revealed non-significant F-tests (P ^ .05). A significant F-test i s not a pre-requisite for the partitioning of the treatment degrees of freedom (Li, 1964a) and the latter may show significance where the former does not. However, analysis of the individual degrees of freedom contrasts revealed no significant treatment effect. PPO activity i s pH dependent and control of browning by decreasing pH i s well known (Eskin et a l . , 1971). Overall treatment effects on pH were not significant" (P y .05) (Table VI). According to the individual degree of freedom contrasts, f r u i t treated with ethephon had higher pH values than f r u i t treated with GA (P < .05) (Table VII). Knapp et a l . (1970) reported that neither GA nor ethephon treatment effected pH of "Early Amber" peaches. Ce l l expansion of peaches (fresh weight growth) (Chalmers and van den Ende, 1975) has been well correlated with GA levels in the mesocarp (Jackson, 1968). Ethephon applied at stage I and II of peach growth has been known to result in increased f r u i t weight, presumably as a result of a thinning effect (Stembridge and Raff, 1973; Paulson, 1973). GA has also been evaluated for peach thinning (Corgan and Widmoyer, 1971; Edgerton, 1966). Chalmers et a l . (1976) reported that exogenously applied GA and ethephon increased sink strength in developing peach f r u i t s . Increased c e l l expansion could dilute the • c e l l constituents involved in browning. Growth regulator treatment affected f r u i t weight (P < .05) (Table VIII). 150 ppm ethephon - 29 -TABLE VI ANALYSIS OF VARIANCE OF pH OF REDHAVEN PEACHES Source of Variation Degrees of Freedom Mean Square F-ratio Treatment 6 0.0062 n.s. Error 21 0.0047 Total 27 n.s. Not significant at P<.05 TABLE VII INDIVIDUAL DEGREES OF FREEDOM FOR THE EFFECTS OF TREATMENTS ON pH Contrast Degrees of Freedom Q 2 F-ratio C/Tr 1 8.57 x 10-3 n.s. E/L 1 4.17 x IO"* n.s. GA/Eth 1 2.30 x IO - 2 * Lo/Hi 1 1.41 x IO - 3 n.s. B x C 1 2.56 x IO - 3 n.s. B x D 1 1.41 x IO - 3 n.s. Treatment 6 3.74 x 10-2 Denotes significance at P<.05 - 30 -treatment appeared to retard f r u i t growth i f applied 21 days after bloom but enhanced f r u i t growth i f applied 46 days after bloom (Table III and IX; Fig. 1). The reverse appeared to be true for the 75 ppm ethephon applications. The reasons for the differing effects on weight with ethephon concentration and application date are not clear. Cell division in peach f r u i t continues for about 30 days after pollination, after which growth i s mostly due to c e l l enlargement (Nitsch, 1970; Jackson, 1968). The 21-day treatments were probably applied during the period of c e l l division, while the 46-day treatments were applied during the period of c e l l enlargement prior to the onset of stage II of "Redhaven" peach f r u i t growth (Looney, 1972). Multiple Regression techniques (Le and Tenisci, 1977) were employed to identify the important experimental factors and measurements contributing to browning. Stepwise multiple regression of browning on o-diphenols, PPO, f r u i t weight, and pH showed that only o-diphenol content was a significant predictor of browning (Table X, column 1). 2 The coefficient of multiple determination (R ) of 0.32 indicates that there i s appreciable variation i n browning after pH, PPO, fr u i t weight, and o-diphenol data have been considered. This i s similar to the results of Gajzago et a l . (1976) who found that 30% of the variation in browning of apricots was explained by o-diphenol content, and 35% by a combined PPO and o-diphenol effect. Qualitative variables can be analyzed by multiple regression through the use of dummy variable or contrast coding (Li, 1964b; Gujarati, 1970; Cohen, 1968). Dummy variables take account of the - 31 -TABLE VIII ANALYSIS OF VARIANCE OF FRESH WEIGHT OF REDHAVEN PEACHES Source of Variation Degrees of Freedom Mean Square F-ratio Treatment 6 1206 * Error 21 410 Total 27 Denotes significance at P <.05 TABLE IX INDIVIDUAL DEGREES OF FREEDOM FOR THE EFFECTS OF TREATMENTS ON FRESH WEIGHT Contrast Degrees of Freedom Q 2 F-ratio C/Tr 1 24.69 n.s. E/L 1 250.26 n.s. GA/Eth 1 126.43 n.s. Lo/Hi 1 352.50 n.s. B x C 1 1641.51 n.s. B x D 1 4840.68 ** Treatment 6 7236.07 Denotes significance at P<.01 - 32 -Figure 1. Effect of Ethephon Concentration and Application Date on Fresh Weight of Redhaven Peaches O Fresh weight means of f r u i t treated with ethephon 21 days after bloom • Fresh weight means of f r u i t treated with ethephon 46 days after bloom - 33 -T A B L E X S T E P W I S E M U L T I P L E R E G R E S S I O N O N B R O W N I N G Independent Regression coefficient Variable 1 2 3 4 5 C/Tr - -0.504* -0.447* -0.439* -0.452** E/L - -2.313** -2.146** -2.364** -2.205** GA/Eth - n.s. n.s. n.s. n.s. Lo/Hi - n.s. n.s. n.s. n.s. B x C - n.s. n.s. n.s. n.s. B x D - n.s. n.s. n.s. n.s. o-Diphenol 6.965** - 6.243** 4.791** 4.623** PPO n.s. - n.s. - 24.879* Weight n.s. - - -0.056** -0.055** pH n.s. - - n.s. Constant 22.978 31.625 23.874 35.231 31.105 t S y 3.319 3.189 2.449 2.103 1.915 ttR2 0.326 0.402 0.662 0.761 0.810 ^Significant at P<.01 * Significant at P <.05 n.s. not significant ( P > .05) t Standard error of estimate tirjoefficient of Multiple Determination - 34 -separate deterministic effects of the treatments on the response, in addition to the variation that may occur due to other variables. The treatment contrasts and multipl iers of Tables I and II were used for this purpose. When treatment contrasts alone were considered as potential independent variables in the regression on browning, only C/Tr and E/L were significant (Table X, column 2). The physical meaning of the negative coefficients of these variables is that treated fruit brown less than control fruit, and fruit treated 46 days after bloom (late treatment) brown less than fruit treated 21 days after bloom (early 2 treatment). The R value of 0.402 indicates that 40.2% of the variation in browning is accounted for by treatment contrasts alone. The result of adding PPO and o-diphenol content as potential independent variables along with the treatment contrasts is shown in Table X, column 3. Only o-diphenol, C/Tr, and E/L were significant. 2 The R value of 0.662 represents a further increase in explanation of variation in browning of 26% by inclusion of o-diphenol data. The addition of fruit weight as a potential independent variable 2 to treatment contrasts and o-diphenol content yields a R value of 0.761 (Table X, column 4), a further increase in explanation of variation in browning of 9.9%. When a l l potential independent variables are included in the regression, C/Tr, E/L, o-diphenol, PPO, and weight are seen to be significant (Table X, column 5). Although the variable PPO was previously not significant, in stepwise multiple regression, the significance of a particular variable depends on the current regression equation (Le and Tenisci, 1977). pH was found to be non-significant. - 35 -The R value of 0.810 represents a 40.8% increase in explanation of variation in browning by inclusion of o-diphenol, PPO, and fruit weight data with the treatment contrasts in the regression equation over treatment contrasts alone. Other possible contributing factors in explanation of browning may be ascorbic acid content of the ripe fruit (Weaver and Charley, 1974; Douglas et al., 1977) and type of o-diphenol (Luh and Phithakpol, 1972). The factors yielding decreased browning of "Redhaven" peaches were growth regulator treatment, treatment 46 days after bloom, decreases in PPO and o-diphenol content of the fruit, and increases in fruit weight. These factors accounted for 81% of the variation in browning. The reason for decreased browning with late but not early treatment application is unknown, but may be related to the stage of fruit development. Environmental factors may also have been important. The spring of 1975 was particularly cold and wet and a light drizzle of rain f e l l within hours of the 21-day application. Whether or not the treatments were washed off the trees by the rain was unknown, but they were not reapplied. The breakdown of ethephon to ethylene as well as uptake of chemicals from a spray application have been seen to be temperature dependent (Lougheed and Franklin, 1972; Leopold and Kriedemann, 1975). Thin Layer Chromatography Knapp et al. (1970) reported slight qualitative differences in the phenolic compounds of "Early Amber" peaches that had been sprayed with GA or ethephon and had decreased enzymatic browning. Ethylene - 36 -has been found to induce the de_ novo synthesis of phenolic compounds not normally present in carrot roots as well as increase the levels of pre-existing phenols, particularly isochlorogenic acid (Sarkar and Ton Phan, 1974). In addition, i t has been shown that certain phenolic compounds such as ferulic acid and coumaric acid inhibit PPO (Walker and Wilson, 1975), while others such as phloroglucinol and resorcinol are competitive inhibitors of PPO but paradoxically are able to increase the rate of browning by reacting with the quinones produced by the enzymatic oxidation of o-diphenolic substrates (Wong et al., 1971b). To determine whether treatment with GA and ethephon had altered the qualitative distribution of "Redhaven" peach phenolic compounds or induced the synthesis of new phenolic compounds, extracts of peach tissue were separated by two-dimentional chromatography on cellulose thin layers. The spots were revealed by ultra-violet light with and without ammonia (Seikel, 1962) and by spraying separate plates with different location reagents (Table XI). Those compounds seen to be PPO substrates were tentatively identified by their mobilities and •behavior with the location reagents. The first plate of each treatment was sprayed with Folin-Ciocalteau reagent. The hydroxyl groups of the phenolic compounds reduce the reagent to a blue color (Ribereau-Gayon, 1972) yielding light-blue to dark-blue spots on a light blue background. Twenty compounds were seen to react with this reagent (Fig. 2). Figures 2-6 were drawn from a representative plate of each reagent. - 37 -TABLE XI. COLOR CHARACTERISTICS3 AND R VALUES OF POLYPHENOLIC COMPOUNDS EXTRACTED FROM "REDHAVEN" PEACHES*30. Rf Location Reagent >ot No. BAW • i — HOAc Folin DPNA Vanillin Molybdate PPO 1 0.71 0.91 fB slT C C c 2 0.57 0.89 fB slT C c c 3 0.61 0.83 B T c 1Y fY 4 0.67 0.79 B T c 1Y fY 5 0.59 0.67 B T c Y Y 6 0.65 0.60 B T c Y Y 7 0.62 0.37 B OT OP 1Y YBr 8 0.61 0.27 fB fYBr c fY C 9 0.66 0.15 fB fYBr c fY C 10 0.53 0.24 fB fYBr c c C 11 0.43 0.26 fB fYBr fp c C 12 0.38 0.37 IB YBr IP fY C 13 0.38 0.47 B YBr OP 1Y Y 14 0.30 0.46 B YBr fp c C 15 0.30 0.30 B YBr fp B C 16 0.36 0.15 fB fYBr fp c C 17 0.44 0.10 B YBr p C C 18 0.58 0.00 B fYBr c C C 19 0.30 0.00 B YBr p YT fYBr 20 0.00 0.00 B YBr p Yt fYBr 21 0.53 0.29 C fYBr fp C C a. B=Blue, Br=Brown, C=Colorless,0=0range, P=Pink, T=Tan, Y=Yellow, f=faint, l=light, sl=slight b. A l l spots were colorless under visible light except spot 17 which appeared pink. c. Mobilities of a l l spots are average values. Figure 2. Peach polyphenols visualized with Folin-Cioucalteau Reagent. 1.0 0.8h II 5% ACETIC ACID Broken lines indicate lighter intensity. - 39 -A second plate was sprayed with DPNA reagent which undergoes a coupling reaction with phenolic compounds giving azo dyes (Ribereau-Gayon, 1972) the colors ranging from tan to orange-brown depending on the nature of the phenol. DPNA rea gent revealed the 20 spots seen with the Folin-Ciocalteau reagent, plus a 21-st spot which was very light orange-brown (Fig. 3). The absence of this spot on the plate sprayed with Folin-Ciocalteau reagent may be due to the light blue background color obscuring the spot or differing sensitivity of the compound to the reagents. A third plate was sprayed with vanillin-HCl reagent which reacts with the "A" ring of catechins and leucoanthocyanidins (Ribereau-Gayon, 1972) yielding spots which are pink to. orange-pink in color. Eleven of the spots reacted with this reagent including spot 21, revealed with DPNA reagent (Fig. 4). To visualize potential browning substrates, a fourth plate was sprayed with 5% Na molybdate which gives a yellow color with o-dihydroxy and vicinal trihydroxy phenolic compounds. Six spots reacted strongly •with this reagent and 7 spots were lighter in color (Fig. 5). A fif t h plate sprayed with crude PPO prepared from control fruit showed that 8 o-diphenolic compounds were oxidized by the enzyme, giving spots ranging in color from very light yellow-brown to intense orange-brown (Fig. 6). The light spots seen with Na molybdate reagent but not seen to be oxidized by PPO were either poor browning substrates or too low in concentration to show a detectable reaction with PPO. No differences were observed in the qualitative distribution of phenolic compounds due to growth regulator treatment. - AO -Figure 3. Peach polyphenols visualized with diazotized p-nitroaniline reagent. 1.01 0.8h II 5% ACETIC ACID Broken lines indicate lighter intensity. - 41 -Figure 4. Peach polyphenols visualized with Vanillin-HCl reagent. l .Oi Col-li 57, ACETIC ACID Broken lines indicate lighter intensity. Figure 5. Peach polyphenols visualized with Na molybdate reagent. 1.01 0.8 0.6 0 ©O ' On°Q « • f l 2 » I 13 ) I ^ w V . — / J19 ( 1 5 ) ( y 1 1 < 1 — — i 1 i _ i t 0 0.2 0.4 0.6 0.8 1.0 115% ACETIC ACID Broken lines indicate lighter intensity. - 43 -Figure 6. Peach polyphenols visualized with polyphenoloxidase. ° « 2 0.4 0.6 0.8 1.0 II 5% ACETIC ACID — Broken lines indicate lighter intensity. - 44 -No attempt was made to conclusively identify each of the phenolic compounds separated, as the primary objective was to determine whether growth regulator treatment qualitatively affected the distribution of peach phenolics, but a tentative identification was made of the PPO reactive compounds. Spots 3, 4, 5 and 6 were identified as chlorogenic acid isomers. They displayed a strong blue fluorescence under UV light which changed to a blue-green fluorescence after fuming with ammonia vapor (Schaller and von Elbe, 1970). They did not react with vanillin-HCl reagent and gave a tan color with DPNA reagent. Spots 4 and 6 are probably the cis and trans isomers respectively of chlorogenic acid as they had similar mobilities to the authentic compounds (Table XII). Spots 3 and 5 are possibly the cis and trans isomers respectively of neochlorogenic acid which have values similar to chlorogenic acid in weak acid systems but lower R^  values in butanol systems than chlorogenic acid (Schaller and von Elbe, 1970). The cis isomers of chlorogenic acid and neochlorogenic acid have higher R^  values than the trans isomers in dilute acid systems on cellulose (Williams, 1955; Roberts, 1956). The trans isomers are the more stable (Walker, 1975) and showed greater intensity of reaction with the location reagents. Chlorogenic acid is known to be a good PPO substrate and has been shown to be present in both freestone (Craft, 1961) and clingstone (Luh et al., 1967) peaches. Neochlorogenic acid has been identified in peaches by Corse (1953). - 45 -TABLE X I I . VALUES OF AUTHENTIC POLYPHENOLIC COMPOUNDS. R Compound f BAW 5% HOAc Caffeic Acid 0.78 0.24 Chlorogenic Acid - cis 0.67 0.81 - trans 0.67 0.60 1-Epicatechin 0.58 0.34 - 46 -Spot 7 was tentatively identified as catechin. It was colorless under UV light before fuming with ammonia but turned dark after fuming. It reacted with DPNA reagent giving the orange-tan color characteristic of catechin and also the characteristic orange-pink to reddish-pink color with vanillin-HCl reagent (Swain and H i l l i s , 1959; Luh et a l . , 1967). Catechin has R^  values slightly greater than epicatechin in both BAW and acetic acid systems on cellulose (El-Sayed and Luh, 1965) as does spot 7 (Tables 13 and 14). Siegelman (1955) showed catechin to be a major browning substrate in pear, and i t has been identified as a browning substrate in peach (Craft, 1961; Luh et a l . , 1967). Spots 13, 19 and 20 resemble leucoanthocyanidins i n mobility (Craft, 1961; Luh et a l . , 1967). They were colorless under UV light with and without ammonia vapor and appeared orange-pink and pink when sprayed with vanillin-HCl reagent, a characteristic of leucoanthocyanidins (Luh et a l . , 1967). These compounds have been shown to be browning substrates in peaches (Craft, 1961; Luh et a l . , 1967; Fel'dman and Kostinskaya, 1970) Caffeic acid was not detected, which i s in agreement with the findings of Craft (1961) for "Elberta" peaches, although i t was present in "Halford" peaches (Luh et a l . , 1967). Only one spot was seen under vi s i b l e light, spot 17, which was pink and thought to be anthocyanin. Hsia et a l . (1965) reported that the major anthocyanin in peaches is cyanidin-3-monoglucoside. The principal polyphenolic compounds in "Redhaven" peaches of the present study oxidized by PPO are tentatively identified as chlorogenic acid isomers, a compound with the characteristics of catechin, and - 47 -leucoanthocyanidins. This is in agreement with the results of Craft (1961), Luh et al. (1967) and Fel'dman and Kostinskaya (1970). The color and intensity of the spots produced with PPO on the chromatograms do not necessarily reflect the true importance of each compound in the browning reaction of the whole fruit. The final color of enzymatic browning is largely the result of non-enzymatic secondary reactions by the quinones after i n i t i a l oxidation (Mathew and Parbia, 1971) under conditions which are not duplicated on the chromatograms. Substrate specificity of PPO is also important. Fel'dman and Kostinskaya (1970) reported that the amount of oxidizable polyphenols in peach varied with the cultivar and type of polyphenol. Browning resulted in a decrease in catechins of 70-100%, leucoanthocyanidins 33-87% and chlorogenic acids 32-40%, depending on cultivar. Craft (1961) found that 75% of the total phenolic compounds and 80% of the leucoanthocyanins in "Elberta" peaches were no longer detected after browning and presumably oxidized. No attempt was made to quantitate individual o-diphenols in the present study, either before or after oxidation. No qualitative changes in peach polyphenols with maturity has been reported except the appearance of anthocyanin with ripening (Craft, 1961; Van Blaricom and Senn, 1967). Ethylene has been shown to stimulate anthocyanin biosynthesis (Craker, 1975) possibly due to stimulation of the enzyme phenylalanine ammonia lyase (PAL) (Camm and Towers, 1973), thought to be a controlling enzyme in the shikimic acid pathway of phenol biosynthesis (Walker, 1975). GA has also been shown to stimulate PAL activity (Camm and Towers, 1973) but Proebsting et al. (1973) found that - 48 -GA treatment decreased cherry anthocyanins. Aoki et al. (1971) found PAL to be present only in the red, anthocyanin containing portions, of mature peaches. Buchanan et al. (1969) noted that ethephon treated peaches had more red color than non-treated or GA treated fruit. Anthocyanin is a poor browning substrate but can take part in coupled oxidations with o-quinones (Mathew and Parbia, 1971) being decolorized in the process. The reaction is similar to the coupled oxidation of ascorbic acid, used in controlling browning (Eskin et al., 1971). There have been no reports on the effect of anthocyanin concentration on rate of enzymatic browning however. - 49 -Polyphenoloxidase Isozymes The effects of plant growth regulators on isozyme formation are many and varied. No reports have been found on their effects on PPO isozymes in particular, but there has been numerous mention made of alterations in peroxidase (PRO) isozymes. Although PRO contains an iron porphyrin as its prosthetic group and PPO contains copper, their isozymes have sometimes been found to be closely associated (Sheen and Calvert, 1969; Srivastava and van Huystee, 1973). The nature of the association is unknown. Galston et al. (1968) reported induction of a PRO isozyme in tobacco tissue culture by IAA. Imaseki et al. (1968) found that ethylene stimulated several PRO isozymes but not others, indicating that ethylene may preferentially affect the synthesis of particular isozymes. Bireka et al. (1976) however, reported no changes in the qualitative isozyme spectrum of PRO from sweet potato tissue treated with ethylene. Lee (1971) found that GA caused increases in three IAA oxidase isozymes in tobacco callus culture, but the effectiveness of GA was dependent on IAA and kinetin. IAA oxidase activity has often been attributed to peroxidases (Shinshi and Noguchi, 1975). Applications of GA to dwarf corn and pea produced no qualitative change in PRO isozyme patterns but quantitatively increased the level of certain isozymes and decreased that of others (Lee, 1972). Inhibition of PRO activity in sugar cane stem tissue by GA produced no change in the isozyme banding pattern (Glasziou et al., 1968). - 50 -It was decided to examine the PPO isozymes in treated and untreated "Redhaven" peaches to determine whether treatment with GA and ethephon affects isozyme number and/or substrate specificity. Preliminary attempts at separating crude PPO preparation by electrophoresis were unsatisfactory due to low protein concentration as well as poor resolution and artifacts probably caused by high pectin content (Frenkel et a l . , 1969). Removal of pectic materials by precipitation with calcium chloride and subsequent concentration by u l t r a f i l t r a t i o n allowed satisfactory separation. Results are shown i n Figure 7. Figure 7A shows the banding pattern of the gels when stained for protein with coomassie blue. Visual observations and densitometric scans of the gels showed very similar banding patterns from control and treated f r u i t . Incubation of gels in catechol, the simplest o-diphenol, revealed up to 11 brown bands after 1.5 hr (Fig. 7B). Bands b, c, d, and j were v i s i b l e after approximately 5 min. The region from a to e had a dark brown background, as indicated by shading. A l l bands except band k were v i s i b l e on gels from each treatment. Band k was only v i s i b l e on gels from peaches treated 21 days after bloom with both GA and ethephon (75 and 150 ppm) and appeared very slowly. Comparison of the mobilities of the bands on the catechol gels with those on the protein gels showed good agreement. The intensity of staining i s not the same, however, as the bands on the catechol gels result from the enzymatic oxidation of a substrate while those on the protein gels resulted from direct staining with coomassie blue. - 51 -F i g u r e 7. Reactions of polyphenoloxidase isozymes w i t h o - d i p h e n o l i c s u b s t r a t e s . 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 + PROT ma d e firm. CAT mm a b d f 9 h i D 7//A CAF CHLOR PROT = p r o t e i n , CAT = c a t e c h o l , CAF = c a f f e i c a c i d , CHLOR = c h l o r o g e n i c a c i d , PG = p y r o g a l l o l - 52 -Using caffeic acid as substrate revealed a banding pattern similar to that with catechol (Fig. 7C). Bands b, d, and j formed within 5 min and the remainder appeared over the course of the 1.5 hr incubation. Immediately noticeable is the disappearance of bands c, e, and k. The background in the region from b to d extended slightly below band d suggesting the presence of another isozyme, but no discrete band was visible. Band g was more diffuse with caffeic acid than with catechol and bands h and i , seen as discrete bands on the catechol gels appeared as one light band with caffeic acid. Band j was more intense with caffeic acid than catechol. None of the growth regulator treatments affected the banding pattern with caffeic acid. Chlorogenic acid, a natural browning substrate in peaches, proved to be unsatisfactory for staining PPO isozymes as the bands formed were very water soluble and quickly diffused, making i t difficult to detect minor bands. A similar effect was noted by Van Loon (1971). A green background quickly formed thus increasing the difficulty. Bands b, d, f, and j were visible with chlorogenic acid (Fig. 7D). Pyrogallol, a vicinal-trihydroxyphenol, was oxidized by only 3 isozymes (Fig. 7E). The mobilities of the bands were similar to bands b, c and e of the catechol gels, but the intensities differed. PPO from different growth regulator treatments showed identical banding patterns with pyrogallol except ethephon (150 ppm, 46-day treatment), in which band b wasn't apparent. Electrophoresis of different amounts of PPO preparation as well as incubation of the gels in differing substrate concentrations at different pH's may give different results (Kahn, 1976; Constantinides and Bedford, 1967). Method of PPO extraction has also been shown to influence - 53 -isozyme pattern (Benjamin and Montgomery, 1973; Kahn, 1977). The lack of knowledge of PPO function in the cell makes i t difficult to determine the functions of the PPO isozymes. A common regulatory feature of branched biosynthetic pathways is the presence of isozymes with differing susceptibilities to end-product control. Constantinides and Bedford (1967) found that PPO isozymes from mushroom had differing susceptibilities to high substrate concentration, suggesting a kind of defence mechanism against product inhibition. In addition, Wong et al. (1971a) noted differences in sensitivities of peach PPO isozymes to heat and chemical inhibitors. It has been suggested that multiple forms of an enzyme are needed to catalyze the same reaction but under different metabolic conditions, cellular locations, and stages of differentiation in order to maximize biological capacity (Markert, 1974). On the other hand, some isozymes may be merely evolutionary accidents with no pressure of natural selection favoring or opposing their existence (Moss and Butterworth, 1974). The differing substrate specificities of peach PPO isozymes may therefore represent alterations in structure which modify the substrate specificities without impairing physiological effectiveness, or they may indicate specific metabolic roles for the isozymes in the ce l l . Markert (1974) suggests that the differences in charge distribution over the surface of the enzyme molecules, which makes electrophoretic separation possible, probably affects the topographic location of the molecule within the cell. This view is interesting as PPO has been shown to exist in forms soluble as well as bound to mitochondria and chloroplasts (Harel et al., 1965; Sato and Hasegawa, 1976). - 54 -The significance of the appearance of band k with catechol in those peaches treated 21 days post-bloom, and the disappearance of band b with pyrogallol in peaches treated with 150 ppm ethephon 46 days post-bloom is presently unknown. As band k was faint and formed slowly with catechol only, i t probably isn't important in the overall browning reaction. The disappearance of band b, however, may indicate a modification of the activity of this isozyme with certain substrates, as i t was seen to oxidize catechol and caffeic acid quite readily. If specific activity of this isozyme with pyrogallol was decreased, a greater amount of PPO preparation added to the gel may cause this band to appear. Band b on the other gels stained with pyrogallol were quite light. It would be necessary to isolate each isozyme and determine their separate kinetic properties with naturally occurring substrates to gain a better understanding of their importance in browning. - 55 -PPO Substrate Specificity The relative activity of crude PPO preparations from control, GA (100 ppm), and ethephon (75 and 150 ppm) treated peaches (46 days after bloom) with various phenolic compounds is shown in Table XIII. It is apparent that treatment with GA and ethephon 46 days after bloom resulted in alterations in the relative activities of the crude PPO preparations with o-diphenolic substrates. The significance of these alterations is not clear; rate and amount of browning may be affected, but i t would be necessary to use naturally occurring peach o-diphenols as substrates to gain a better understanding. Although caffeic acid was oxidized by many isozymes, the relative PPO activities with this substrate are low. Pyrogallol, on the other hand, was only oxidized by 3 isozymes yet relative activity with this substrate is high. The disappearance of isozyme b with pyrogallol may be related to the low activity of PPO from ethephon (150 ppm) treated peaches toward this substrate. None of the PPO preparations had activity with p-cresol or quinol, even after the addition of a small amount of catechol (Whitaker, 1972). Luh and Phithakpol (1972) found PRO from "Halford" peaches to be active with o-diphenols only, but Reyes and Luh (1960) and Harel et al. (1970) found slight PPO activity with quinol. Harel and Mayer (1970) attributed this activity to a single isozyme. TABLE XIII. RELATIVE ACTIVITY OF PPO FROM TREATED AND UNTREATED "REDHAVEN" PEACHES WITH PHENOLIC COMPOUNDS AT pH 6.3. PPO Source Ethephon Substrate Configuration Concentration Control GA 75ppm 150ppm (mM) (late) (late) (late) Catechol o-diphenol 9.3 100 100 100 100 Pyrogallol o-diphenol 9.3 121 124 102 94 Chlorogenic acid o-diphenol 4.2 59 31 33 64 Caffeic acid o-diphenol 9.3 17 14 13 18 p-Cresol monophenol 9.3 0 0 0 0 Quinol p-diphenol 9.3 0 0 0 0 - 57 -PPO pH Optima The effect of pH on activity of the PPO preparations just discussed i s shown in Figure 8. Two pH optima were seen with PPO from each source; pH 4.4 and 6.2 for PPO from control and GA treated peaches, and pH 4.4 and 6.6 for PPO from ethephon treated peaches (both 75 and 150 ppm). The reason for the shift in pH optimum (from pH 6.2 to 6.6) with ethephon treatment is unknown. Jen and Kahler (1974) reported a shift in pH optima of "Redhaven" peach PPO with ripening from a single optimum at pH 6.2 to double optima at pH 6.0 and 6.5. Although the peaches in the present study were harvested at the same maturity, the effect of ethephon in advancing peach maturity may have also accelerated ripening after harvest over control and GA treated f r u i t . The decrease in relative activity at pH 4.4 in the treated peaches may indicate a lower PPO activity with natural substrates at the pH of the peach slurries, leading to decreased browning. No reports have been found of peach PPO with a pH optimum near 4.4. Effect of Substrate Concentration The effect of varying catechol concentration on activity of a crude PPO preparation from control peaches was studied. The Michaelis constant (Km) was determined by least squares treatment of the straight line obtained by plotting 1/substrate cone, versus 1/v^ (Lineweaver and 100 h 90 U 80 70 60 50 40 30 20 co • A © A Control GA (46 days) Ethephon (75 ppm,46 days) Ethephon (150ppm,46 days) 10 4.0 Figure 8. 4.5 5.0 5.5 6.0 pH 6.5 7.0 Effect of pH on Polyphenoloxidase Activity from Untreated and Treated (46-day application) "Redhaven" Peaches. 7.5 - 59 -Burke, 1934) (Figure 9), and was found to be 9.1 x 10 M (catechol) at pH 6.3, and 25°C. The Km value i s a measure of the a f f i n i t y of the enzyme for the substrate and represents the substrate concentration when V q i s half of the maximum velocity of the enzyme. Smaller Km values represent greater a f f i n i t y for the substrate. The Km value i s one of the characteristics of an enzyme; similar Km values under similar conditions indicate similar enzyme characteristics (Jen and Kahler, 1974). Km values for peach PPO from different sources have been reported to be: 15 mM catechol for "Halford" peaches (Luh and Phithakpol, 1972), 29 mM catechol for "Redhaven" peaches (Jen and Kahler, 1974), and 120 mM for "Elberta" peaches (Reyes and Luh, 1960). Wong et a l . (1971a) reported that peach PPO isozymes have differing Km values. The differing characteristics of "Redhaven" peach PPO of the present study compared to that of Jen and Kahler (1974) may indicate differences in area, growing conditions and rootstalk. Figure 9. Double Reciprocal Plot of Crude Polyphenoloxidase from Untreated "Redhaven" Peaches. - 61 -SUMMARY AND CONCLUSIONS Redhaven peaches treated with gibberellic acid (100 ppm) or ethephon (75 or 150 ppm), 21 or 46 days after f u l l bloom, were evaluated for enzymatic browning in the ripe fruit. Treated fruit had less browning than untreated fruit, and fruit treated 46 days after bloom had less browning than fruit treated 21 days after bloom. Fruit pH and fresh weight were affected by treatment, but not o-diphenol content or PPO activity. Stepwise multiple regression revealed that 81% of the variation in browning was explained by differences in treatment, treatment application time, o-diphenol content, PPO activity, and fresh weight. Unlike previous reports, the reduction in browning observed in the present study could not be attributed to any single factor. Qualitative analysis of "Redhaven" peach polyphenol compounds by 2-dimentional thin layer chromatography showed the presence of 21 spots on the TLC plates, eight of which were oxidized by a crude PPO preparation from control peaches. These were tentatively identified as 4 chlorogenic acid isomers, a compound with properties similar to catechin, and 3 leucoanthocyanidin-like compounds. There were no differences observed between treatments in qualitative distribution of phenolic compounds, ruling out the possibility of a treatment induced phenolic PPO inhibitor or the disappearance of a PPO substrate in low browning peaches. Possible quantitative changes in amount of each PPO substrate were not investigated, nor the importance of each type of PPO substrate in the browning reaction. - 62 -Analysis of pa r t i a l l y purified peach PPO preparations by polyacrylamide disc-gel electrophoresis showed the presence of up to 11 isozymes with activity toward catechol. The isozymes had differing substrate s p e c i f i c i t i e s and were present in differing amounts. Treatment 21 days after bloom with both GA and ethephon (75 and 150 ppm) seemed to induce the appearance of a minor catechol reactive isozyme. This isozyme had no activity with the other substrates tested. Treatment with 150 ppm ethephon 46 days after bloom appeared to decrease the substrate specificity of one isozyme. The importance of these changes on the degree of enzymatic browning were not determined. Crude PPO preparations from control f r u i t and f r u i t s treated 46 days after bloom with GA and ethephon (75 and 150 ppm) were analyzed for substrate s p e c i f i c i t i e s . Enzymatic activity was seen with o-diphenolic compounds only; no activity was seen with either a mono-phenol or a p-diphenol. Activity toward o-diphenols relative to catechol showed slight variations with treatment which may indicate alterations in reactivity with naturally occurring PPO substrates in peach. The same PPO preparations exhibited two pH optima in phosphate-citrate buffer. PPO from ethephon treated f r u i t had pH optima of 6.6 and 4.4 while that from GA treated f r u i t and control f r u i t had pH optima of 6.2 and 4.4. 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