Physiological Roles of Peroxidase in Postharvest Fruits and Vegetables

9 Physiological Roles of Peroxidase in Postharvest Fruits and Vegetables

Downloaded by GEORGE MASON UNIV on February 29, 2016 | http://pubs.acs.org Publication Date: June 1, 1977 | doi: 10.1021/bk-1977-0047.ch009

NORMAN F. HAARD Department of Biochemistry, Memorial University of Newfoundland, St. Johns, Newfoundland, Canada, A1C5S7

Peroxidase (E.C. 1.11.1.7, hydrogen donor oxidoreductase) was initially reported as a c o n s t i t u e n t of mushroom e x t r a c t s i n 1855 (1) and was l a t e r named by L i n o s s i e r i n 1898 (2). Since t h i s time i t has been the subject of continued i n t e r e s t , experimentat i o n and s p e c u l a t i o n (3). Peroxidase appears to be ubiquitous to the living s t a t e having been i d e n t i f i e d i n animal, p l a n t , microbial and viral systems. Peroxidase is a common c o n s t i t u e n t of higher p l a n t s and may occur a t concentrations up to s e v e r a l percent on a f r e s h weight b a s i s . There is extensive l i t e r a t u r e d e s c r i b i n g changes in peroxidase a c t i v i t y and isoenzyme spectra as a f u n c t i o n of ontogenic change, c o n d i t i o n s of s t r e s s (e.g. water deficit, freeze i n j u r y , chill i n j u r y , h y p e r s e n s i t i v i t y , pathogen i n t e r a c t i o n , hyperoxygenicity, etc.) and as a s p e c i f i c c h a r a c t e r i s t i c of plant v a r i e t i e s and c u l t i v a r s . Although thousands of experimental studies with peroxidase have been publ i s h e d , we have a relatively poor grasp of the f u n c t i o n ( s ) and metabolic c o n t r o l ( s ) of t h i s enzyme in v i v o . Today, there a r e many who b e l i e v e that peroxidase is a v e s t i g e of the past, having no e s s e n t i a l f u n c t i o n in higher p l a n t s . A l t e r n a t i v e l y , other i n d i v i d u a l s f i n d it tempting to link peroxidase with a myriad of important events which include r e s p i r a t o r y c o n t r o l , gene c o n t r o l , hormone metabolism and the b i o s y n t h e s i s and biodegradation of a wide range of secondary plant metabolites. There have been ext e n s i v e s t u d i e s d e s c r i b i n g the a c t i o n of peroxidase on substances which y i e l d b r i g h t c o l o r s on o x i d a t i o n , but which have no apparent p h y s i o l o g i c a l r o l e . Unfortunately, there has been f a r too little done to understand the p h y s i o l o g i c a l l y r e l e v a n t subs t r a t e s f o r t h i s enzyme. I r r e s p e c t i v e of t h e i r p h y s i o l o g i c a l r o l e , it has been w e l l e s t a b l i s h e d that peroxidase can c o n t r i b u t e to d e t e r i o r a t i v e changes i n f l a v o r , texture, c o l o r and n u t r i t i o n i n properly processed f r u i t s and vegetables (4-6). I t has g e n e r a l l y been observed that peroxidase is q u i t e s t a b l e to adverse c o n d i t i o n s encountered during food processing - such as elevated temperature, f r e e z i n g , i o n i z i n g r a d i a t i o n , dehydration and even when inacti143 In Enzymes in Food and Beverage Processing; Ory, Robert L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


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vated by such treatments i s capable of r e - n a t u r a t i o n (7). Regene r a t i o n of peroxidase a c t i v i t y can pose a s e r i o u s l i m i t a t i o n to a food p r o c e s s i n g o p e r a t i o n . For example, f r u i t s and vegetables subjected to h i g h temperature - short time treatments (HTST) are p a r t i c u l a r l y prone to peroxidase r e g e n e r a t i o n and a s s o c i a t e d q u a l i t y change during storage. Regeneration of peroxidase occurs w i t h i n hours or days f o l l o w i n g thermal processing and may occur even a f t e r s e v e r a l months i n f r o z e n f r u i t s and vegetables. Peroxidase regeneration appears to i n v o l v e r e f o l d i n g of the polypeptide chain (8) and may r e l a t e to the f a c t that peroxidase contains a conjugated carbohydrate moiety. The r e s i s t a n c e of peroxidase to t h e r mal i n a c t i v a t i o n , together with i t s u b i q u i t y and d i r e c t i n v o l v e ment i n d e t e r i o r a t i o n of food q u a l i t y , has l e d to the wide use of peroxidase a c i t i v i t y as an index of processing e f f i c a c y by blanching and other heat, treatments. Peroxidase i s a l s o of u t i l i t y to the food t e c h n o l o g i s t . For example, i t i s used e x t e n s i v e l y i n conjunction with glucose o x i dase f o r the d e t e c t i o n and determination of glucose. In t h i s r e a c t i o n the hydrogen peroxide generated by the glucose oxidase c a t a l y z e d conversion of glucose to g l u c o n i c a c i d i s used i n the p e r o x i d a t i c r e a c t i o n to produce a r e a d i l y measurable chromophore from a hydrogen donor such as O - d i a n i s i d i n e . The coupled r e a c t i o n of glucose oxidase and peroxidase may a l s o f i n d use i n food processes designed to r i d a system of r e s i d u a l glucose or oxygen (9). A l s o , the f i n d i n g that isoenzyme p r o f i l e s of peroxidase may be h i g h l y s p e c i f i c f o r d i f f e r e n t p l a n t t i s s u e s a l s o leads to the sugg e s t i o n that peroxidase be used as a means of checking a d u l t e r a t i o n or contamination i n p l a n t e x t r a c t i v e s such as f l o u r s and protein isolates. In t h i s t r e a t i s e , I w i l l examine s e v e r a l l i n e s of experiment a t i o n designed to understated the p h y s i o l o g i c a l r o l e of peroxidase i n postharvest f r u i t s and vegetables. In most case, harvested f r u i t s and vegetables undergo ontogenic change s i m i l a r to that which occurs on the parent p l a n t . A c c o r d i n g l y ; t h e r e a c t i o n s to be discussed are somewhat d i f f e r e n t from those o c c u r r i n g i n processed foods where the s t r u c t u r e and f u n c t i o n a l c a p a b i l i t y of the t i s s u e has been g r o s s l y d i s r u p t e d . I t i s c l e a r that at l e a s t part of the mystery surrounding peroxidases i s due to the presence of unusua l l y l a r g e numbers of isoenzyme species and to the observation that peroxidase can c a t a l y z e a v a r i e t y of r e a c t i o n s , i n some cases with apparently low s u b s t r a t e s p e c i f i c i t y . For t h i s reason i t i s important to review some general information on t h i s enzyme. Classification Peroxidases have been c l a s s i f i e d as i r o n c o n t a i n i n g p e r o x i dases and f l a v o p r o t e i n peroxidases ( 3 ) . The i r o n enzymes are f u r ther subgrouped i n t o ferriprotoporphyrin peroxidases and verdoperoxidases. The f i r s t group a l l c o n t a i n f e r r i p r o t o p o r p h y r i n I I I as a p r o s t h e t i c group and e x h i b i t a red-brown c o l o r when h i g h l y p u r i -

In Enzymes in Food and Beverage Processing; Ory, Robert L., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1977.


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f i e d . The f e r r i p r o t o p o r p h y r i n type peroxidases are common i n higher p l a n t s and have a l s o been i d e n t i f i e d i n animals (e.g. t r y p tophan p y r r o l a s e , t h y r o i d i o d i n e peroxidase) and microorganisms (e.g. yeast cytochrome C p e r o x i d a s e ) . The verdoperoxidases c o n t a i n an i r o n porphyrin p r o s t h e t i c group which d i f f e r s from f e r r i p r o t o porphyrin I I I and which i s not removed on treatment with a c i d i c acetone as occurs with the former group of peroxidases. Examples of t h i s enzyme are lactopeioxidase found i n m i l k and myeloperoxidase from myelocytes. F l a v o p r o t e i n peroxidases, c o n t a i n FAD as p r o s t h e t i c group, and have been p u r i f i e d from microorganisms (e.g. Streptococcus f a e c a l i s ) a n d animal t i s s u e s . At t h i s time we know r e l a t i v e l y l i t t l e about the l a t t e r two groups of p e r o x i dase i n higher p l a n t s . However, the f e r r i p r o t o p o r p h y r i n I I I peroxidases from h o r s e r a d i s h , Japanese r a d i s h and t u r n i p have been f a i r l y w e l l c h a r a c t e r i z e d . The h o r s e r a d i s h enzyme has a molecul a r weight of approximately 40,000 and contains one f e r r i p r o t o porphyrin I I I group per molecule. Four of the s i x c o o r d i n a t i o n bonds are taken up i n i n t e r a c t i o n with the p y r r o l e r i n g n i t r o g e n s . One of the remaining c o o r d i n a t i o n bonds appears to be a s s o c i a t e d with a c a r b o x y l group of the p r o t e i n and the other i s coordinated to an amino group or to a water molecule. The h o r s e r a d i s h enzyme and apparently other sources of peroxidase (10) c o n t a i n conjugated carbohydrate which appears to impart unusual s t a b i l i t y to the molecule. The h o r s e r a d i s h enzyme i s s t a b l e i n s o l u t i o n from pH 4 to 12, r e t a i n s 50% of i t s a c t i v i t y a f t e r h e a t i n g at 100 C f o r 12 minutes and i s s t a b l e to low water a c t i v i t y and f r e e z i n g . Because of t h e i r c h a r a c t e r i s t i c a b s o r p t i o n maxima i n the U.V. and i n the Soret r e g i o n the peroxidases are o f t e n c h a r a c t e r ized by the r a t i o of a b s o r p t i o n at 403 and 275 nm or the R.Z. value ( R e i n h e i t s z a h l ) . A h i g h l y p u r i f i e d isoenzyme of h o r s e r a d i s h peroxidase may have an R.Z. value greater than 3.0. Catalytic Properties Four general types of c a t a l y t i c a c t i v i t y have been found i n a s s o c i a t i o n with peroxidases. These are the p e r o x i d a t i c , o x i d a t i c , c a t a l a t i c and h y d r o x y l a t i o n r e a c t i o n s . The p e r o x i d a t i c r e a c t i o n , more g e n e r a l l y thought to be of most p h y s i o l o g i c a l s i g n i f i c a n c e , has been studied more e x t e n s i v e l y than the other three reactions. P e r o x i d a t i c Reaction. In g e n e r a l , p e r o x i d a t i c r e a c t i o n s occur i n the presence of a wide v a r i e t y of hydrogen donors, i n c l u d i n g p - c r e s o l , q u a i c o l , r e s o r c i n o l , benzidine and 0 - d i a n i s i dine. C e r t a i n peroxidases appear to have a greater a f f i n i t y f o r s p e c i f i c hydrogen donors such as NADH, g l u t a t h i o n e or cytochrome C (11). One approach to e l u c i d a t e hydrogen donors of p h y s i o l o g i c a l importance i s the use of a f f i n i t y chromatography (12). The p r e f e r r e d oxidant f o r the p e r o x i d a t i c r e a c t i o n i s hydrogen peroxide although other peroxides are e f f e c t i v e s u b s t r a t e s . In post-


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harvest t i s s u e s the p e r o x i d a t i c r e a c t i o n of most obvious importance at t h i s time i s l i g n i f i c a t i o n which can profoundly i n f l u e n c e the toughening of vegetables such as beans and asparagus. I t has a l s o been suggested that the p e r o x i d a t i c r e a c t i o n f u n c t i o n s to protect the c e l l u l a r m i l i e u from peroxides which may cause an imbalance i n redox p o t e n t i a l and damage membranes, enzymes, e t c . (13). O x i d a t i c Reaction. The o x i d a t i c r e a c t i o n r e q u i r e s the presence of molecular oxygen and a s u i t a b l e hydrogen donor. Examples of s o - c a l l e d redogenic hydrogen donors are i n d o l e - 3 - a c e t i c a c i d (IAA), o x a l a c e t i c a c i d , dihydroxyfumaric a c i d , a s c o r b i c a c i d and hydroquinone. The IAA oxidase f u n c t i o n of peroxidase appears to be extremely important i n postharvest f r u i t s and vegetables. There i s a l s o recent evidence that c y t o k i n i n s are o x i d i z e d by peroxidase. I t may a l s o be that the o x i d a t i c f u n c t i o n may cont r i b u t e to d e t e r i o r a t i v e r e a c t i o n s such as membrane l i p i d o x i d a t i o n and o x i d a t i o n of e s s e n t i a l t h i o l groups. Treatment of peroxidases which are e f f i c i e n t i n p e r o x i d a t i c r e a c t i o n with c e r t a i n s u l f h y d r y l reagents w i l l convert them to forms capable of o x i d a t i c c a t a l y z e d o x i d a t i o n of hydrogen donors such as membrane l i pids (14). I t i s tempting to speculate that peroxidase f u n c t i o n s i n such a d e t e r i o r a t i v e way i n senescing or s t r e s s e d f r u i t s and vegetables s i n c e increases i n peroxidase a c t i v i t y i n v a r i a b l y precede or accompany the h y p e r s e n s i t i v e r e a c t i o n . C a t a l a t i c and Hydroxylation Reactions. In the absence of hydrogen donor, peroxidase can convert hydrogen peroxide to water and oxygen although t h i s r e a c t i o n i s some 1000 times slower than the p e r o x i d a t i c and o x i d a t i c r e a c t i o n s . F i n a l l y i n the presence of c e r t a i n hydrogen donors, such as dihydroxyfumaric a c i d , and molecular oxygen, peroxidase can c a t a l y z e h y d r o x y l a t i o n of a v a r i e t y of aromatic compounds notably t y r o s i n e , phenylalanine, p - c r e s o l and benzoic a c i d . The metabolism of phenolic substances i s of p a r t i c u l a r importance to the q u a l i t y of postharvest f r u i t s and vegetables i n that they may act as e f f e c t o r s of hormone metabo l i s m , intermediates i n l i g n i n b i o s y n t h e s i s and may r e s u l t i n d i s c o l o r a t i o n r e s u l t i n g from t h e i r enzymic or non-enzymic oxidation. Isoenzymes The occurrence of m u l t i p l e forms of peroxidase was f i r s t noted by T h e o r e l l (15) working with h o r s e r a d i s h r o o t s . This t i s sue contains seven major isoenzyme forms, which are s i m i l a r i n molecular weight and amino a c i d composition but may be separated by i o n exchange chromatography or e l e c t r o p h o r e s i s (16). The c a t i o n i c species c o n t a i n greater numbers of a r g i n i n e residues while the a n i o n i c species are r i c h i n glutamic a c i d , phenylalanine and t y r o s i n e . Other sources of isoperoxidases are s i m i l a r to the



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horseradish species i n t h e i r constancy of molecular weight and d i f f e r e n c e s i n e l e c t r o p h o r e t i c behavior. Peroxidase from r i p e n ing banana f r u i t i s composed of at l e a s t twelve isoenzymes which have i s o e l e c t r i c p o i n t s ranging from approximately 3.3 up to 9.5 (17). T h i s wide range i n P j makes i t impossible to i s o l a t e and p u r i f y a l l isoenzymes by one technique s i n c e c e r t a i n species are i n v a r i a b l y l o s t by p r e c i p i t a t i o n or adsorption phenomena. There are many s t u d i e s showing changes i n isoperoxidase with r e spect to growth, d i f f e r e n t i a t i o n , age and r e a c t i o n to environment a l s t r e s s e s . We w i l l come back to some s p e c i f i c examples of such change i n postharvest systems. While isoenzyme forms o f t e n d i f f e r i n c a t a l y t i c e f f i c i e n c y (18), f a r too l i t t l e work has been done to e l u c i d a t e the physiol o g i c a l s i g n i f i c a n c e of these m u l t i p l e forms. One must c e r t a i n l y t e s t the p o s s i b i l i t y that isoperoxidases are an a r t i f a c t of the i s o l a t i o n and p u r i f i c a t i o n procedures employed (19). I t has a l s o been shown that c e r t a i n isoenzymes may be formed from p r e e x i s t i n g species during plant development i n v i v o or i n v i t r o during ext r a c t i o n (20). There have been, however, numerous experiments employing i n h i b i t o r s of t r a n s c r i p t i o n and t r a n s l a t i o n , deuterium oxide l a b e l and i n c o r p o r a t i o n of labeled-amino a c i d s which demonstrate the de nova synthesis of new isoperoxidases during the p l a n t ' s l i f e c y c l e (21,22). Cellular Localization There are s e v e r a l r e p o r t s shwoing that peroxidase i s l o c a l ized i n v a r i o u s s e c t o r s of the c e l l i n c l u d i n g ribosomes (23), nucleus (24), nucleolus (24), mitochondria (25), c e l l w a l l s (26) and i n the i n t e r c e l l u l a r spaces (27). Peroxidase may be bound to given c e l l p a r t i c u l a t e s by i o n i c i n t e r a c t i o n or by covalent bonding (26). In most cases, only c e r t a i n isoenzyme species are found i n a s s o c i a t i o n with a c e l l u l a r o r g a n e l l e or f r a c t i o n . Unf o r t u n a t e l y i t i s o f t e n d i f f i c u l t to d i s t i n g u i s h l o c a l i z a t i o n r e p r e s e n t a t i v e of the n a t i v e c e l l and that which a r i s e s as a r e s u l t of c e l l u l a r d i s r u p t i o n . I t i s , however, i n t r i g u i n g to spec u l a t e that nature's r a t i o n a l e f o r m u l t i p l e molecule forms of peroxidase l i e s i n t h e i r a f f i n i t y f o r s p e c i f i c microenvironments w i t h i n and between the c e l l s . Such microcompartmentation may provide an added dimension of s p e c i f i c i t y to the peroxidases. In a d d i t i o n , i t i s known that a s s o c i a t i o n of peroxidase with c e l l surfaces can profoundly a f f e c t t h e i r c a t a l y t i c p r o p e r t i e s . This phenomenon, c a l l e d a l l o t o p i c c o n t r o l , w i l l be i l l u s t r a t e d i n the f o l l o w i n g case s t u d i e s . C o n t r o l of L i g n i n B i o s y n t h e s i s L i g n i f i c a t i o n may occur i n postharvest f r u i t s , vegetables, c e r e a l s and pulses and have a profound i n f l u e n c e on the e d i b l e q u a l i t y of the t i s s u e . The presence of l i g n i n and a s s o c i a t e d

American Chemicaf Society Library 1155 16th St. H. W. In Enzymes in Food and Beverage Processing; Ory, Robert L., et al.; Washington, D. C. 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1977.

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TABLE I . Comparison of browning and g r i t c e l l formation i n d i f f e r e n t pear c u l t i v a r s .


Cultivar

TABLE I I .

Browning

Sclereids

40.0

++ +

++ +

52.5

++ +

++ +

97.5

++++++

+

22.5

+

Total polyphenolics

+++++ +

T o t a l recoverable peroxidase from developing pear fruit. Peroxidase a c t i v i t y , assayed with hydrogen peroxide as oxidant and O - d i a n i s i d i n e as H-donor, has the u n i t s A 460 nm/min/mg Ν a t 25 C. 'Yuzuhada f r u i t e x h i b i t excessive l i g n i f i c a t i o n r e l a t i v e to ' B a r t l e t t ' f r u i t . Data from (34). 1

peroxidase days before harvest

'Bartlett'

'Yuzuhada'

0.62

TABLE I I I .

35

1.19

28

1.28

1.18

21

0.84

0.82

14

0.89

0.52

7

1.10

0.63

0

1.18

0.82

Soluble peroxidase from developing pear f r u i t . Peroxidase a c t i v i t y , assayed with hydrogen perox ide as oxidant and O - d i a n i s i d i n e as Η-donor, has the u n i t s A 460 nm/min/mg Ν a t 25 C. 'Yuzuhada' f r u i t have excessive l i g n i f i c a t i o n compared t o ' B a r t l e t t f r u i t . Data from (34). 1

peroxidase 'Bartlett'

'Yuzuhada'

21

0.65

0.39

14

0.81

0.29

7

1.08

0.37

0

1.18

0.57

days before harvest



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f i b r o u s m a t e r i a l s can reduce the d i g e s t a b i l i t y c o e f f i c i e n t of prot e i n s by as much as eighty percent. Toughening of vegetables, such as asparagus and beans, i s due to l i g n i f i c a t i o n of f i b r o v a s c u l a r t i s s u e which occurs s h o r t l y a f t e r harvest (28). Simil a r l y , c e r t a i n f r u i t s c o n t a i n aggregates of h i g h l y l i g n i f i e d c e l l s ( s c l e r e i d s ) which impart a g r i t t y t e x t u r e to the pulp (29). The extent of l i g n i f i c a t i o n i n such cases i s markedly i n f l u e n c e d by v a r i e t y or c u l t i v a r type, c u l t i v a t i o n p r a c t i c e s and postharvest h a n d l i n g . Peroxidase i s involved with c o n j u n c t i o n of phenylpropanoid u n i t s during the d e p o s i t i o n of l i g n i n (30). While other enzymes p a r t i c i p a t e i n l i g n i n b i o s y n t h e s i s , evidence p o i n t s to the peroxidase c a t a l y z e d r e a c t i o n as an important l e v e l of c o n t r o l (31). T i s s u e c u l t u r e s t u d i e s have shown that l i g n i n formation occurs only when peroxidase i s a s s o c i a t e d with c e l l surfaces and that calcium ions e l i c i t s o l u b l i z a t i o n of the w a l l bound enzyme (32, 33). The concept that peroxidase involvement i n l i g n i f i c a t i o n i s subject to a l l o t o p i c c o n t r o l provided a c l u e to understanding and s o l v i n g a problem encountered by f r u i t growers. "Yuzuhada D i s o r d e r " . Pear f r u i t may e x h i b i t a p h y s i o l o g i c a l d i s o r d e r c a l l e d "Yuzuhada" which i s symptomized by excessive s c l e r e i d development (29). "Yuzuhada" i s transmitted as a hered i t a r y c h a r a c t e r i s t i c but may develop i n any c u l t i v a r which i s subjected to adverse growing c o n d i t i o n s . Our survey of some f o r t y s e l e c t i o n s of pear f r u i t i n d i c a t e d an i n v e r s e r e l a t i o n s h i p between f r e e p h e n o l i c substances, noteably c h l o r o g e n i c a c i d , and the degree of s c l e r e i d formation (Table 1) (34). This observation suggested any given pear s e l e c t i o n w i l l i n v a r i a b l y e i t h e r be h i g h l y s u b j e c t to enzymic browning or to l i g n i f i c a t i o n and the most acceptable s e l e c t i o n s were intermediate i n l e v e l s of f r e e phenoli c s and i n l i g n i n content. From these f i n d i n g s we hypothesized that m i n e r a l n u t r i t i o n and l o c a l i z a t i o n i n the developing f r u i t determine the extent of peroxidase a s s o c i a t i o n with the l i g n i n template(s) which, i n t u r n , d e l i m i t s the conjugation of phenylpropanoid u n i t s i n t o l i g n i n . The t h e s i s was supported by our study of peroxidase d i s t r i b u t i o n i n s o l u b l e and p a r t i c u l a t e p o o l s . Comparison of peroxidase i n a pear s e l e c t i o n extremely prone to s c l e r e i d development with a l e s s s u s c e p t i b l e v a r i e t y at v a r i o u s stages of f r u i t development revealed that peroxidase l o c a l i z a t i o n and not i t s net a c t i v i t y were p o s i t i v e l y r e l a t e d to s c l e r e i d f o r mation. Although t o t a l peroxidase was c o n s i s t e n t l y higher i n the pear s e l e c t i o n e x h i b i t i n g minimal l i g n i f i c a t i o n (Table 2), i t was p r i m a r i l y l o c a l i z e d i n the s o l u b l e phase (Table 3 ) . A l t e r n a t i v e l y , a r e l a t i v e l y h i g h f r a c t i o n of peroxidase was a s s o c i a t e d with c e l l p a r t i c u l a t e s i n the pear s e l e c t i o n e x h i b i t i n g "Yuzuhada" d i s o r d e r (Table 4). Histochemical examination of these f r u i t f o r peroxidase confirmed the observation that wall-bound peroxidase was more extensive i n the f r u i t e x h i b i t i n g excessive l i g n i f i c a t i o n . The calcium content of the pulp was a l s o c o n s i s t e n t with


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TABLE IV. C e l l w a l l and membrane-bound peroxidase from developing pear f r u i t . Peroxidase a c t i v i t y assayed with hydrogen peroxide as oxidant and O - d i a n i s i d i n e as H-donor has the u n i t s A 460 nm/min/mg Ν at 25°C. "Yuzuhada f r u i t e x h i b i t excessive l i g n i f i c a t i o n r e l a t i v e t o B a r t l e t t f r u i t . Data from (34). 11

f

f

peroxidase


days before harvest

TABLE V.

'Yuzuhada'

'Bartlett'

21

0.19

0.43

14

0.09

0.23

7

0.02

0.26

0

0.00

0.23

Calcium content of developing pear f r u i t . C a l cium was determined by atomic absorption spec trophotometry on pulp. Note lower l e v e l s of calcium during e a r l y stages of development where l i g n i f i c a t i o n i s i n i t i a t e d . Data from (34). calcium ( p p m ) 'Yuzuhada'

days before harvest

'Bartlett'

41

46

16

34

56

30

27

68

32

21

56

40

14

54

36

7

48

36

0

52

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our t h e s i s (Table 5). The lower calcium content of "Yuzuhada" f r u i t throughout e a r l y development may have provided an environment conducive to peroxidase a s s o c i a t i o n with l i g n i n template. The i n f l u e n c e of calcium on peroxidase r e l e a s e from c e l l w a l l s i s shown i n F i g u r e 1. Although a d d i t i o n a l study i s needed t o confirm t h i s model i t provides an a t t r a c t i v e explanation r e l a t i n g mineral n u t r i t i o n of the t r e e to important q u a l i t y parameters of the fruit. F i b e r Formation i n Postharvest Asparagus. Asparagus spears undergo texture changes that occur during maturation, e i t h e r i n the f i e l d or i n storage, and these changes progress from the butt end of the stem t o the t i p . Toughening of asparagus i s due t o l i g n i f i c a t i o n of f i b r o v a s c u l a r t i s s u e and occurs w i t h i n hours a f t e r harvest i n spears stored a t ambient c o n d i t i o n s (35). In view of the apparent involvement of peroxidase-wall a s s o c i a t i o n s i n developing pear f r u i t , we thought i t would be of i n t e r e s t to see whether s i m i l a r c o n t r o l s are o p e r a t i v e i n the asparagus stem. If so, b r i e f treatment of spears with s o l u t i o n s c o n t a i n i n g c a l cium s a l t s would be expected t o prevent l i g n i f i c a t i o n a f t e r harv e s t . Attempts by us to achieve such b e n e f i c i a l e f f e c t s were uns u c c e s s f u l apparently because of the greater t e n a c i t y of asparagus peroxidase f o r the f i b r o v a s c u l a r bundles. Levels of calcium e f f e c t i v e i n a r r e s t i n g l i g n i n d e p o s i t i o n (0.5M C a C l ) i n v a r i a b l y caused extensive t i s s u e damage which was followed by pathogen i n v a s i o n . The s o l u b l i z a t i o n of peroxidase from asparagus t i s s u e r e q u i r e d approximately f i v e - f o l d higher l e v e l s of Ca++ than was observed f o r pear f r u i t (Figure 2)(28). While we observed no d i f ferences i n the degree of w a l l binding of peroxidase and r e l a t i v e l y l i t t l e d i f f e r e n c e i n e x t r a c t a b l e a c t i v i t y during aging of the spears (Figure 3 ) , the i n i t i a t i o n of r a p i d l i g n i f i c a t i o n was c l o s e l y p a r a l l e l e d by the emergence o f new isoperoxidase species (Figure 4). The i n d u c t i o n of new isoperoxidase species and l i g nin d e p o s i t i o n occurred w i t h i n a few hours a f t e r c u t t i n g and were dependent on p r o t e i n s y n t h e s i s . The emergence of new i s o p e r o x i dase species progresses from the butt end of the spear to the t i p . A s i m i l a r p r o g r e s s i o n from the butt to the t i p i s observed i n the toughening process. The emergence of the prédominent new i s o peroxidase species c o r r e l a t e d c l o s e l y with l i g n i n d e p o s i t i o n i n the three segments of the spear (Figure 5). These f i n d i n g s suggest that and 72 hr (Ί -1; Ο - Ο ). Spears were cut at 5 cm above ground level (A-A; W~M) ground level (O-O; -\ \-). Each data point represents an average of readings from four spears (29). o r a t


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ground l e v e l produce ethylene at a greater r a t e than those cut below ground l e v e l (Table 6). Moreover, treatment of spears with exogenous ethylene a c c e l e r a t e s the emergence of new isoperoxidase species and, as w e l l , the r a t e of spear toughening (28). Our suggested r o l e of ethylene i n l i g n i f i c a t i o n a l s o lends n i c e l y to previous observations that c o n t r o l l e d atmospheres employing e l e vated carbon d i o x i d e depress the onset of f i b e r development (36). Carbon d i o x i d e i s a competitive i n h i b i t o r of a l l known ethylene mediated events i n p l a n t t i s s u e (37).


B i o s y n t h e s i s of S t r e s s Metabolites Sweet potato roots accumulate a v a r i e t y of metabolites i n response to s t r e s s (38). These i n c l u d e phenolic substances, coamarin d e r i v a t i v e s and a f a m i l y of f i f t e e n and nine carbon furanoterpenoids (Figure 7 ) . These furanoterpenoid s t r e s s metabo l i t e s are of i n t e r e s t to us because they accumulate at low l e v e l s i n marketable sweet potatoes (39) and are t o x i c to mammals (40). Ipomeamorone and ipomeamaronal are hepatotoxins and the nine c a r bon compounds 4-ipomeanol, 1-ipomeanol, 1.4-ipomeadiol and i p o meanine are lung and kidney t o x i n s . Evidence has been provided that these terpenes are f u n g i s t a t i c agents which c o n t r i b u t e to disease r e s i s t a n c e of the sweet potato root (38). E a r l i e r s t u d i e s showed that on i n f e c t i o n with the b l a c k r o t organism (Cerat o c y s t i s f i m b r i a t a ) r o o t s accumulate peroxidase i n the healthy l a y e r of c e l l s adjacent to the zone of i n c i p i e n t i n f e c t i o n (41). These f i n d i n g s l e d to the n o t i o n that a s p e c i f i c isoperoxidase species acts as an e f f e c t o r of p r o t e i n synthesis by v i r t u e of i t s a b i l i t y to modify l y s i n e residues i n h i s t o n e p r o t e i n s , and t h e r e by a c t s to e l i c i t the formation of furanoterpenoids (42). I f t h i s concept i s c o r r e c t i t may help us understand why root t i s s u e accumulates low l e v e l s of furanoterpenoids as a r e s u l t of s t r e s s e s imposed during storage and handling. We a l s o had the idea that a unique isoperoxidase may be used as a simple i n d i c a t o r f o r the presence of s t r e s s metabolites i n root t i s s u e . Ethylene can increase the r e s i s t a n c e of sweet potatoes to i n f e c t i o n by £. f i m b r i a t a (43). Such r e s i s t a n c e i s a l s o a s s o c i ated with increased peroxidase and polyphenoloxidase a c t i v i t y (38). E a r l i e r s t u d i e s i n d i c a t e d that ethylene r e l e a s e d from sweet potato t i s s u e by i n v a s i o n of C. f i m b r i a t a took part i n i n c r e a s i n g peroxidase a c t i v i t y (44). Matsuno and U r i a n i (45) reported that sweet potato r o o t s c o n t a i n 4 major and 3 minor isoperoxidase spec i e s . Black r o t i n f e c t e d species e x h i b i t e d no change i n i s o peroxidase numbers while c u t - i n j u r e d and ethylene t r e a t e d root s l i c e s contained a new c a t i o n i c species c a l l e d component H. Ind u c t i o n of component H was accompanied by the formation of a l i g n i n - l i k e substance on the cut s u r f a c e . These workers provided a d d i t i o n a l evidence that component H was e f f i c i e n t i n the conjugation of phenylpropanoid monomers i n t o l i g n i n . Thus, while cut



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i n j u r y and ethylene l e d to increases of c o n s t i t u t i v e enzymes and i n d u c t i o n of a new isoperoxidase, i n f e c t i o n only r e s u l t e d i n i n creases i n the constitutive-enzymes. While these data appear to negate any hypothesis suggesting the involvement of a unique i s o peroxidase i n the e l i c i t o r response we continued with t h i s work because previous s t u d i e s d i d not account f o r isoperoxidases ass o c i a t e d with the c e l l u l a r p a r t i c u l a t e f r a c t i o n s . Our study showed that sweet potato roots c o n t a i n i o n i c a l l y bound peroxidase which increased s l i g h t l y as a r e s u l t of cut i n j u r y and q u i t e d r a m a t i c a l l y as a r e s u l t of ethylene treatment and i n f e c t i o n (Figure 8 ) . The r a t e at which peroxidase a c t i v i t y increased a f t e r i n o c u l a t i o n was dependent on the v i r u l e n c e of the mold. That i s , a more v i r u l e n t c u l t u r e of

Physiological Roles of Peroxidase in Postharvest Fruits and Vegetables

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