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“Cloud” or vascularbrowning in tomatoesElsa B. Kidson aa Soil Bureau, Department of Scientific andIndustrial Research , Seconded to CawthronInstitute , Nelson , New ZealandPublished online: 06 Jan 2012.

To cite this article: Elsa B. Kidson (1958) “Cloud” or vascular browning intomatoes, New Zealand Journal of Agricultural Research, 1:6, 896-902, DOI:10.1080/00288233.1958.10422392

To link to this article: http://dx.doi.org/10.1080/00288233.1958.10422392

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896 NEW ZEALAND JOURNA1. OF AGRICULTURAL RESEARCH (DEC.

"CLOUD" OR VASCULAR BROWNING IN TOMATOES IV. POL YPHENOL OXIDASE ACTIVITY IN

CLOUD-SUSCEPTIBLE FRUIT

By ELSA B. KmsoN, Soil Bureau, Department of Scientific and Indmtrial Research, Seconded to Cawthron InstItute, Nelson.

(Received for publication, 26 June 1958)

Summary

The browning in the vicinity of the vascular system characteristic of cloud appears to be polyphenol oxidase browning.

Polyphenol oxidase has been found to be active in abnormally ripening cloud-affected wall tissue, but appears to be inactive in normally ripening wall tissue even in cloud-affected fruit.

High polyphenol oxidase activity has been found in very young fruits, diminishing and finally disappearing with increasing maturity.

It is suggested that cloud··affected fruits show an abnormal re­tention of polyphenol oxidase activity in the blotched areas. This activity persists in cloud tissue ripened to the orange-red stage on the plant.

INTRODUCTION

"Cloud" in tomatoes, a form of uneven ripening, has been described in a previous paper of the series (Kidson and Stanton 1953a). It has been reported in many parts of New Zealand, particularly in glasshouse fruit. It appears to be identical with a fonn of blotchy ripening described by Bewley and White (1926) and with vascular browning as described by Conover (1949). It has been identified by the author in Britain where it is frequently of widespread occurrence. There is evidence that the same disorder causes serious loss in the Netherlands and in parts of North America.

Cloud is characterised by the appearance of green or brownish­green areas on the ripening fruit. The most shaded parts of cloud­susceptible fruits, i.e., parts touching the main stem or other fruits, are those most noticeably affected and are often the only areas so affected. Underlying the blotches there are dark veins following the vascular system. The browning appears to develop as the fruit approaches the colouring stage (Kidson and Stanton 1953c). Recent work has shown that the browning takes place in the parenchyma cells in the vicinity of the vascular strand rather than in the vascular tissue itself, observations agreeing with those of Seaton and Gray (1936) for blotchy ripening. The brown colour is usually associated with a collapse 6£ the affected cells, in extreme cases resulting in the formation of thannels through the flesh.

There are marked varietal differences in susceptibility to cloud. Stanton testing dwarf varieties on outdoor sterilised soil found that whereas one variety averaged 18 to 26 cloud fruits per plant, five others averaged less than 5 cloud fruits per plant and one variety was free

N.Z. ]. agric. Res. 1: G96-902.

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1958) KmsoN-"CLOUD" IN TOMATOES 897

from the disorder (unpublished data, 1954). Massey and Winsor (1957) examined the incidence of blotchy ripening in ten varieties and found that Potentate was much the most susceptible, with 20.7% of affected approaches the colouring stage (Kidson and Stanton 1953c). Recent work by the author has produced further evidence of striking varietal differences.

Cloud is increased by soil sterilisation, heavy watering, and wet, cool seasons (Kidson and Stanton 1953a, b). It has been induced in water-culture experiments by conditions of low light and high humidity (Kidson 1956). It can be much reduced or prevented in soil-grown plants by liberal drel'sings of fertiliser during the season (Kidson and Stanton 1953a) and'l;>y a reduced water supply. In water-cultures under cloud-inducingcQnditions it has been almost eliminated by trebling the osmotic pressure of the nutrient solution either by increased quantities of major elements or by the addition of sodium sulphate (Kidson, unpublished data). Hall and Dennison (1955) have found that cool temperatures, shade, and mist increased vascular browning.

Percentage of dry-matter and of total carbohydrate appear to be below normal in cloud-affected fruit (Kidson and Stanton 1953b, c), and treatments reducing cloud have raised the percentage of these con­stituents in the fruit. Massey and Winsor (1956, 1957) found blotchy ripening to be associated with a reduced percentage of total solids, reducing sugars, acids, and nitrogenous compounds. Cloud-affected tissue has been found by the author to have a lowered acid content, as measured by titratable acidity.

THE FUNCTION OF POLYPHENOL OXIDASE IN THE RESPIRATION OF

TOMATO FRUITS

It is known that most browning of fruits is produced by the action of polyphenol oxidase. Polyphenols with ortho di-phenolic groups are oxidised by this enzyme to corresponding quinones. The browning takes place by what is referred to by MacDougal and Dufrenoy (1946) as the decompensated action of polyphenol oxidase. Normally the oxidation is reversed by some reducing system, but in its absence, brown compounds are formed as a result of further oxidation and polymerisa­tion. The oxidation products of the quinones may unite with constituents of the cytoplasm (MacDougal and Dufrenoy 1946).

Polyphenol

O-OH

I-OH~----~ H

oxidase

o , O brown = 0 ____ O=-___ ~ compounds

~ =0 ~ polymerlsatlon

The browning usually occurs as a result of injury by mechanical damage or invasion by a pathogen. In fruits such as apple, pear, or quince, any damage to the flesh will result in a rapid appearance of polyphenol oxidase browning. Normal tomatoes do not behave in this fashion, at least at the ripening stage. There is no rapid development of brown colour with mechanical damage. If the enzyme is present, the system is protected from complete oxidation and browning.

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898 NEW ZEALAND JOURNAL OF AGRICULTURAL RESEARCH (DEc.

It appears that polyphenol oxidase may function as a respiratory enzyme in many but not all plant tissues (Bonner and Galston 1955). The polyphenol-quinone oxidase system may act in a chain of reactions transferring electrons from certain respiratory substrates to the oxygen of the air (W. D. Bonner 1956), or in the last step as a terminal oxidase (Bonner and Galston 1955). Observations by W. D. Bonner (1955) with mushrooms and mung beans have suggested that the enzyme functions anaerobically in respiration. He concludes that polyphenol oxidase reacts directly with oxygen only under conditions of high oxygen tension and that under normal conditions it acts as an electron transport system using cytochrome C as an acceptor.

The function of polyphenol oxidase in the respiratory system of the tomato fruit has been investigated by Marh and Feljdman (1956). They suggested that in the two different varieties tested, the part played by polyphenol oxidase in oxygen absorption in the oxidising systems of tomato fruits was very considerable (more than 50% of the total respiration).

Work reported in the present paper has confirmed the presence of this enzyme in local tomato varieties and has indicated differences in polyphenol oxidase activity between healthy and blotched areas in cloud-affected tomatoes.

Polyphenol Oxidase Activity in Cloud-affected and Healthy Wall Tissue

Blotched and normally ripening areas in fruits of the variety Potentate have been compared for their ability to oxidise catechol to the brown stage. The fruit wall plus skin was disintegrated in a blendor with an equal weight of water. The disintegrated material was filtered through plastic gauze to remove the coarsest particles and to the suspension passing the filter was added a solution of catechol. It was found that cloud-affected (blotched) tissue, i.e. tissue from the areas showing abnormal ripening and brown veins, invariably produced a brown colour with catechol indicating polyphenol oxidase activity. The test was repeated many times, and in no case did samples of normally ripening, cloud-free areas of cloud-affected fruits give this brown colour with catechol, nor did the reaction take place in similarly treated walls of healthy ripening fruits. If slices of blotched tissue were treated with catechol after the removal of the brown veins many cells not formerly affected developed the brown colour. Unlike the fruit walls, ground seeds of both healthy and cloud-affected fruits gave a strong reaction with catechol, most marked in the seed coat.

Site of Polyphenol Oxidase Activity

It was found that browning originated in the solid particles of disintegrated tissue and that the activity of the blotched suspension was much reduced by filtering methods which removed a large proportion of the insoluble material. Slices of blotched tissue treated with catechol developed a widespread browning which appeared to be strongest where the chloroplasts were most numerous and, in many large parenchyma cells, appeared to be confined to the vicinity of the nucleus where most or all of the chloroplasts were concentrated. The usual distribution of

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1958) KIDSON--"CLoun" IN TOMAToES 899

browning was heavy in and near the vascular bundles, less concen­trated in the larger parenchyma cells and occasionally fairly heavy in cells near the inner surface of the fruit wall.

Inhibiting Effect of Extract's of Healthy Tissue

Healthy fruit-wall tissue had an inhibiting action on the polyphenol oxidase browning of disintegrated cloud tissue. If a large enough proportion of healthy suspension was added, no browning occurred. Smaller proportions prevented the full development of the colour. Wall tissue of fruits from cloud-free treatments was more effective in this way than that of healthy areas of cloud fruit. The inhibitory action appeared to be present in the water-soluble fraction, a clear filtered solution retaining its effectivenesfi.

Effect of Washing

It was found that the treatment of slices of blotchy tissue with certain solvents greatly accelerated the speed and extent of the catechol browning. This effect was shown by slices washed with BO% acetone, with 95% ethyl alcohol, both followed by water, or by water alone. The acetone had the most pronounced effect and the water alone the least effect. In contrast with cloud tissue which browned heavily, slices of wall of normally ripening areas of cloud fruit gave only slight traces of browning or no browning at all with catechol after treatment with these solvents. As might be expected, such treatment resulted in loss of organic acids as measured by titratable acidity.

Effect of Organic Acids and Glucose

Ascorbic acid was very effective in preventing browning in suspen­sions of blotched tissue, and its action was related apparently more to its reducing power than its effect on pH. There was evidence that citric acid and malic acid had some inhibiting effect. At low concen­trations where pH was not a factor, the inhibiting action of citric and malic acids was slower and less effective than that of equivalent amounts of ascorbic acid. Glucose inhibited the browning if present in sufficient quantity. The addition of this sugar increased the acidity of the suspension.

Effect of pH and of Buffer Solutions

Attempts to determine the effect' of pH on the polyphenol oxidase browning were complicated by apparent specific effects of certain buffers. With a phosphate-citrate buffer of pH 3.70 to 4.70 the browning of disintegrated blotched tissue did not occur below 4.25 and increased with rising pH. Healthy areas of cloud fruit gave no brown colour over this range. Ground seeds treated with a phosphate-citrate buffer did not change colour below pH 4.BO. Tests on disintegrated cloud tissue from fruits of the variety Pearl Harbour with a phosphate­citrate buffer, an acetate buffer and a buffer of phosphate alone, all at a pH of approximately 5.0, showed a strong polyphenol oxidase browning with the acetate buffer and a trace only with the phosphate­citrate and phosphate buffers. The colour with the acetate buffer

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900 NEW ZEALAND JOURNA~, 0:; AGIUCULTURAL RESEARCH (DEC.

developed faster and was stronger than that with water alone at a similar pH. This action of acetate buffer was observed also with disintegrated seeds. With a phosphate-citrate buffer in the pH range of 4.45 to 6.45 no catechol browning of seed particles took place below 4.80 and above that figure there was slight reaction increasing with rising pH. With an acetate buffer of pH 4.25 to 5.65, on the other hand, there was a strong reaction over the whole pH range. Seed particles with acetate at a pH of 5.0 gave a darker catechol browning than with water, even though the pH of the water suspension was as high as 6.50.

The tests indicated that the citrate-phosphate and phosphate buffers inhibited the browning to a large extent, a reaction which may be related to an ability to form complexes with copper, the metal activator of polyphenol oxidase. Acetate buffer had the opposite effect.

Whereas acetate-buffered cloud-affected tissue and seeds produced a strong oxidation of catechol to the brown stage, wall tissue from normally ripening areas of cloud fruits did not brown catechol in the presence of acetate, even when the tissue had previously been washed with acetone and water.

Age of Fruit and Polyphenol Oxidase Activity

It has been noticed that very young tomato fruits darken easily on bruising or cutting, and that this effect is much reduced or absent in more mature fruits. Tests with tomatoes at different stage's of development showed that the skin and vascular systems of the walls of very young fruits had a high polyphenol oxidase activity as measured by the speed and extent of the development of brown colour in acetate buffer on the addition of catechol. The reaction diminished with increasing maturity and was not shown by normal fruits at the colour­ing stage. The same general trend of decreasing polyphenol oxidas~ activity with increasing maturity as measured by this method was shown by a number of different varieties, some of them grown out­doors in conditions not conducive to cloud.

DISCUSSION

The tests have shown the presence of polyphenol oxidase in abnormally ripening areas of cloud fruit and suggest that the browning is a polyphenol oxidase browning. So far it has not been possible to demonstrate that this enzyme is active in the wall tissue of ripening fruits not affected by the disorder. Moreover, normally ripening wall areas of cloud fruit have shown no polyphenol oxidase activity except for an occasional slight trace. It is suggested that, in healthy fruits, polyphenol oxidase is not present or is inactivated at the onset of ripening, and its occurrence in cloud-affected areas is an abnormality. Very young fruits had a high polyphenol oxidase activity as measured by the rapidity with which they oxidised catechol to the brown stage. This activity was found to diminish and finally disappear with increas­ing maturity. A drop in the activity of polyphenol oxidase as ~l respiratory enzyme with increasing age of the fruit has been found by

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1958) KIDSON--"CLOUD" IN TOMATOES 901

Marh and Feljdman (1956) for two different varieties tested. With increasing maturity there was a continuous fall in the intensity of respiration including that controlled by polyphenol oxidase. This was accompanied by a decrease in the fraction of respiration catalysed by metal-containing enzymes (including polyphenol oxidase) and an increase in that catalysed by the flavin enzymes. It appears that in cloud-affected fruits the blotched areas retain a juvenile characteristic of high polyphenol oxidase activity. That this is not merely an indication of unripe tissue is shown by the fact that the power to brown catechol is retained by cloud-affected tissue ripened on the plant to the orange-red stage.

Not only is the enzyme retained in the blotched tissue, but the oxidising action is inadequently compensated by a reducing mec~'lanism, at least in certain cells. The inhibiting action in vitro of citric and malic acids, both acids of the Krebs cycle, and the increase in brown­ing produced by acetate are of interest from the point of view of the dehydrogenase systems which may function in this respect. Acetate has been found by Laties (1949) to have a strong inhibitory action on respiration, probably by competition with one of the first intermediates in the oxidation of pyruvate. Ascorbic acid has been very effective in preventing the browning in vitro but whether it functions in this way in the plant is in some doubt. Appreciable quantities of ascorbic acid (10-15 mg per 100 g of fresh tissue) have been found in cloud-affected patches and also of total organic acids as measured by titratable acidity, though both tend to be lower in blotched than in healthy areas of the same fruit. No examination has been made of the organic acid content of the cells in the immediate vicinity of the browning and these may possibly show greater differences from healthy tissue. Whatever the direct cause of the browning, environmental conditions that tend to produce it, viz, low light, high humidity, high available water, are those that tend to reduce the content of sugars and their derivatives either by reduced photosynthesis or increased water uptake.

ACKNOWLEDGEMENTS

The author wishes to thank Professor V. W. MacFarlane of the University of Queensland for suggesting the line of work, Dr K. Strzemienski of the Soil Bureau for much help and advice, Miss Anne Hole for conscientious and able assistance with water-cultures and laboratory work, Mr R. L. Closs of the Dominion Physical Lab:)ratory for the provision of a humidity control apparatus, and Mr D. J. Stanton without whose willing co-operation this investigation would have been seriously handicapped.

REFE~ENCES

BEWLEY, W. F.; WHITE, H. L. 1926: Ann. appl. Ko!. 13: 323.

BONNER, J.; GALSTON, A. W. 1955: "Principles of Plant Physiology". Freem:lll, San Francisco.

BONNER, W. D. 1955: Plant Physiol. Proc. Suppl. 30: xxx.

----1956: Ibid. 31: xli.

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902 NEW ZEALAND JOURNAL OF AGRICULTURAL RESEARCH (DEC.

CONOVER, R. A. 1949: Plant Dis. Reptr 33: 283.

HALL, C. B.; DENNISON, R. A. 1955: Proc. Amer. Soc. hort. Sci. 65: 353.

KIDSON, E. B. 1956: Cawthron Inst. annu. Rep. 1955-56. p. 48.

KIDSON, E. B.; STANTON, D. J. 1953a: N.z. J. Sci. Tech. A34: 52l.

-----1953b: Ibid. A35: l.

----------1953c: Ibid. A35: 368.

LATIES, G. G. 1949: Arch. Biochem. 20: 284.

MACDoUGAL, D. T.; DUFRENOY, JEAN 1946: Plant Physiol. 21: l.

MARH, A. T.; FELDJ MAN, A. L. 1956: Biokhimija 21: 33.

MASSEY, D. M.; WINSOR, G. W. 1956: Rep. expo Res. Sta. Cheshunt, 1954 p. 50.

-------------1957: Glasshouse Crops Res. Inst. annu. Rep. 1954-55.

SEATON, H. L.; GRAY, G. F. 1936: J. agric. Res. 52: 217.

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