Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1978_A6_7 P39.pdf [ 3.62MB ]
Metadata
JSON: 831-1.0094297.json
JSON-LD: 831-1.0094297-ld.json
RDF/XML (Pretty): 831-1.0094297-rdf.xml
RDF/JSON: 831-1.0094297-rdf.json
Turtle: 831-1.0094297-turtle.txt
N-Triples: 831-1.0094297-rdf-ntriples.txt
Original Record: 831-1.0094297-source.json
Full Text
831-1.0094297-fulltext.txt
Citation
831-1.0094297.ris

Full Text

THE EFFECT OF GIBBERELLIC ACID AND ETHEPHON ON ENZYMATIC BROWNING OF REDHAVEN PEACHES  by  ALLAN THOMAS PAULSON B.Sc.(Agr.), University of B r i t i s h 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 t h i s thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA March, 1978 A l l a n Thomas Paulson, 1978.  6  In presenting this thesis in partial fulfilment of the requirements f o r an advanced degree at the University of B r i t i s h Columbia, I a g r e e  that  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 B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5  Date  MARCH  W78  - ii-  ABSTRACT  Redhaven peaches treated with g i b b e r e l l i c acid (GA, 100  ppm)  and ethephon (75 and 150 ppm) 21 and 46 days a f t e r f u l l bloom were evaluated f o r enzymatic browning i n the r i p e f r u i t .  Treated f r u i t  had l e s s browning than untreated f r u i t , and f r u i t treated 46 days a f t e r bloom had l e s s browning than f r u i t treated 21 days a f t e r bloom. F r u i t pH and fresh weight were affected by treatment, but o-diphenol content and polyphenoloxidase (PPO) a c t i v i t y were not.  Forward  stepwise multiple regression on browning showed that 81% of the v a r i a t i o n i n browning was explained by differences i n treatment, treatment a p p l i c a t i o n date, o-diphenol content, PPO a c t i v i t y , and fresh weight. Twenty-one polyphenolic compounds from Redhaven peaches were separated by two-dimentional t h i n layer chromatography.  Eight were  oxidized by PPO, and were t e n t a t i v e l y i d e n t i f i e d as four chlorogenic acid isomers, three leucoanthocyanidins, and catechin.  No differences  i n q u a l i t a t i v e d i s t r i b u t i o n s of phenolic compounds were observed i n peaches receiving the d i f f e r e n t treatments. Polyacrylamide disc g e l electrophoresis of peach PPO preparations showed the presence of up to eleven isozymes with a c t i v i t y toward catechol.  The isozymes had d i f f e r e n t substrate s p e c i f i c i t i e s and  were present i n d i f f e r e n t amounts.  PPO from peaches treated 21 days  a f t e r bloom appeared to have a catechol reactive isozyme not present i n untreated peaches or peaches treated 46 days a f t e r bloom.  One  PPO isozyme from peaches treated 46 days a f t e r bloom with 150 ppm  - iii -  ethephon appeared to have decreased substrate s p e c 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 r e l a t i v e a c t i v i t i e s of the PPO preparations with these compounds varied with treatment. optima; pH 4.4 and 6.2  The same PPO preparations exhibited two  f o r untreated and GA treated peaches (46-day  treatment), and pH 4.4 and 6.6 (75 or 150 ppm,  f o r peaches treated with ethephon  46-day treatment).  PPO  from the treated peaches had  a lower proportion of t o t a l a c t i v i t y at pH 4.4 than PPO peaches.  pH  from untreated  The Michaelis constant f o r PPO from untreated peaches was  9.1 x 10~ M. 3  -  iv -  TABLE OF CONTENTS  ABSTRACT .  Page  • •  TABLE OF CONTENTS  i i  • • •  iv  LIST OF TABLES . . . . LIST OF FIGURES  vi •  v i i  ACKNOWLEDGEMENTS  viii  INTRODUCTION  1  LITERATURE REVIEW  3  Enzymatic Browning Reaction  3  Polyphenoloxidase  4  Browning Substrates .  7  G i b b e r e l l i c Acid and Ethephon EXPERIMENTAL A p p l i c a t i o n of Growth Regulators . . . . . . . . . . . . . .  .9 12 .12  Enzymatic Browning  13  Polyphenoloxidase E x t r a c t i o n and Assay  13  Extraction and Assay of o-Diphenolic Compounds  14  S t a t i s t i c a l Methods  15  Thin Layer Chromatography of Peach Phenolic Compounds . . . . 16 Electrophoresis  19  PPO Substrate S p e c i f i c i t y  24  PPO pH Optima  24  E f f e c t 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 E f f e c t of Substrate Concentration  •  .57 •  57  SUMMARY AND CONCLUSIONS  6 1  LITERATURE CITED  6 4  -vi -  Table  LIST OF TABLES  Page  I  Treatment Contrasts  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  V  17  27  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 . Analysis of Variance of Fresh Weight of Redhaven Peaches  VIII  29 31  IX  Individual Degrees of Freedom for the Effects of Treatments on Fresh Weight  X  Stepwise Multiple Regression on Browning . . . . . . 33  XI  Color Characteristics and R Values of Polyphenolic Compounds Extracted from Redhaven Peaches  XII XIII  .31  f  37  Values of Authentic Polyphenolic Compounds. . . . 45 Relative Activity of PPO from Treated and Untreated Redhaven Peaches with Phenolic Compounds at pH 6.3. .56  - vii-  LIST OF FIGURES Figure  1  Page  Effect of Ethephori Concentration and Application Date on Fresh Weight of Redhaven Peaches  32  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  2  9  Double Reciprocal Plot of Crude Polyphenoloxidase from Untreated Redhaven Peaches . . . . . 60  -viii -  ACKNOWLEDGEMENTS  The author wishes to express h i s g r a t i t u d e to Dr. J . Vanderstoep, Research Supervisor, f o r h i s help and encouragement during t h i s study, to Dr. S.W. P o r r i t t f o r assistance i n the f i e l d work, and the Canada A g r i c u l t u r e Research S t a t i o n , Summerland, B.C., f o r making a v a i l a b l e the Redhaven peach trees used i n t h i s study.  S p e c i a l thanks go to  Dr. G.W. Eaton f o r h i s 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 i n browning control represent a large cost to the food industry. Traditional methods of controlling enzymatic browning are based on controlling some aspects of i t s 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 a l . , 1969). This was found to be due to a decrease in polyphenoloxidase, the browning enzyme, of over 90% (Knapp et a l . , 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 t h i s to a decrease i n polyphenolic substrate.  GA and ethephon at  50 ppm had no e f f e c t and "Fairhaven" peaches receiving the same treatment displayed no change i n browning.  Porritt  (1974) however,  found that "Redhaven" peaches receiving 75 ppm ethephon sprays 46 days a f t e r f u l l bloom had decreased enzymatic browning at maturity. I t a l i a n 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 i n t e r n a l browning, and applies sprayed with ethephon ten days before harvest had lower l e v e l s of polyphenoloxidase and were more r e s i s t a n t 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 to determine the e f f e c t of GA  (100 ppm)  undertaken  or ethephon (75 or 150  ppm),  applied at e i t h e r of two a p p l i c a t i o n dates, on enzymatic browning of Redhaven peaches.  - 3 -  LITERATURE REVIEW  Enzymatic Browning Reaction  The fundamental  step i n the enzymatic browning of peaches i s  the oxidation of ortho-dihydroxyphenolic compounds (o-diphenols) to o-quinones catalyzed by the enzyme polyphenoloxidase (Luh and Phithakpol, 1972).  (PPO)  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 i s n ' t  known with c e r t a i n t y .  Data from d i f f e r e n t 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  a c t i o n of PPO's have suggested that they possess separate binding s i t e s f o r 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 a f t e r such complexing.  PPO undergoes  - 4 -  "reaction i n a c t i v a t i o n " due to the formation of a covalent linkage between the enzyme molecule and a quinone at or near the active s i t e (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 s p a c i a l l y separated (Ponting, 1960).  There has been l i t t l e concrete evidence  demonstrating the s u b - c e l l u l a r l o c a t i o n of PPO and phenolic compounds however (Anderson, 1968).  After the c e l l s are damaged such as by  impact or cutting, the enzyme and substrate are free to mix and i n the presence of oxygen the browning reaction can proceed. The function of the browning complex i s uncertain.  I t has been  suggested that PPO functions as a terminal oxidase i n r e s p i r a t i o n (Boswell, 1963) but i t has been found to compete poorly with the r e s p i r a t o r y chain at low p a r t i a l pressures of oxygen (Anderson, 1968). I t has also been implicated i n 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 l e v e l , substrate l e v e l , and a combination of both factors (Kahn, 1975), but browning of peaches was most c l o s e l y related to l e v e l s of polyphenols (Guadagni et a l . , 1949; Nakabayashi  et a l . , 1963).  Polyphenoloxidase  Polyphenoloxidase (o-diphenol: oxygen oxidoreductase EC 1.14.18.1 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 very l i t t l e i n the Cu  ++  form.  +  ions, with  They suggested that a Cu  +  -C^  i n t e r a c t i o n would a c t i v a t e molecular oxygen and the reaction would occur according to the scheme of Mason (1957). I t also appears to involve an active s i t e with a high a f f i n i t y for the aromatic r i n g and a basic group which promotes phenol to phenolate i o n i z a t i o n (Bright et a l . , 1963;  Duckworth and Coleman, 1970).  I t i s assumed, based on  studies of the e f f e c t s of i n h i b i t o r s and r i n g s u b s t i t u t i o n on reaction v e l o c i t y that oxidation occurs v i a an e l e c t r o p h i l i c attack  (Lanzarini  et a l . , 1972). PPO's from d i f f e r e n t sources usually have differences i n such properties as substrate  s p e c i f i c i t i e s , pH optima, and reaction k i n e t i c s .  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 n e g l i g i b l e a c t i v i t y with monophenols (Luh and Phithakpol, Reyes and Luh, been reported et a l . , 1970).  1960).  S l i g h t a c t i v i t y with the p-diphenol quinol  and v a r i e s with maturity (Reyes and Luh, PPO  the hydroxylation  1972;  1960;  has  Harel  from some sources has the a b i l i t y to catalyze both  of monophenols to o-diphenols and t h e i r subsequent  oxidation to o-quinones (Mason, 1957). (1967) resolved mushroom PPO a c t i v i t y with monophenols.  Constantinides and Bedford  into nine isozymes, three of which had 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 i n d i f f e r e n t enzymes.  - 6 -  The pH optimum of peach PPO ranges from 5.9 to 6.3 depending on the type of buffer used Luh, 1960).  (Luh and Phithakpol, 1972; Reyes and  Jen and Kahler (1974) found that as "Redhaven" peaches  matured the PPO pH optimum changed from a single optimum at pH to  double optima  at pH 6.0 and 6.5,  isozymes with maturity.  6.2  suggesting the synthesis of new  Wong et a l . (1971a) separated PPO  from  "Cortez" peaches into four isozymes which d i f f e r e d from each other i n pH optima, substrate s p e c i f i c i t y , rate constants, and susceptability to i n h i b i t o r s and heat.  Harel and Mayer (1970) e l e c t r o p h o r e t i c a l l y  separated PPO from "Salvey" peaches into f i v e bands, one of which was a c t i v e with quinol, a c h a r a c t e r i s t i c of the enzyme laccase, not normally found i n peaches. The sub-cellular l o c a t i o n of PPO i s poorly understood.  Sections  of mature peach f r u i t stained f o r PPO with catechol showed the enzyme to be present i n l o c a l i z e d patches of parenchyma c e l l s (Reeve, 1959). Harel et a l . (1970) found peach PPO to be present i n both the p a r t i c u l a t e and soluble form, which were present i n d i f f e r i n g amounts at d i f f e r e n t -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 p a r t i c u l a t e PPO can often be s o l u b i l i z e d by treatment with  various detergents, which i s often accompanied by PPO a c t i v a t i o n (Sato and Hasegawa, 1976).  Soluble PPO  sometimes also e x i s t s i n a latent  form that can be activated by storage, temperature  change, detergent  treatment, or denaturing agents, contributing a d d i t i o n a l PPO (Kahn, 1977).  activity  - 7 -  Due  to the complexity of PPO  i t i s d i f f i c u l t to ascribe  single r o l e i n c e l l u l a r metabolism. for PPO  i n biosynthesis  of phenolic  any  Conn (1964) suggested a r o l e compounds, but any  explanation  of function must take into account the demonstrated m u l t i p l i c i t y of forms.  Browning Substrates  The  ortho-dihydroxyphenolic configuration i s e s s e n t i a l for  a c t i v i t y , but not a l l compounds with such a configuration are and those that are exhibit d i f f e r e n t rates of oxidation.  PPO  oxidized,  Substituents  on the benzene r i n g have been shown to influence reaction rate depending on p o s i t i o n and electron donating or a t t r a c t i n g character  (Lanzarini  et a l . , 1972). Natural browning substrates  found i n food are usually cinnamic  acid derivatives which a r i s e from the shikimic acid pathway, and flavonoid compounds of which the "A" malonate pathway and  r i n g i s derived from the  acetate-  the "B" r i n g from cinnamic acid derivatives (Hess,  1975). Of the cinnamic acid derivatives the most important i n enzymatic browning i s chlorogenic  acid (3-caffeoylquinic acid) a 3-depside of  quinic acid with c a f f e i c acid.  Chlorogenic acid content has been  related to browning of several v a r i e t i e s of applies Flavonoids involved i n browning include catechins, anthocyanins, and flavonols.  (Walker, 1962). leucoanthocyanidins,  Anthocyanins are not primarily s i g n i f i c a n t  - 8 -  as PPO  substrates but have been shown to be involved i n secondary  reactions (Mathew and Parbia, 1971). The main polyphenols  involved i n enzymatic browning of peaches  were found to be leucoanthocyanidins, and catechins (Craft, 1961; 1967).  chlorogenic acid isomers,  Fel'dman and Kostinskaya, 1970;  Luh et a l . ,  Craft (1961) observed no q u a l i t a t i v e change i n polyphenolic  pattern with ripening and the r e l a t i v e proportions remained constant. Phenolic compounds were seen to increase i n concentration during the early stages of f r u i t growth, reach a maximum at pit-hardening, then slowly decline u n t i l harvest  (Craft, 1961;  The amount of phenols on a whole f r u i t basis was  and  Harel et a l . , 1970). seen to increase  however (Craft, 1961). L i et a l . (1972) found that both f l a v o r and color of peaches were negatively c o r r e l a t e d with t o t a l phenols.  Astringency of f r u i t s  has been associated with polyphenolic concentration, p a r t i c u l a r l y catechins and leucoanthocyanidins 1961).  (Goldstein and Swain, 1963;  Craft,  Peaches show a decrease i n astringency during ripening (Craft,  1961), but the reasons f o r t h i s are unknown. The functions of plant phenols are obscure perhaps because they are secondary products and play no r o l e i n metabolism.  Anthocyanins,  flavones, and f l a v o n o l s , due to t h e i r c o l o r a t i o n , probably play a r o l e i n a t t r a c t i n g insects (Salisbury and Ross, 1969). cinnamic acids are believed to be important (Van Buren, 1970). i s at pit-hardening.  Hydroxylated  precursors of l i g n i n  In peach, the highest t o t a l phenolic concentration Phenolic compounds have also been suggested to  - 9 -  control auxin concentrations  i n some plants through t h e i r e f f e c t s  on the enzyme indole a c e t i c acid (IAA) oxidase.  Monophenols have  been shown to enhance IAA oxidase a c t i v i t y while o-diphenols i n h i b i t the enzyme (Nitsch, 1970).  G i b b e r e l l i c Acid and Ethephon  G i b b e r e l l i c acid (abbreviated  GAorGA^), best known f o r i t s  stimulating e f f e c t s on plant growth, i s one of many d i f f e r e n t s t r u c t u r a l v a r i a t i o n s of the plant growth hormones known as g i b b e r e l l i n s . As w e l l as stimulating growth, g i b b e r e l l i n s have been found to have regulatory e f f e c t s on plant development.  Plants have been found to  have s e l e c t i v i t y of response to the d i f f e r e n t forms.  The term  " g i b b e r e l l i n " i s often used rather loosely i n the l i t e r a t u r e as being synonymous with g i b b e r e l l i c a c i d (Stuart and Cathey, 1961).  Recent  reviews of the g i b b e r e l l i n s have been made by Lang (1970) and Jones (1973). Ethephon (2-chloroethylphosphonic acid) breaks down i n the plant releasing the plant hormone ethylene (Yang, 1969).  Ethylene i s best  known f o r i t s e f f e c t i n t r i g g e r i n g ripening of c l i m a c t e r i c f r u i t but has also been found to be important i n regulation of plant development. The physiology  of ethylene has recently been reviewed by Abeles (1972).  GA and ethylene often have s i m i l a r e f f e c t s (e.g. breaking of seed dormancy) as well as opposing e f f e c t s (e.g. ethylene promotes but GA delays ripening and senescence i n many types of f r u i t ) Kriedmann, 1975).  (Leopold and  -  10 -  Peach f r u i t growth Is characterized by two stages of rapid growth (Stages I and I I I ) 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 ( p i t 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 p h y s i o l o g i c a l sinks, a t t r a c t i n g nutrients at the expense of the remainder of the tree.  Both GA and ethylene are  thought to be involved i n t h i s e f f e c t (Nitsch, 1970; Chalmers, et a l . , 1976).  Jackson (1968) found g i b b e r e l l i n i n the seeds of peaches  immediately a f t e r f u l l bloom, and l a t e r i n the mesocarp and endocarp. G i b b e r e l l i n was found to be c l o s e l y associated with the rate of c e l l expansion i n each t i s s u e but not with c e l l d i v i s i o n .  Ogawa (1965)  reported that g i b b e r e l l i n s i n peach seeds began to increase 35 days post-bloom, reached t h e i r maximum amount by 50 days post-bloom, and decreased r a p i d l y thereafter.  Looney et a l .  (1974) found an association  between ethylene l e v e l and growth rate i n Stage I of peach growth. Applied GA and ethephon have varying e f f e c t s on peach growth.  GA  has been shown to induce parthenocarpy (seedlessness), stimulate vegetative growth (Jerie and Taylor, 1971), achieve a thinning e f f e c t 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 I I of peach growth Induced early onset of Stage I I I . Looney et a l . (1974), however, found no e f f e c t of ethephon application  - l i -  on length of Stage I I . Other ethephon induced e f f e c t s 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 a l t e r a t i o n s i n c e l l u l a r membranes (Leopold and Kriedmann, 1975). The manner i n which a s i n g l e early season a p p l i c a t i o n of a growth regulator can induce a response months l a t e r 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 l i n g e r i n g increase i n ethylene l e v e l i n immature peach f r u i t which p e r s i s t s u n t i l the f r u i t develop s u f f i c i e n t l y 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 t h i s could be removed by washing.  They l a t e r found, however, that ethephon binds r a p i d l y to  sugars i n the peach mesocarp and suggested that the formation of stable sugar-ethephon complexes may be involved i n long-term responses, rather than release of ethylene at the f r u i t surface (Lavee and Martin, 1975).  This i s s i m i l a r to the binding of GA as glycosides, which may  be a storage form of t h i s 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. Twentyeight 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.  F u l l 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 l i t e r s per tree) to three of the seven groups. spreader-activator was used as a wetting agent.  0.1% Rhodes R - l l 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 i n cold storage (0°C) immediately after picking.  Five peaches from each tree were removed from cold storage  and allowed to ripen at 21°C f o r determination of enzymatic browning and pH.  The remainder of the f r u i t s were transported to Vancouver,  ripened at 21°C, halved, p i t t e d , vacuum packaged with a nitrogen backflush i n aluminum f o i l pouches, then placed i n frozen storage (-35°C) u n t i l used.  Enzymatic Browning  One-third sectors of f i v e r i p e peaches from each tree were pureed for  20 seconds at h a l f speed i n a Waring blendor at 21°C. The degree  of browning was measured with a Hunterlab D-25 Color Difference Meter as the difference i n Rd (lightness) between readings taken at one minute and t h i r t y minutes a f t e r blending.  A yellow t i l e  (Rd =68.7,  a =-3.8, b = 25.2) was used f o r 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 f o r 4 hours before extraction.  Twenty-five grams from each of the  3 halves were blended with 150 ml cold acetone (-35°C) f o r 25 seconds at h a l f speed plus an a d d i t i o n a l 5 seconds at f u l l speed i n a s t a i n l e s s s t e e l S o r v a l l Omnimixer j a r .  The r e s u l t i n g s l u r r y was allowed to stand  - 14 -  for  5 minutes i n an i c e 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 i n 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 i n a S o r v a l l RC-2  r e f r i g e r a t e d centrifuge.  The  supernatant was c o l l e c t e d as the crude enzyme solution f o r PPO assay and the p e l l e t was discarded. The spectrophotometric method of Wong et a l . assay of PPO a c t i v i t i e s .  (1971a) was used f o r  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 i n v e s t i g a t i o n had found t h i s to be the  optimum pH of "Redhaven" peach PPO i n Na phosphate buffer.  To insure  that oxygen was not l i m i t i n g , the catechol solution was aerated by bubbling oxygen through i t f o r 5 min. at  25°C.  420 nm  Reaction temperature was maintained  The enzymatic formation of benzoquinone was followed at  (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 i n absorbance  was used as a measure of PPO a c t i v i t y , and was recorded as PPO * min  A Abs^QO. 1ml  The reaction was performed i n duplicate with PPO from each  tree and the r e s u l t s averaged.  Extraction and Assay of o-Diphenolic Compounds  Twenty-five grams from each of 3 frozen peach halves per tree were macerated i n 300 ml b o i l i n g 80% ethanol for 3 minutes at half speed  - 15 -  i n an Osterizer blender under a nitrogen atmosphere.  The s l u r r y  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 a i d .  The f i l t e r cake was  washed with 2 aliquots of 300 ml b o i l i n g 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 v i c i n a l - t r i h y d r o x y phenolic compounds i n the ethanol extract were determined by a modification of the method of Mapson et a l . (1963).  This method measures the yellow color r e s u l t i n g from a complex  formed between 2 polyphenol molecules and one molybdate and Paragamian,  1960).  ion (Haight  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 t h i s 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 f o r measuring t h i s reaction.  The reaction was performed i n duplicate and the values  averaged.  The concentration of o-dihydroxy and v i c i n a l - t r i h y d r o x y  phenolics was determined from a standard curve prepared using 0.02-0.2 mg catechol/ml.  The r e s u l t s were expressed as mg catechol/g peach t i s s u e .  S t a t i s t i c a l Methods  Data were analyzed by analysis of variance. To obtain information regarding s p e c i f i c treatment e f f e c t s , the treatment sums of squares  - 16 -  and degrees of freedom were partitioned using the i n d i v i d u a l degree of freedom technique ( L i , 1964a).  The s i x treatments and the control  were arranged as s i x treatment contrasts (Table I) and an orthogonal set m u l t i p l i e r s obtained (Table I I ) . The f a c i l i t i e s of the U.B.C. Computing Center Triangular Regression Package (Le and T e n i s c i , 1977) were used for m u l t i p l e regression analyses.  Thin Layer Chromatography of Peach Phenolic Compounds  Q u a l i t a t i v e analyses of peach phenolic compounds were accomplished by 2-dimentional t h i n layer chromatography (TLC).  Peaches from one  r e p l i c a t e of each treatment and control were used.  To improve the  p o s s i b i l i t y of detecting treatment e f f e c t s , the r e p l i c a t e from each treatment was chosen on the basis of low browning and/or low o-diphenol content, and the untreated r e p l i c a t e 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 f o r browning. Twenty grams from each of 5 frozen peach halves were blended with 300 ml b o i l i n g 95% ethanol f o r 3 min under a nitrogen atmosphere i n an Osterizer blender.  The s l u r r y 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 a i d .  The  cake was washed with an a d d i t i o n a l 300 ml b o i l i n g 95% ethanol. yellow f i l t r a t e was  filter The  evaporated on a f l a s h evaporator at 32° - 35° C  u n t i l an aqueous s o l u t i o n 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  Control vs. Treated  b.  E/L  Early Treatment (21 days) vs. Late Treatment (46 days)  c.  GA/Eth  Treatment with GA vs. Treatment with Ethephon  d.  Lo/Hi  Low Ethephon Treatment (75 ppm) vs. High Ethephon Treatment (150 ppm)  e.  B x C  E/L x GA/Eth  f.  B x D  E/L x Lo/Hi  TABLE I I ORTHOGONAL MULTIPLIERS FOR TREATMENT CONTRASTS  Treatment  Application Time (days)  Treatment Contrast GA/Eth Lo/Hi BxC  C/Tr  E/L  —  -6  0  0  0  0  0  Ethephon (75ppm)  21  1  -1  1  -1  -1  1  Ethephon (150ppm)  21  1  -1  1  1  -1  -1  G i b b e r e l l i c Acid  21  1  -1  -2  0  2  0  Ethephon (75ppm)  46  1  1  1  -1  1  -1  Ethephon (150ppm)  46  1  1  1  1  1  1  G i b b e r e l l i c Acid  46  1  1  -2  0  -2  0  Control  BxD  - 18 -  paper.  The f i l t r a t e 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 l e f t 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 f i r s t 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, a i r dried, then developed in the second direction with 5% acetic acid (5% HOAc) u n t i l the solvent front was 1 cm from the top of the plate. The a i r 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 a l . , 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 - n i t r o a n i l i n e (DPNA) reagent, prepared by mixing i n an i c e bath 0.5% p - n i t r o a n i l i n e In 2N HCI, 5% NaN0  2>  and 20% sodium acetate (w/v) i n a r a t i o of  1:10:30 (v:v:v) (Luh et a l . , 1967).  This reagent gives c h a r a c t e r i s t i c  colors with phenolics. A t h i r d chromatogram was sprayed with f r e s h l y 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 f r e s h l y prepared solution of 5% Na molybdate i n 0.1 M Na phosphate buffer (pH 7.0).  Phenolic  compounds containing an o-dihydroxy or v i c i n a l - t r i h y d r o x y configuration form yellow complexes with t h i s reagent  (Haight and Paragamian, 1960)  and were v i s u a l i z e d 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 chamber f o r 2 hours.  (see Electrophoresis) and incubated i n a humid 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, c a f f e i c acid, and 1-epicatechin ( a l l 5 mg/ml ethanol) were also chromatographed as described above and R^ values were determined.  Electrophoresis  Polyacrylamide g e l 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 . , Harel and Mayer, 1970).  1971;  To determine the e f f e c t of growth regulator  treatment on PPO isozymes, one tree from each treatment was selected on the basis of low browning and/or low PPO a c t i v i t y f o r electrophoresis, (Table I I I ) .  The control r e p l i c a t e was chosen on the basis of high  browning and high PPO For  activity.  preparation of PPO f o r electrophoresis, 10 g of frozen peach  tissue from each of 5 peach halves were blended with 100 ml cold acetone (-35°C) f o r 3 bursts of 10 seconds each at high speed i n an Osterizer blender and allowed to stand at 4°C f o r 5 min.  The s l u r r y 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 i n 100 ml of 0.1 M Na  phosphate buffer (pH 7.0) by s t i r r i n g f o r 1.5 hr at 4°C.  The suspension  was centrifuged at 33,000 x G f o r 15 min i n a S o r v a l l RC2 refrigerated centrifuge at 0°C.  The supernatant (crude PPO preparation) was collected  and the p e l l e t discarded. To p r e c i p i t a t e pectins, 0.5 M C a C ^ 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 p r e c i p i t a t e was removed by centrifugation at 15,000 x G and 0°C f o r 10 min i n a S o r v a l l centrifuge.  RC-2  Twenty ml of the supernatant was d i l u t e d 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 of  membrane, Amicon Corp.) under a pressure  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 c r y s t a l l i n e bovine serum albumin as standard.  (1969) using  This modification  allows f o r the estimation of protein i n the presence of phenolic compounds. For electrophoresis, 150-200 u l of enzyme s o l u t i o n (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 g e l (5.0 cm long) and 1.25%  stacking gel (1.0 cm long).  Model GE-4  A l l gels were run i n a Pharmacia  electrophoresis apparatus.  running pH was  9.5.  The s t a r t i n g pH was  A current of 3.5 mA per tube was  and  the  employed u n t i l  the tracking dye migrated close to the end of the tube. phoresis chamber was  8.3  The e l e c t r o -  kept cool with c i r c u l a t i n g cold tap water.  The  p o s i t i o n of the tracking dye was marked on the gel with a needle containing India ink. One  g e l was  stained f o r protein by immersion for 1.5 hr i n  0.25%  Coomassie blue dissolved i n a mixture of 7% methanol and 5% a c e t i c acid. The g e l was  destained by soaking i n several changes of 7% methanol-5%  a c e t i c a c i d solution u n t i l a clear background was  obtained.  The p o s i t i o n  and i n t e n s i t y of the bands were recorded v i s u a l l y and then by densitometry using a Gelman Gelscan densitometer. Four other gels were soaked for 30 min phenylenediamine (MPD)  i n a solution of 0.1%  i n 0.1 M Na phosphate buffer (pH 6.3)  and then  placed i n a solution of catechol (0.03 M), p y r o g a l l o l (0.03 M), acid  m-  caffeic  (0.03 M), or chlorogenic acid (0.01 M) a l l i n Na phosphate buffer  - 22 -  (pH 6.3).  The gels were vigorously aerated by bubbling oxygen  through the solutions for 5 min.  MPD  i s a coupling agent which  reacts with the quinones produced at the s i t e of substrate oxidation by PPO  (van Loon, 1971;  Harel et a l . , 1965).  varied depending on the substrate used.  The color of the bands  Band development was  i n 1.5 hours and t h e i r p o s i t i o n and i n t e n s i t y recorded.  complete  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 S p e c i f i c i t y  Several compounds with polyphenolic or monophenolic were used for t h i s study.  configurations  2.8 ml of catechol (lOmM), p y r o g a l l o l (lOmM),  p-cresol (lOmM), quinol (lOmM), or chlorogenic acid (4.5mM) i n 0.1M  Na  phosphate buffer (pH 6.3) was aerated and then r a p i d l y mixed with 0.2 ml of a crude PPO preparation (see E l e c t r o p h o r e s i s ) . of the substrate s o l u t i o n was  25 C.  The reaction was  The temperature  followed at 420  nm  with a Beckman DB spectrophotometer with Photovolt recorder and the data f o r each substrate, with each PPO preparation,was recorded as a c t i v i t y r e l a t i v e to a c t i v i t y with catechol.  PPO pH Optima  A c t i v i t y of the crude PPO preparations r e l a t i v e to pH was  determined  - 23  -  as above by r a p i d l y mixing 0.2 ml of crude PPO preparation with 2.8 ml of 10 mM  catechol i n 0.1 M citrate-0.2 M Na phosphate buffer  over a pH range of 4.0  to 7.4.  A c t i v i t y at each pH was plotted as  per cent of maximum a c t i v i t y attained.  E f f e c t of Substrate Concentration  0.2 ml of crude PPO preparation from control peaches (see Electrophoresis)  was  r a p i d l y mixed with 2.8 ml of catechol i n 0.1 M Na  phosphate buffer (pH 6.3)  to f i n a l concentrations i n the reaction  mixture ranging from 9.3 mM as above.  to 28 mM,  and PPO a c t i v i t y determined  The data were p l o t t e d as 1/substrate concentration  versus 1 / i n i t i a l v e l o c i t y (v ).  (M)  _ 24 -  RESULTS AND DISCUSSION  Enzymatic Browning  Treated f r u i t had l e s s browning at harvest than untreated fruit  (Tables IV and V).  F r u i t treated 46 days a f t e r bloom had  l e s s browning than f r u i t treated 21 days a f t e r 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 o r i g i n a l PPO a c t i v i t y but  t o t a l amount of browning depended on polyphenol content. The f a i l u r e of "Early Amber" peaches to undergo enzymatic  browning a f t e r early season applications of GA and ethephon was due to a reduction i n PPO a c t i v i t y by over 90% i n the treated f r u i t , with . s l i g h t reductions i n o-diphenol content (Knapp et a l . , 1970).  Paulson  (1973) attributed the reduction i n browning of "Redhaven" peaches a f t e r a post-bloom a p p l i c a t i o n of GA to a reduction i n available substrate.  Sal'kova et a l . (1977) found that apples treated with  ethephon had lower l e v e l s of PPO, peroxidase (PRO), and ascorbic acid oxidase, and were more r e s i s t a n t to browning on cutting.  GA applied  to West Indian cherries was seen to cause a marked reduction i n PPO and ascorbic acid oxidase a c t i v i t i e s (Srinivasan et a l . , 1973).  TABLE I I I ENZYMATIC BROWNING, O-DIPHENOL CONTENT,  PPO ACTIVITY, FRESH WEIGHT, AND pH OF  "REDHAVEN" PEACHES RECEIVING GROWTH REGULATOR TREATMENTS  Treatment  Application Time(days)  Tree No.  Browning ARd(29min)  -  1 2 3 4«b  36.6 33.6 32.2 36.2  .1.027 1.253 1.067 1.840  1 2 3 4  33.6 32.1 31.0 35.9  1 2 3 4b 1 2 •3 a  Control  Ethephon(75ppm)  Ethephon(150ppm)  21  21  b  a  a  Gibberellic Acid (100 ppm)  21  4  b  o-diphenols PPO A c t i v i t y mg catechol/g tissue A A b s ^ g 0.1 m l l min~l  Fresh Weight g/fruit  pH  0.163 0.160 0.163 0.183  170.3 • 148.4 181.6 177.6  3.70 3.80 3.75 3.70  1.507 1.053 1.347 1.693  0.135 0.280 0.145 0.190  164.6 201.7 191.4 177.7  3.85 3.70 3.80 3.80  34.4 33.7 34.4 32.8  1.013 1.600 1.000 0.840  0.170 0.145 0.150 0.230  178.0 150.3 151.3 154.2  3.85 3.80 3.85 3.80  30.9 34.5 32.6 35.3  1.027 1.507 0.987 1.533  0.170 0.178 0.175 0.155  185.5 189.2 198.5 162.1  3.85 3.75 3.85 3.60  -  Continued  TABLE I I I  Treatment  (Continued)  Application Time(days)  Ethephon(75ppm)  46  Tree No.  Browning ARd(29min)  o-diphenols mg catechol/g tissue  PPO A c t i v i t y AADS420  0*  1  ~  Tal  1  min  - 1  Fresh Weight g/fruit  pH  1 ab 3 4  31.6 21.4 27.4 29.3  1.640 0.653 0.747 1.280  0.165 0.148 0.125 0.170  141.9 193.2 158.9 123.2  3. 75 3. 85 3. 90 3. 75  2  Ethephon(150ppm)  46  1 2ab 3 4  28.9 22.1 26.8 29.4  1.333 0.840 0.880 1.667  0.188 0.138 0.135 0.133  168.0 232.4 208.3 185.2  3. 85 3.80 3. 85 3. 75  Gibberellic (100  46  1  32.9 33.4 26.3 36.2  1.280 1.467 1.080 1.600  0.200 0.140 0.170 0.255  147.6 133.5 174.6 160.2  3.80 3. 65 3. 75 3. 70  Acid ppm)  b 4 3  a  T r e e s chosen f o r PPO isozyme electrophoresis Trees chosen f o r TLC  TABLE IV ANALYSIS OF VARIANCE OF BROWNING OF REDHAVEN PEACHES  Source of V a r i a t i o n  Mean Square  Degrees of Freedom 6  40.30  Error  21  8.74  Total  27  Treatment  F-ratio  **  ** Denotes s i g n i f i c a n c e at P<.01  TABLE  V  INDIVIDUAL DEGREES OF FREEDOM FOR THE EFFECTS OF TREATMENTS ON BROWNING  '. Degrees of Freedom  Contrast  Q  2  F-ratio  42.70  *  1  128.34  **  GA/Eth  1  32.34  n.s.  Lo/Hi  1  0.00  n.s.  B xC  1  36.75  n.s.  B xD  1  1.69  n.s.  Treatment  6  241.82  C/Tr  1•  E/L  Denotes s i g n i f i c a n c e at P<.01  * Denotes Note:  s i g n i f i c a n c e at P^.05 Q  2  = (MiT! + M T + . . . + M T ) 2  n(Mi + 2  where  M Q  2  M  k  2 2  2  k  . . . + Mfc ) 2  =  (X MT) nlM  2  2  = M u l t i p l i e r s (from Table I I ) 2  = An independent component of the treatment sum of squares  T  = Treatment t o t a l  n  = Number of observations per treatment  - 28 -  Analysis of variance of PPO present  and o-diphenol data from the  study revealed non-significant F-tests (P ^ .05).  A  s i g n i f i c a n t F-test i s not a pre-requisite f o r the p a r t i t i o n i n g of the treatment degrees of freedom ( L i , 1964a) and the l a t t e r show s i g n i f i c a n c e where the former does not.  may  However, analysis  of the i n d i v i d u a l degrees of freedom contrasts revealed no  significant  treatment e f f e c t . PPO  a c t i v i t y i s pH dependent and control of browning by  decreasing  pH i s w e l l known (Eskin et a l . , 1971). Overall treatment e f f e c t s on pH were not s i g n i f i c a n t " (P y .05)  (Table VI).  According  to the  i n d i v i d u a l 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. C e l l expansion of peaches (fresh weight growth) (Chalmers and van den Ende, 1975)  has been well correlated with GA l e v e l s i n the  mesocarp (Jackson, 1968).  Ethephon applied at stage I and II of peach  growth has been known to r e s u l t i n increased f r u i t weight, presumably as a r e s u l t of a thinning e f f e c t (Stembridge and Raff, 1973; 1973).  GA has also been evaluated  Widmoyer, 1971;  Paulson,  f o r peach thinning (Corgan and  Edgerton, 1966). Chalmers et a l . (1976) reported  that  exogenously applied GA and ethephon increased sink strength i n developing  peach f r u i t s .  Increased  c e l l expansion could d i l u t e the  • c e l l constituents involved i n browning. affected f r u i t weight (P < .05)  Growth regulator treatment  (Table VIII).  150 ppm  ethephon  - 29 -  TABLE VI ANALYSIS OF VARIANCE OF pH OF REDHAVEN PEACHES  Mean Square  F-ratio  6  0.0062  n.s.  Error  21  0.0047  Total  27  Source of V a r i a t i o n  Degrees of Freedom  Treatment  n.s.  Not s i g n i f i c a n t at P<.05  TABLE VII INDIVIDUAL DEGREES OF FREEDOM FOR THE EFFECTS OF TREATMENTS ON pH  Contrast  Degrees of Freedom  Q  F-ratio  2  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 I O  - 2  Lo/Hi  1  1.41 x I O  - 3  n.s.  B x C  1  2.56 x I O  - 3  n.s.  B x D  1  1.41 x I O  - 3  n.s.  Treatment  6  3.74 x 10-2  Denotes s i g n i f i c a n c e at P<.05  *  - 30 -  treatment appeared to retard f r u i t growth i f applied 21 days a f t e r bloom but enhanced f r u i t growth i f applied 46 days after bloom (Table I I I and IX; F i g . 1).  The reverse appeared to be true f o r the  75 ppm ethephon a p p l i c a t i o n s . The reasons f o r the d i f f e r i n g e f f e c t s on weight with ethephon concentration and a p p l i c a t i o n date are not clear.  C e l l d i v i s i o n i n peach f r u i t continues for about 30 days a f t e r  p o l l i n a t i o n , a f t e r 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 d i v i s i o n , while the 46-day treatments were applied during the period of c e l l enlargement p r i o r to the onset of stage I I of "Redhaven" peach f r u i t growth (Looney, 1972). M u l t i p l e Regression techniques (Le and T e n i s c i , 1977) were employed to i d e n t i f y 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 s i g n i f i c a n t predictor of browning (Table X, column 1). 2 The c o e f f i c i e n t of multiple determination (R ) of 0.32 indicates that there i s appreciable v a r i a t i o n i n browning a f t e r pH, PPO, f r u i t weight, and o-diphenol data have been considered.  This i s s i m i l a r to the  r e s u l t s of Gajzago et a l . (1976) who found that 30% of the v a r i a t i o n i n browning of apricots was explained by o-diphenol content, and 35% by a combined PPO and o-diphenol e f f e c t . Q u a l i t a t i v e v a r i a b l e s can be analyzed by multiple regression through the use of dummy v a r i a b l e or contrast coding ( L i , 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 V a r i a t i o n  Degrees of Freedom  Mean Square  F-ratio  6  1206  *  Error  21  410  Total  27  Treatment  Denotes s i g n i f i c a n c e 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 s i g n i f i c a n c e at P<.01  - 32 -  Figure 1.  E f f e c t 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 a f t e r bloom  •  Fresh weight means of f r u i t treated with ethephon 46 days a f t e r bloom  - 33 -  T A B L E  S T E P W I S E  M U L T I P L E  X  R E G R E S S I O N  O N  B R O W N I N G  Independent Variable  1  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.  4.791**  o-Diphenol  Regression c o e f f i c i e n t 2 3 4  6.965**  -  6.243**  PPO  n.s.  -  n.s.  Weight  n.s.  -  pH  n.s.  Constant  -  5  4.623** 24.879*  -  -0.056**  -0.055**  -  -  n.s.  22.978  31.625  23.874  35.231  31.105  tSy  3.319  3.189  2.449  2.103  1.915  ttR2  0.326  0.402  0.662  0.761  0.810  ^ S i g n i f i c a n t at P < . 0 1 * S i g n i f i c a n t at P <.05 n.s.  not s i g n i f i c a n t  ( P > .05)  t Standard error of estimate t i r j o e f f i c i e n t 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 i n 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 i s that treated fruit brown less than control f r u i t , and f r u i t 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 i s accounted for by treatment contrasts alone. The result of adding PPO and o-diphenol content as potential independent variables along with the treatment contrasts i s 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 f r u i t 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 a l . , 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 i s 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 a l . (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 i n 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 a l . , 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 f i r s t 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 CHARACTERISTICS AND R VALUES OF POLYPHENOLIC COMPOUNDS EXTRACTED FROM "REDHAVEN" PEACHES* . 3  30  R •i— BAW HOAc  Location Reagent  f  >ot No.  Folin  DPNA  Vanillin  Molybdate  PPO  1  0.71  0.91  fB  slT  C  C  2  0.57  0.89  fB  slT  C  c  c c  3  0.61  0.83  B  T  1Y  fY  4  0.67  0.79  B  T  1Y  fY  5  0.59  0.67  B  T  Y  Y  6  0.65  0.60  B  T  c c c c  Y  Y  7  0.62  0.37  B  OT  OP  1Y  YBr  8  0.61  0.27  fB  fYBr  fY  C  9  0.66  0.15  fB  fYBr  fY  C  10  0.53  0.24  fB  fYBr  c 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  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 FolinCioucalteau 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 (RibereauGayon, 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 i n 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 i n color (Fig. 5). A f i f 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 i n 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 i n the qualitative distribution of phenolic compounds due to growth regulator treatment.  - AO -  Figure 3.  Peach polyphenols v i s u a l i z e d with p-nitroaniline  reagent.  1.01  0.8h  II 5% ACETIC ACID Broken l i n e s indicate l i g h t e r i n t e n s i t y .  diazotized  - 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 v i s u a l i z e d with Na molybdate reagent.  1.01  0.8  ©O  0 0.6  «•  fl2»  I ^  w  (y  I  13 ) V . — /  (15)  J19  0  ' On°Q  1  1  0.2 115% ACETIC ACID  <  1  0.4  —  —  i  1  0.6  Broken l i n e s indicate l i g h t e r i n t e n s i t y .  i _  i  0.8  t  1.0  - 43 -  Figure 6.  °«2 II 5% ACETIC ACID  Peach polyphenols v i s u a l i z e d with polyphenoloxidase.  0.4  0.6  —  Broken l i n e s indicate l i g h t e r i n t e n s i t y .  0.8  1.0  - 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 i n 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 i n 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 i s known to be a good PPO substrate and has been shown to be present i n both freestone (Craft, 1961) and clingstone (Luh et a l . , 1967) peaches. in peaches by Corse (1953).  Neochlorogenic  acid has been identified  - 45 -  TABLE X I I .  VALUES OF AUTHENTIC POLYPHENOLIC COMPOUNDS.  R  Compound BAW  5% HOAc  0.78  0.24  - cis  0.67  0.81  - trans  0.67  0.60  0.58  0.34  Caffeic Acid Chlorogenic Acid  1-Epicatechin  f  -  46  -  Spot 7 was t e n t a t i v e l y i d e n t i f i e d as catechin.  It was  colorless  under UV l i g h t before fuming with ammonia but turned dark a f t e r fuming. It reacted with DPNA reagent giving the orange-tan color c h a r a c t e r i s t i c of catechin and also the c h a r a c t e r i s t i c orange-pink to reddish-pink color with v a n i l l i n - H C l reagent (Swain and H i l l i s , 1959; 1967). BAW  Luh et a l . ,  Catechin has R^ values s l i g h t l y greater than epicatechin i n both  and acetic acid systems on c e l l u l o s e (El-Sayed and Luh, 1965)  does spot 7 (Tables 13 and 14).  Siegelman  as  (1955) showed catechin to  be a major browning substrate i n pear, and i t has been i d e n t i f i e d as a browning substrate i n peach (Craft, 1961; Luh et a l . , 1967). Spots 13, 19 and 20 resemble leucoanthocyanidins i n mobility 1961; Luh et a l . , 1967).  (Craft,  They were c o l o r l e s s under UV l i g h t with and  without ammonia vapor and appeared orange-pink and pink when sprayed with v a n i l l i n - H C l reagent, a c h a r a c t e r i s t i c of leucoanthocyanidins (Luh et a l . , 1967).  These compounds have been shown to be browning  substrates i n peaches (Craft, 1961; Luh et a l . , 1967;  Fel'dman and Kostinskaya,  1970) C a f f e i c acid was not detected, which i s i n agreement with the findings of Craft  (1961) f o r "Elberta" peaches, although i t was present  i n "Halford" peaches (Luh et a l . , 1967). Only one spot was  seen under v i s i b l e l i g h t , spot 17, which was  pink and thought to be anthocyanin. the major anthocyanin i n peaches  Hsia et a l . (1965) reported that  i s cyanidin-3-monoglucoside.  The p r i n c i p a l polyphenolic compounds i n "Redhaven" peaches of the present study oxidized by PPO are tentatively i d e n t i f i e d as chlorogenic acid isomers, a compound with the c h a r a c t e r i s t i c s of catechin, and  - 47 -  leucoanthocyanidins.  This i s in agreement with the results of Craft  (1961), Luh et a l . (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 f r u i t .  The final color  of enzymatic browning i s 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 i s also important. Fel'dman and Kostinskaya (1970) reported that the amount of oxidizable polyphenols i n peach varied with the cultivar and type of polyphenol.  Browning resulted in  a decrease i n 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 i n  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 a l . (1973) found that  - 48 -  GA treatment decreased cherry anthocyanins.  Aoki et a l . (1971) found  PAL to be present only in the red, anthocyanin containing portions, of mature peaches.  Buchanan et a l . (1969) noted that ethephon treated  peaches had more red color than non-treated or GA treated fruit. Anthocyanin i s 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 i s similar to the coupled oxidation of  ascorbic acid, used in controlling browning (Eskin et a l . , 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 i n particular, but there has been numerous mention made of alterations in peroxidase (PRO) isozymes. Although PRO contains an iron porphyrin as i t s 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 i s unknown. Galston et a l . (1968) reported induction of a PRO isozyme in tobacco tissue culture by IAA.  Imaseki et a l . (1968)  found that ethylene stimulated several PRO isozymes but not others, indicating that ethylene may preferentially affect the synthesis of particular isozymes.  Bireka et a l . (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 i n PRO isozyme patterns but quantitatively increased the level of certain isozymes and decreased that of others (Lee, 1972).  Inhibition  of PRO activity i n sugar cane stem tissue by GA produced no change in the isozyme banding pattern (Glasziou et a l . , 1968).  - 50 -  It was  decided to examine the PPO  isozymes i n treated and  untreated "Redhaven" peaches to determine whether treatment with GA and ethephon a f f e c t s isozyme number and/or substrate s p e c i f i c i t y . Preliminary attempts at separating crude PPO  preparation by  electrophoresis were unsatisfactory due to low protein concentration as w e l l as poor r e s o l u t i o n and a r t i f a c t s probably caused by high pectin content  (Frenkel et a l . , 1969).  Removal of p e c t i c materials  by p r e c i p i t a t i o n with calcium chloride and subsequent concentration by u l t r a f i l t r a t i o n allowed s a t i s f a c t o r y separation.  Results are  shown i n Figure 7. Figure 7A shows the banding pattern of the gels when stained for p r o t e i n with coomassie blue.  V i s u a l observations and  densitometric  scans of the gels showed very s i m i l a r banding patterns from control and treated f r u i t .  Incubation of gels i n catechol, the simplest  o-diphenol, revealed up to 11 brown bands a f t e r 1.5 hr (Fig. 7B). Bands b, c, d, and j were v i s i b l e a f t e r approximately  5 min.  The region  from a to e had a dark brown background, as indicated by shading. bands except band k were v i s i b l e on gels from each treatment. was  All  Band k  only v i s i b l e on gels from peaches treated 21 days a f t e r bloom with  both GA and ethephon (75 and 150 ppm)  and appeared very slowly.  Comparison of the m o b i l i t i e s of the bands on the catechol gels with those on the protein gels showed good agreement.  The i n t e n s i t y of  s t a i n i n g i s not the same, however, as the bands on the catechol gels r e s u l t from the enzymatic oxidation of a substrate while those on the protein gels resulted from d i r e c t staining with coomassie blue.  - 51 -  F i g u r e 7.  Reactions  of polyphenoloxidase  o-diphenolic  isozymes w i t h  substrates.  D 0 0.1  0.2  a b  ma  d firm.e  0.3  d  7//A  f  0.4  mm 9  0.5  hi  0.6 0.7  0.8 0.9 1.0  +  PROT  PROT = p r o t e i n ,  CAT  CAF  CAT = c a t e c h o l ,  CHLOR = c h l o r o g e n i c a c i d ,  CHLOR  CAF = c a f f e 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. and k.  Immediately noticeable i s the disappearance of bands c, e,  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. was more intense with caffeic acid than catechol.  Band j  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 d i f f i c u l t to detect minor bands.  A similar effect was noted by Van Loon (1971).  background quickly formed thus increasing the d i f f i c u l t y .  A green  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 c e l l makes i t d i f f i c u l t to determine the functions of the PPO isozymes.  A common  regulatory feature of branched biosynthetic pathways i s 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 a l . (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 c e 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 c e l l . This view i s interesting as PPO has been shown to exist in forms soluble as well as bound to mitochondria and chloroplasts (Harel et a l . , 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 i s 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. pyrogallol were quite light.  Band b on the other gels stained with 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 i s 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 a l . (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 (mM)  Control  GA (late)  75ppm (late)  150ppm (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 e f f e c t of pH on a c t i v i t y of the PPO preparations just discussed i s shown i n Figure 8.  Two pH optima were seen with PPO  from each source; pH 4.4 and 6.2 f o r PPO from control and GA treated peaches, and pH 4.4 and 6.6 f o r PPO from ethephon treated peaches (both 75 and 150 ppm).  The reason f o r the s h i f t i n pH optimum  (from pH 6.2 to 6.6) with ethephon treatment i s unknown.  Jen and  Kahler (1974) reported a s h i f t i n 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 i n the present study were harvested  at the same maturity, the e f f e c t of ethephon i n advancing peach maturity may have also accelerated ripening a f t e r harvest over control and GA treated f r u i t . The decrease i n r e l a t i v e a c t i v i t y at pH 4.4 i n the treated peaches may i n d i c a t e a lower PPO a c t i v i t y with natural substrates at the pH of the peach s l u r r i e s , leading to decreased browning.  No reports have been  found of peach PPO with a pH optimum near 4.4.  E f f e c t of Substrate Concentration  The e f f e c t of varying catechol concentration on a c t i v i t y of a crude PPO preparation from control peaches was studied.  The Michaelis constant  (Km) was determined by least squares treatment of the straight l i n e obtained by p l o t t i n g 1/substrate c o n e , versus 1/v^  (Lineweaver and  100  h  90 U  80 70  60 co  50  40  •  Control  A  GA (46 days)  ©  Ethephon (75 ppm,46 days)  A  Ethephon (150ppm,46 days)  30  20 10  4.0  4.5  5.0  6.0  5.5  6.5  7.0  pH Figure 8.  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 at pH 6.3, and 25°C.  M (catechol)  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 v e l o c i t y of the enzyme.  Smaller Km  values represent greater a f f i n i t y f o r the substrate. The Km value i s one of the c h a r a c t e r i s t i c s of an enzyme; similar Km values under s i m i l a r conditions i n d i c a t e similar enzyme c h a r a c t e r i s t i c s (Jen and Kahler, 1974).  Km values for peach PPO from d i f f e r e n t  sources  have been reported to be: 15 mM catechol for "Halford" peaches (Luh and Phithakpol, 1972),  29 mM catechol f o r "Redhaven" peaches (Jen and  Kahler, 1974), and 120 mM f o r " E l b e r t a " peaches (Reyes and Luh, 1960). Wong et a l . (1971a) reported that peach PPO isozymes have d i f f e r i n g Km values. The d i f f e r i n g c h a r a c t e r i s t i c s of "Redhaven" peach PPO of the present study compared to that of Jen and Kahler  (1974) may indicate differences  i n area, growing conditions and r o o t s t a l k .  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 i n 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 i n treatment, treatment application time, o-diphenol content, PPO activity, and fresh weight.  Unlike previous reports,  the reduction in browning observed i n 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 preparation from control peaches.  by a crude PPO  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 i n 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 i n the browning reaction.  - 62 -  Analysis of p a r t i a l l y p u r i f i e d  peach PPO preparations by  polyacrylamide d i s c - g e l electrophoresis showed the presence of up to 11 isozymes with a c t i v i t y toward catechol.  The isozymes had  d i f f e r i n g substrate s p e c i f i c i t i e s and were present i n d i f f e r i n g amounts.  Treatment 21 days a f t e r 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 a c t i v i t y with the other  substrates tested.  Treatment with 150 ppm ethephon 46 days a f t e r  bloom appeared to decrease the substrate s p e c i f i c i t y 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 a f t e r 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 a c t i v i t y was seen with  o-diphenolic compounds only; no a c t i v i t y was seen with either a monophenol or a p-diphenol. A c t i v i t y toward o-diphenols r e l a t i v e to catechol showed s l i g h t v a r i a t i o n s with treatment which may indicate a l t e r a t i o n s i n r e a c t i v i t y with n a t u r a l l y occurring PPO substrates i n peach. The same PPO preparations exhibited two pH optima i n phosphatec i t r a t e 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.  PPO from GA and ethephon treated f r u i t s had lower  amounts of t o t a l a c t i v i t y at pH 4.4 than control f r u i t .  As pH 4.4  i s closer to the f r u i t pH than pH 6.3, at which PPO a c t i v i t y was measured,  63 -  decreased PPO activity at this pH optimum may be reflected in reduced enzymatic browning in the peach tissue.  - 64 -  LITERATURE CITED  Abeles, F.B. 1972. Biosynthesis and mechanism of action of ethylene. Ann. Rev. Plant Physiol. 23:259. Anderson, J.W. 1968. Extraction of enzymes and subcellular organelles from plant tissues. Phytochem. 7:1973. Aoki, S., Araki, C., Kaneko, K. and Katayama, 0. 1971. Occurrence of L-Phenylalanine ammonia lyase activity in peach fruit. Agr. Biol. Chem. 35:784. Benjamin, N.D. and Montgomery, M.W. 1973. Polyphenol oxidase of Royal Anne cherries: purification and characterization. J i Food Sci. 38:799. Bireka, H., Catalfamo, J . and Urban, P. 1976. Isoperoxidases in sweet potato plants in relation to mechanical injury and ethylene. Plant Physiol. 57:74. Boswell, J.G. 1963. Plant polyphenol oxidases and their relation to other oxidase systems in plants. In "Enzyme Chemistry of Phenolic Compounds". Ed. Pridham, J.B., p.25. Permagon Press, New York. Bright, H.J., Wood, B.J.B., Ingraham, L.L. 1963. Copper, tyrosinase and the kinetic stability of oxygen. Ann. N.Y. Acad. Sci. 100:965. Buchanan, D.W., Hall, C.B., Biggs, R.H. and Knapp, F.W. 1969. Influence of Alar, Ethrel and gibberellic acid on browning of peaches. Hort. Science 4:302. Byers, R.E. and Emerson, F.H. 1973. Effect of SADH and ethephon on peach f r u i t growth and maturation. Hort. Science 8:48. Byers, R.E., Dostal, H.C. and Emerson, F.H. 1969. Regulation of fruit growth with 2-chloroethylphosphonic acid. Bioscience 19:903. Camm, E.L. and Towers, G.H.N. 1973. Phytochem. 12:961.  Phenylalanine Ammonia Lyase.  Chalmers, D.J. and van den Ende, B. 1975. A reappraisal of the growth and development of peach fruit. Aust. J . Plant Physiol. 2:263.  - 65 ~  Chalmers, D.J., van den Ende, B. and J e r l e , P.H. 1976. The e f f e c t of (2-chloroethyl) phosphonic acid on the sink strength of developing peach (Prunus Perslca L.) f r u i t . Planta 131:203. Cohen, J . 1968. M u l t i p l e regression as a general data-analytic system. Psych. B u l l . 70:426. Conn, E.E. 1964. Enzymology of phenolic biosynthesis. In "Biochemistry of Phenolic Compounds," Ed. Harborne, J.B., Ch. 10. Academic Press, New York. Constantinides, S.M. and Bedford, C.L. 1967. M u l t i p l e forms of phenoloxidase, J . Food S c i . 32:446. Corgan, J.N. and Widmoyer, F.B. 1971. The e f f e c t s of g i b b e r e l l i c acid on flower d i f f e r e n t i a t i o n , date of bloom, and flower hardiness of peach. J . Am. Soc. Hort. S c i . 96:54. Corse, J . 1953. A new isomer of chlorogenic a c i d from peaches. 172:771.  Nature  Craft, C.C. 1961. Polyphenolic compounds i n E l b e r t a peaches during storage and ripening. J.Am.Soc.Hort.Sci. 78:119. Craker, L.E. 1975. E f f e c t of ethylene and metabolic i n h i b i t o r s on anthocyanin biosynthesis. Phytochem. 14:151. Davis, B.J. 1964. Disc electrophoresis. I I Method and a p p l i c a t i o n to human serum proteins. Ann. N.Y. Acad. S c i . 121:404. Douglas, M.A., Vanderstoep, J . and Paulson, A. T. 1977. E f f e c t of g i b b e r e l l i c acid and ethephon on ascorbic acid content and ascorbic acid oxidase a c t i v i t y of Redhaven peaches. Can.Inst.Food S c i . Technol.J. 10:233. Duckworth, H. and Coleman, J.E. 1970. Physicochemical and k i n e t i c properties of mushroom tyrosinase. J . B i o l . Chem. 245:1611. Edgerton, L . J . 1966. Some e f f e c t s of g i b b e r e l l i n and growth retardants on bud development and cold hardiness of peach. Proc.Am.Soc. Hort.Sci. 88:197. El-Sayed, A.S. and Luh, B.S. 1965. Polyphenolic compounds i n apricots. J . Food S c i . 30:1017. Eskin, N.A.M., Henderson, H.M. and Townsend, R.J. of Foods". Academic Press, New York.  1971. "Biochemistry  - 66 -  Fel'dman, A.L. and Kostinskaya, L.I. 1970. Peach polyphenols and t h e i r r o l e i n color changes i n the f r u i t . P r i k l . Biochim. i Mikrobiol. 6:442. Frenkel, C , K l e i n , I. and D i l l e y , D.R. 1969. Methods for the study of ripening and protein synthesis i n i n t a c t pome f r u i t s . Phytochem. 8:945 Gajzago, I., Vamos-Vigyazo, L. and Nadudveri-Markus, V. 1976. Investigations into the enzymic browning of apricot c u l t i v a r s . Acta. Alimentaria 6:95. Galston, A.W., Vance, S. and Siegel, B.Z. 1968. The induction and repression of peroxidase isozymes by 3-IAA. In "Biochemistry and Physiology of Plant Growth Substances," Ed. Wightman, F. and S e t t e r f i e l d , G., p.455. Runge Press, Ottawa, Canada. Glaszion, K.T., Gaylor, K.R. and Waldon, J.C. 1968. E f f e c t s of auxin and g i b b e r e l l i c acid on the regulation of enzyme synthesis i n sugar-cane stem t i s s u e . In "Biochemistry and Physiology of Plant Growth Substances," Ed. Wightman, F. and S e t t e r f i e l d , G., p.433. Runge Press, Ottawa, Canada. Goldstein, J.L. and Swain, T. 1963. Changes i n tannins i n ripening f r u i t s . Phytochem. 2:371. Grice, M.R., Brown, H.D. and B u r r e l l , R.C. 1952. V a r i e t a l c h a r a c t e r i s t i c s influence browning of frozen peaches. Food Eng. p.131. Guadagni, D.G., Sorber, D.G. and Wilber, J.S. 1949. Enzymatic oxidation of phenolic compounds i n frozen peaches. Food Tech. 3:359. Gujarati, D. 1970. Use of dummy v a r i a b l e s i n t e s t i n g f o r equality between sets of c o e f f i c i e n t s i n l i n e a r regressions: a generalization. Amer. Stat. December, p.18. Haight, G.P. J r . and Paragamian, V. 1960. Color complexes of catechol with molybdate. A n a l y t i c a l Chem. 32:642. Harel, E. and Mayer, A.M. 1970. The use of a fungal pectate lyase i n the p u r i f i c a t i o n of laccase from peaches. Phytochem. 9:2447. Harel, E., Mayer, A.M. and Lerner, H.R. 1970. Changes i n the l e v e l s of catechol oxidase and laccase i n developing peaches. J . S c i . Fd. A g r i c . 21:542. Harel, E., Mayer, A.M. and Shain, Y. 1965. P u r i f i c a t i o n and m u l t i p l i c i t y of catechol oxidase from apple chloroplasts. Phytochem. 4:738. Hess, D.  1975. "Plant Physiology'.' Springer-Verlag, New York.  - 67 -  Hsia, C.L., Luh, B.S. and Chichester, CO. 1965. Anthocyanin i n freestone peaches. J . Food S c i . 30:5 Imaseki, H., Uchiyama, M. and U r i t a n i , I . 1968. E f f e c t of ethylene on the i n d u c t i v e increase i n metabolic a c t i v i t i e s i n s l i c e d potato r o o t s . Agr. B i o l . Chem. 32:387. Jackson, D.I. 1968. G i b b e r e l l i n and the growth of peach and a p r i c o t f r u i t s . Aust. J . B i o l . Sciences 21:209. Jen, J . J . and Kahler, K.R. 1974. C h a r a c t e r i z a t i o n of polyphenol oxidase i n peaches grown i n the southeast. Hort.Science 9:590. J e r i e , P.H. and T a y l o r , B.K. 1971. Influence of f o l i a r sprays of growth r e g u l a t i n g m a t e r i a l s on the v e g e t a t i v e growth of one-year-old peach t r e e s . Hort.Res. 11:136. Jones, R.  1973. G i b b e r e l l i n s : t h e i r p h y s i o l o g i c a l r o l e . Plant P h y s i o l . 24:571.  Ann. Rev.  Kahn, V.  1975. Polyphenol oxidase a c t i v i t y and browning of three avocado v a r i e t i e s . J . S c i . Fd. A g r i c . 26:1319.  Kahn, V.  1976. Polyphenol oxidase isozymes i n avocado. 15:267.  Kahn, V.  1977. Latency p r o p e r t i e s of polyphenol oxidase i n two avocado c u l t i v a r s d i f f e r i n g i n t h e i r r a t e of browning. J . S c i . Fd. A g r i c . 28:233.  Phytochem.  Knapp, F.W., H a l l , C.B., Buchanan, W.D. and Biggs, R.H. 1970. Reduction i n polyphenoloxidase a c t i v i t y i n peaches sprayed w i t h A l a r , E t h r e l , or g i b b e r e l l i c a c i d . Phytochem. 9:1453. Krebs, K.G., Heusser, D. and Wimmer, H. 1969. Spray reagents. In "Thin-layer Chromatography", ed. S t a h l , E., p. 854. SpringerV e r l a g , New York. Lang, A.  1970. G i b b e r e l l i n s : s t r u c t u r e and metabolism. Plant P h y s i o l . 21:537.  Ann. Rev.  L a n z a r i n i , G., P i f f e r i , P.G. and Zamorani, A. 1972. S p e c i f i c i t y of an o-diphenol oxidase from prunus avium f r u i t s . Phytochem. 11:89. Lavee, S. and M a r t i n , G.C. 1974. Ethephon {1,2- *C(2-Chloroethyl) phosphonic acid} i n Peach F r u i t s . I . P e n e t r a t i o n and P e r s i s t e n c e . J . Am. Soc. Hort. S c i . 99:97. 1/  Lavee, S. and M a r t i n , G.C. 1975. Ethephon { 1,2- ^C(2-Chloroethyl) phosphonic a c i d } i n peach (Prunus p e r s i c a L.) f r u i t s . I I I . S t a b i l i t y and p e r s i s t e n c e . J . Am. Soc. Hort. S c i . 100:28. 1  - 68 -  Le, C. and T e n i s c i , T. 1977. UBC TRP Triangular Regression Package, Computing Centre, the University of B r i t i s h Columbia, Vancouver,B.C. Lee, T.T. 1971. Increase of IAA oxidase by GA i n tobacco c a l l u s cultures. Can. J . Bot. 49:687. Lee, T.T. 1972. Interaction of cytokinin, auxin, and g i b b e r e l l i n on peroxidase isoenzymes i n tobacco tissues cultured i n v i t r o . Can. J . Bot. 50:2471. Leopold, A.C. and Kriedemann, P.E. 1975. "Plant Growth and Development", McGraw-Hill, Toronto. Lerner, H.R. and Mayer, A.M. 1976. Reaction mechanism of grape catechol oxidase: a k i n e t i c study. Phytochem. 15:57. L i , J.C.R. 1964a. " S t a t i s t i c a l Inference I". Edwards Brothers Inc., Ann Arbor, Mich. L i , J.C.R. 1964b. " S t a t i s t i c a l Inference I I " . Edwards Brothers Inc., Ann Arbor, Mich. L i , K.C.,  Boggess, T.S. J r . and Heaton, E.K. 1972. Relationship of sensory ratings with tannin components of canned peaches. J.Food Sci.37:177.  Lineweaver, H. and Burke, D. 1934. The determination of enzyme d i s s o c i a t i o n constants. J . Am. Chem. Soc. 56:658. Looney, N.E. 1972. E f f e c t s of succinic acid 2,2-dimethylhydrazide, 2-chloroethylphosphonic acid, and Ethylene on r e s p i r a t i o n , ethylene production, and ripening of "Redhaven" peaches. Can. J . Plant S c i . 52:73. Looney, N.E., McGlasson, W.B. and Coombe, B.G. 1974. Control of f r u i t ripening i n peach, Prunus persica: action of Succinic acid -2, 2-dimethylhydrazide and (2-chloroethyl) phosphonic acid. Aust. J . Plant Physiol. 1:77. Loughheed, E.C. and Franklin, E.W. 1972. E f f e c t s of temperature on ethylene evolution from ethephon. Can.J. Plant S c i . 52:769. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. 1951. Protein measurement with the F o l i n phenol reagent. J . B i o l . Chem. 193:265. Luh, B.S.  and Phithakpol, B. 1972. C h a r a c t e r i s t i c s of polyphenoloxidase related to browning i n c l i n g peaches. J . Food S c i . 37:264.  Luh, B.S.,  Hsu, E.T. and Stachowicz, K. 1967. Polyphenolic compounds i n canned c l i n g peaches. J . Food S c i . 22:807.  Mapson, L.W., Swain, T. and Tomalin, A.W. 1963. Influence of v a r i e t y , c u l t u r a l conditions and temperature of storage on enzymic browning of potato tubers. J . S c i . Fd. A g r i c . 14:673. Markert, C L . 1974. Biology of isozymes. In: "Isozymes: I Molecular Structure," Ed. Markert, C L . p.11. Academic Press, New York.  - 69 -  Mason, H.S. 1957. Mechanisms of oxygen metabolism. 19:131.  Adv. Enz.  Mathew, A.G. and Parbla, H.A.B. 1971. Food browning as a polyphenol reaction. Adv. Food Res. 19:75. Moss, D.W. and Butterworth, P.J. 1974. "Enzymology and Medicine", p.28, Copp Clark Pub. Co., Toronto, Ontario. Nakabayashi, T. and Uhai, N. 1963. Browning of peaches by polyphenolase. Japanese J . Food Tech. 10:211. Nitsch, J.P. 1970. Hormonal factors i n growth and development. In "The Biochemistry of F r u i t s and Their Products". Vol. I. Ed. Hulme, A . C , Ch. 15, Academic Press, New York. Ogawa, Y.  1965. Changes i n the content of g i b b e r e l l i n - l i k e substances i n the seed of Prunus persica Bot. Mag. 78:412.  Paulson, A. 1973. E f f e c t of f o l i a r sprays of E t h r e l and g i b b e r e l l i c acid on enzymatic browning of Fairhaven and Redhaven peaches. Unpublished undergraduate thesis, Dept. of Food Science, U.B.C. Ponting, J.D. 1960. Control of enzymatic browning of f r u i t s . In "Food Enzymes", ed. Schultz, H.W. Ch. 9 A v i Publishing Company, Westport, Conn. Ponting, J.D. and Joslyn, M.A. 1948. Ascorbic acid oxidation and browning i n apple tissue extracts. Arch. Biochem. 19:47. P o r r i t t , B. 1974. E f f e c t of storage treatments and f o l i a r sprays of ethephon and g i b b e r e l l i c acid on Redhaven peaches for processed r e f r i g e r a t e d peach s l i c e s . Unpublished undergraduate thesis, Dept. of Food Science, U.B.C Potty, V.H. 1969. Determination of proteins i n the presence of phenols and pectins. A n a l y t i c a l Bioc. 29:535. Proebsting, E.L. J r . and M i l l s , H.H. 1966. E f f e c t of g i b b e r e l l i c acid and other growth regulators on q u a l i t y of Early I t a l i a n prunes (Prunus domestica L.) Proc. Am. Soc. Hort. S c i . 89:135. Proebsting, E.L. J r . and M i l l s , H.H. 1969. E f f e c t s of 2-chloroethane phosphonic acid and i t s i n t e r a c t i o n with g i b b e r e l l i c acid on q u a l i t y of Early I t a l i a n prunes. J . Am. Soc. Hort. S c i . 94: 443. Proebsting, E.L. J r . , Carter, G.H. and M i l l s , H.H. 1973. Quality improvement i n canned Rainier cherries (P_. avium L.) with g i b b e r e l l i c acid. J . Am. Soc. Hort. S c i . 98:334.  - 70 -  Reeve, R.M. 1959. Histological and histochemical changes in developing and ripening peaches. I The catechol tannins. Am. J. Bot. 46:210. Reyes, P. and Luh, B.S. 1960. Characteristics of browning enzymes in Fay Elberta freestone peaches. Food Tech. 14:570. Ribereau-Gayon, P.  1972. "Plant Phenolics", Hafner Pub. Co., New York.  Rivas, N. and Whitaker, J.R. 1973. Purification and some properties of two polyphenoloxidases from Bartlett pears. Plant Physiol. 52:501. Roberts, E.A.H. 1956. The chlorogenic acids of tea and mate. and Industry, p.985.  Chemistry  Rom, R. and Scott, K. 1971. The effect of 2-chloroethylphosphonic acid (ethephon) on maturation of a processing peach. Hort. Science 6:134. Salisbury, F.B. and Ross, C. 1969. Pub. Co., Belmont, Cal.  "Plant Physiology",  p.398, Wadsworth  Sal'kova, E.G., Zuyagintseva, Y.V., Kuliev, A.A. and Akhundov, R.M. 1977. (Effect of Ethrel on biochemical processes during fruit ripening). Prikl. Biokhim. i Mikrobiol. 13:97 (Abstract). Sarkar, S.K. and Ton Phan, C. 1974. Effect of ethylene on the qualitative and quantitative composition of the phenol content of carrot roots. Physiol. Plant. 30:72. Sato, M. and Hasegawa, M. 1976. The latency of spinach chloroplast phenolase. Phytochem. 15:61. Schaller, D. and von Elbe, J. 1970. Polyphenols i n Montmorency cherries. J. Food Sci. 35:762. Siegelman, H.W. 1955. Detection and identification of polyphenoloxidase in apple and pear skins. Arch. Biochem. Biophys. 56:97. Seikel, M.K. 1962. Chromatographic methods of separation, isolation, and identification of flavanoid compounds. In "The Chemistry of Flavanoid Compounds", p. 34, Ed. Geissman, T.A. Macmillan Pub. Co., New York. Sheen, S.J. and Calvert, J. 1969. Studies on polyphenol content, activities and isozymes of polyphenol oxidase and peroxidase during air-curing i n three tobacco types. Plant Physiol. 44:199.  - 71 -  Shinshi, H. and Noguchi, M. 1975. Relationships between peroxidase, IAA Oxidase and Polyphenol Oxidase. Phytochem. 14:1255. Srinivasan, C , Pappiah, CM. and Doraipandian, A. 1973. Effect of gibberellic acid on ascorbic acid, sugar content and oxidative enzyme activity of West Indian Cherry (Malpighia punicifola) f r u i t . J. Exp. Biol. 11:469. Srivastava, O.P. and van Huystee, R.B. 1973. Evidence for close association of peroxidase, polyphenol oxidase, and IAA oxidase of peanut suspension culture medium. Can. J. Bot. 51:2207. Stembridge, G.E. and Raff, J.W. Hort. Science 8:500.  1973.  Ethephon and peach fruit development.  Stuart, N.W. and Cathey, H.M. 1961. Applied aspects of the gibberellins. Ann. Rev. Plant Physiol. 12:369. Swain, T. and H i l l i s , W.E. 1959. The phenolic constituents of Prunus domestica. I. The quantitative analysis of phenolic constituents. J. Sci. Food Agric. 10:63. Taneja, S.R. and Sachar, R.C 1974. Separate monophenolase and o-diphenolase enzymes i n Triticum Aestivum. Phytochem. 13:1367. Van Blaricom, L.O. and Senn, T.L. 1967. Anthocyanidin pigments i n freestone peaches grown in the Southeast. Proc. Am. Soc. Hort. Sci. 90:541. Van Buren, J. 1970. Products". New York.  Phenolics.In "The biochemistry of Fruits and Their Vol. I. Ed. Hulme, A.C, Ch. 11, Academic Press,  Van Loon, L.C. 1971. Tobacco polyphenoloxidases: a specific staining method indicating non-identity with peroxidases. Phytochem. 10:503. Walker, J.R.L. 1962. Studies on the enzymic browning of apple fruit. N.Z.J.Sci. 5:316. Walker, J.R.L. 1975. "The Biology of Plant Phenolics". Edward Arnold (Publishers) Ltd. London. Walker, J.R.L. and Wilson, E. 1975. Studies on the enzymatic browning of applies. Inhibition of apple o-diphenol oxidase by phenolic acid. J. Sci. Food Agric. 26:1825. Weaver, C. and Charley, H. 1974. J. Food Sci. 39:1200.  Enzymatic browning of ripening bananas.  - 72 -  Whitaker, J.R. 1972. "Principles of Enzymology for the Food Sciences." p. 577. Marcel Dekker, Inc., New York. Williams, A.H. .1955. Paper chromatography of cinnamic acid derivatives. Chemistry and Industry, p.120. Wong, T.C., Luh, B.S. and Whitaker, J.R. 1971a. Isolation and characterization of polyphenol oxidase isozymes of clingstone peach. Plant Physiol. 48:19. Wong, T.C., Luh, B.S. and Whitaker, J.R. 1971b. Effect of phloroglucinol and resorcinol on the clingstone peach polyphenol oxidase catalyzed oxidation of 4-methyl catechol. Plant Physiol. 48:24. Yang, S.F. 1969. Ethylene evolution from 2-chloroethylphosphonic Plant Physiol. 44:1203.  acid.  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0094297/manifest

Comment

Related Items