SOME-2 A$PECTS OF THE PHYSIOLOGY AND {DEVELOPMENT OF JONATHAN 5967 EN AFPLE mm Time“: for flu Dogma of DE. D. MICHIGAN STATE UNIVERSITY Amos Edwin Richmond 1963 TH ESlS This is to certify that the thesis entitled Some aspects of the physiology and development of Jonathan spot in apple fruits. presented by Amos Edwin Richmond has been accepted towards fulfillment of the requirements for Ph.D. degree in Horticulture r"4(afl(‘k / : l \gfi}kfc . Major professor I Date @:\“QZ 4/ / 7(3 0-169 LIBRARY Michigan State University ABSTRACT SOME ASPECTS OF THE PHYSIOLOGY AND DEVELOPMENT OF JONATHAN SPOT IN APPLE FRUITS by Amos Edwin Richmond Researches were conducted to determine the causal sequence involved in the development of Jonathan spot. a physiological disorder affecting ripe apple fruits of the Jonathan cultivar. Morphological and anatomical observations revealed that the dis- order commonhy termed as Jonathan spot consists of two types. One occurs in association with an aperture in the fruit epidermis. and was named in this study “lenticel spot“. The other; or "true” Jonathan spot, occurs without relation to lenticels or other apertures in the epidermis. Once the spot types were distinguished. the researches were confined to Jonathan spot. Study of harvest and storage factors affecting Jonathan spot showed that early picking. storage in 60°F or above and as low as 0.7% 602 in the atmospheres surrounding the fruits reduced or inhibited spot forma- tion. Relative humidity did not affect the disorder. various analyses of the spotted tissue were conducted. The pH of spotted tissue homogenates ranged between h.“ and 5.2. which was 0.5 to 0.7 units higher than the pH of normal tissue. The buffering capacity of this homogenate all along the titration curve was approximately double that of normal. Mineral accumulation in the spotted tissue was confirmed. To Amos Edwin Richmond - 2 ascertain a possible sequential relation between spot development and K accumulation, 63137 was introduced into the fruits some 3 weeks before harvest. Autoradiographs showed the isotope to be uniformly distributed in the fruit peel immediately after harvest. The few spots that devel- oped in early January were free of 05137; however. accumulation of the tracer was observed in several spots that developed later in the storage season. Nonvolatile acid extraction and fractionation revealed an approxi- mate two-fold increase in total acidity in the spotted tissue. as well as an increase in the percentage of citrate. The observed changes in cations and total acidity were shown to be related to the raised pH and the titrational characteristics of the spotted homogenate. Jonathan spot characteristically appeared earlier and to a higher extent on the red areas of the apple skin. The anthocyanin destruction that accompanied spot deve10pment increased with aging of the spots. Thus, it was speculated that degradated anthocyanin served as a precursor for the synthesis of the brown pigment which is typical to the disorder. However, since mature green-colored apples eventually developed exten- sive spotting. anthocyanin would be only one of several possible precur- sore. Respiratory measurements of peel discs in a warburg respirometer showed that endogenous 02 uptake was close to normal for newly spotted tissue. declining to one-half with the aging of the spots. 02 uptake of normal and spotted peel discs upon the addition of catechol and ascorbic acid was identical. suggesting that the lower endogenous 02 uptake of old spotted tissue was due to a lack of substrate. Addition of malate Amos Edwin Richmond - 3 revealed that spotted discs partially lost the capacity to decarboxylate malate. A possible hypothesis for Jonathan spot development is that some cells in the periphery of the receptacle of the young fruit undergo modification early in the ontogeny of the fruit. With maturation. cations and phosphorus accumulate in the spot-susceptible cells. The cation influx is then responsible for the higher total acidity and higher pH observed for spotted tissue. The rise in pH is suggested as the ini- tial causal factor promoting anthocyanin breakdown and the channeling of various oarbonylic compounds and polyphenols into the synthesis of the brown pigment that gives the typical discoloration associated with Jonathan spot. SOME ASPECTS OF THE PHYSIOLOGY AND DEVELOPMENT OF JONATHAN SPOT IN APPLE FRUITS By Amos Edwin Richmond A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1963 G.1Hx mung. am 0M :.a MA OH mm mm Nd awed. NM. HH. Now as. mummm cmzumcom mm on N.H o 0 mm 0H Nu nwo. 5N. HH. :3. mm. 050:9“: mm on N.H HA w mm 0H on who. dw. NH. mm. mm. macawoom Hood N: om o.H 5 OH wN 0H mm mNH. 3N. mm. mo.H on. macaw canpm:05 wN 0a N.H ma om mm mm ow mNH. 3N. mm. oo.H on. maficawacoo Nmmd honflooen :m o mH.H we. mcowuoom Heom & z comma. mama. Ohm onen— 03 e 838 8H < see 88830 3 m 22on 2388 Hood no as me: ed at m 8H 55 8538 38m osmosis. 823:8 2338 Heed to 38m 2362 be :e .8385 H265: .HHQ 3m: 8 0H m . o m 1%an H mm ewe. no. 8.. 8 4 H828 memH HHH em NH e. 0 HH NR NH 8 m3. 3. NN. mN.H 38m 5528 p.33 8 n n . o a in 0 NH Re. No . No. on . H828 NoeH HH Ne NH o.H 0 0H «NM NH 8. 2H. S. 3. ON.H 38m seepage refined NN 0H m. m HH $0. No. No. 8. H828 HRH H R NH eh. e 3 WE .mH MH EH. 2. mH. 8. H 3.. Seem fieeeeoe enema. 1e. He. e: ee 5 oz ez d 8 Jae e eleoeHelll 881.418 .llqezui 28 £8 nee hoses 38.3 38m some: be no 682.9 Hood HeEoz one 8325 do 2235 HepeeHz .Hdn mam: 41 TABLE XV. The Average K. Mg. Ca. Peel Tissues and P Content of Spotted and Normal ‘_ 4‘ Averagefll AverageQ/ meq Per cent Fold Per cent Per cent per of Increase on on 100 gm Total Cation in Dry Weight Fresh Weight Fresh Weight Equivalents Spotted Basis Basis Tissue Normal r”t: 1 Normal Spgt Normal Sm} Normal Smt K .54 1.26 .0940 .2260 2.41 5.80 74.1 60.8 2.4 Mg .04 .18 .0069 .0320 .58 2.68 17.8 28.1 4.6 Ca .03 .12 .0052 .0210 .26 1.05 8.0 111.0 4.0 P .059 .167 .0103 .0299 1.00 2.91 _ _ 2.9 TOtal Cations 3025 9053 2.9 Total minerals 4.25 12.44 2.9 é/ Average based on analyses I. II. and III in Table x11. 9/Computed on the basis of 17.4 and 1749 per cent dry weight for normal and spotted peel tissues. respectiVely. Autoradiograpgy Autoradiographs of peel sections that were obtained from apples in ”Lot B" some two weeks after the apples were harvested showed that 09.137 was evenly distributed in the skin. The different shade inten- sities of the autoradiograph reflect the variable thicknesses of the sections. Thus the darkest area of one section measured 17 units (B. Figure 5 left). whereas the lightest sections (C and D in Figure 5 left). measured only 5 units of thickness each. Autoradiographs that were prepared on Jan. 2. 1963. when a few Jonathan spots were first observed in the Cs-treated apples showed that the tracer moved out of the spotted tissue. (Right. top and bottom in 42 Figure 5). Autoradiographs of spotted peel sections obtained on March 26 from apples of Lot "A" were very light and showed no conclusive evidence as to the distribution of 03137. Figure 6 shows autoradiographs of peel sections prepared on May 20. 1963 from apples in lots "A” and "B". As reflected in these autoradio- graphs. the distribution of C3137 in the sections was extremely variable: Corresponding to the spots in the upper and bottom sections shown in photograph A. there appear white areas in the respective autoradiograph B. pointing to the loss of 03137 from the spotted tissue. The opposite may be observed for some spots (marked with arrows) shown in photograph C. corresponding to which there are dark areas in autoradiograph D. pointing to an accumulation of Cs in the spots. Similarly. corresponding to the middle spot in the upper section of photograph E there is a dark spot in autoradiograph F, and corresponding to the Spots marked with arrows on the lower section in E,there are dark spots in F. The situation is obscure in the middle section in F, where only very faint shades corre- spond to the spots shown in E. No relationship seems to exist between the spots photographed in 0 and the uneven C3137 distribution portrayed in auto radiograph H. IV. Studies of Basic Respirations - 002 Output and 02 Uptake The introduction of malic acid into warburg reaction flasks con- taining peel discs obtained from the new crop apples accelerated a marked 002 evolution. This malate effect. however. was more pronounced with normal peel discs than with spotted peel discs. With peel tissues ob- tained from l8-month-old-apples (Figure 7. II). the quantitative Figure 5. Left: 43 Autoradiographs and photographs of peel sections prepared from Cs137-treated Jonathan apples. Autoradiographs of peel section taken on October 4. 1962 shortly after fruits were harvested. Arrows indicate position where the thickness of the section was meas- ured in a calibrated microscopic field. A=6. B=l7. C=l3. D=5. E=5. units of relative thickness. top and bottom: photographs of peel sections secured when spots initially appeared on January 3. 1963. top and bottom: the respective autoradiographs. Arrows in the middle. lower photo indicate the loca- tion of spots which become barely visible when the sec- tions were dried. Figure 6. Photographs and the corresponding autoradiographs of "spotted peel sections obtained from apples in Lots ”A" and "B" on May 20. 1963. A. C. E. and G are photographs of the peel sec- tions; B. D. F. and H are the respective autoradio- graphs. U . ‘ 1 .‘ _ . . . 7' . 1* .3 1'. 1' 4- 1" 333' 4r“! .1. 1" - f 2? ' g A Figure 7. I. II. 4? Endogenous respiration and malate decarboxylation of spotted (dashed curve) and normal (full curve) tissues. Apples held 6 months in storage, spots ca. 2 months old. Apples held 18 months in storage. spots ca. 14 months old. 0.5 m1 of 0.1M malate dipped at 60 minutes (shown by arrow). 3825.... v 25... 3055:; 06¢ 0n. ON. om ow On 0 09 ON. cm 8 On 0 _ H H H a _ H q d _ a \ A \ 1 Cc .4 1 14 1 00 4 1 ON. 1 10w. Nocx 1 no L 00.0. L No 1 1 0.5 can»: 3:on III 1 03mm: _OF=OZ III. ICON N00 1 1 .1on H in up - 6w om 13d (uounpona) 30;) .10 (axmdn) 30 11" “9 differences in 002 evolution between normal and spotted peel discs were greater. spotted discs exhibited a nearly complete loss of capacity to decarboxylate malate. while normal tissue showed an increase in this capacity. Thusly. total 002 evolution by old normal tissue (Figure 7, II) with the introduction of malate was 210 1u/100 mg dry wt/hour. while the evolution by old spotted tissue was 60 ul. The straight line that describes the course of’evolution of C02 by old spotted tissue (Figure 7, II) indicates little malate effect. Endogenous oxygen up- take (up to the introduction of malate} was also greatly diminished in the old spotted tissue, becoming ca. 50% of the old normal tissue. Whereas a slight increase in 02 uptake accompanied the introduction of malate in the old normal tissue, a slight decrease in 02 uptake occurred in the old spotted tissue. The 02 uptake of spotted and normal discs increased remarkedly and to the same extent upon the addition of 0.5 cc of either 0.1M ascor- bic acid or catechol (Figure 8, C and D). Endogenous 02 uptake of spotted tissue was ca. 50% of the adjacent normal tissue. 50 Figure 8. Cumulative oxygen uptake of spotted and normal tissues. 0.5 cc of 0.1M substrates,forming a final substrate concn cf .GléM, were added at 60 minutes as indicated by arrow. Cumulative oxygen uptake, uI/IOO mg dry wt. skin tissue 51 200 — Catechol r Ascorbic 180— __ Normal tissue » ————— Spotted tissue IGO /CafeCh0’ / Ascorbic I40 / / / / / / IZO / I00 80 60 40 20 L #1 Gluta thione IZO '00 G I ycola te /6Iutathione 60 / / _____.....---Glycolate ” 4O 20 1 1 1 1 J O 30 60 90 120 I50 I80 Time (minutes) DISCUSSION Early in this investigation it became apparent that the disorder must be delineated in respect to the terminology employed in the litera- ture and of symptoms describing the disorder and the relation of these to previous researches concerning Jonathan spot. Much of the evidence in the literature seems controversial and could not be verified. Eor example; Tomana's report (1959) that spotting was more prevalent in the early harvest and the observation by Plagge and Haney (192h) that short exposures to high humidity caused an immediate high incidence of spot- ting. Humidity was found to have no effect on spot formation. and late harvest rather than early harvest highly accelerated the degree and time of appearance of the disorder. Also. this study showed that a spot centered around a lenticel could be quickly'developed in certain apple lots at 70°F, at which temperature Jonathan spots did not form. Plagge and Maney's (l92h) suggestion that the two types of spots they recog- nized were identical,being caused by the same conditionthherefore seems unfounded. The photomicrograph of a type of lenticel spot which rapidly develops in room temperature and high humidity (C in Fig. 3) clearly portrays the break in the sub-epidermal layer associated with an open lenticel (Clements. 1935). No such breaks were found in epidermal tissue affected by Jonathan spot. A lenticel spot may be superficially similar to Jonathan spot but it has dissimilar anatomical features and may form rapidly under very different temperature conditions. There can be little doubt that Tomana, Plagge and Haney. Smock (1950) and Krapf 52 53 (1961). among possible others. have been including more than one or altogether different. distinct physiological disorders under the name “Jonathan spot.' Many factors could account for the acceleration of spotting of fruits from late and delayed harvests. Starting with the time of bloom. the respiration rate curve of the attached Jonathan apples reaches its low point sometime around the commercial harvest period. It rises again after that date. reaching its post harvest peak in late October or early November (Ezell and Gerhardt l9b2). Concomitantly. the pro- tein content rises (Hulme 1958) in association with the climacteric rise. The buildup in protein may. to a certain extent. be qualitative. providing the necessary catalyst for some possible limiting stages in the biogenesis of the spot. Likewise. a limiting substrate may be formed or some reaction rates changed to favor spot formation. In no case was the disorder observed immediately after harvest and thus there can be little doubt that a certain preparatory period must elapse before Jonathan spots appear. The accelerated development of spots after delayed harvest or storage as well as after being placed in non-refrigerated storage where the temperature declines only with the advent of winter. all point to a certain high temperature optimum in this preparatory period. Since spots did not form at a temperature above 60°F and in one year (1962) did not form above 50°. it is suggested that the sequence of Jonathan spot development includes at least two temperature phases. with the optimal temperature of the second phase being lower than that of the first phase. The failure in 1963 to bracket the optimal temperature of the second phase indicates. in the author's opinion. that this optimal may vary from season to season and 5n possibly from one lot of fruit to another. This possibility shall be elaborated shortly. The data obtained in the temperature experiments of 1962 indicate that Jonathan spot may be classed as a “chilling disorder". being possibly a modified case in that optimal development favoured an initial exposure to a certain high temperature. Here it becomes clear that un- less a distinction is made between Jonathan and lenticel spots. the temperature effect on Jonathan spot formation becomes rather obscure. especially in fruits having a pronounced tendency to develop lenticel spot. Oxygen tension. at least in the range of 3 to 17%. seemed unim- portant as a causal factor'in Jonathan spot formation. since little difference in spotting was observed between a treatment of 3% 002; 3% 02, and 3% CO2 and 17% 02. It is likely. then. that the complete control of Jonathan spot obtained in controlled atmosphere storage is not a result of the lowered oxygen tension and decreased respiration rate. but is probably a direct affect of the increased 002 level. Surprisingly. a concentration as low as 0.7% CO2 effectively inhibited spot development. proving whatever factor is involved to be extremely sensitive to an in- crease in the 002 tension. Noteworthy. however. the reported effect of 002 on spot formation in this study as well as in the literature is in itself only an assump- tion. Other apple volatiles are also very probably present in higher concentrations in controlled atmosphere chambers and may be. like 002. absorbed by hydrated lime. the material used to scrub the C02 in the chamber down to the desired level. In view of this possibility. the effect of 002 was observed in the 1963 experiment by passing a 55 commercially prepared gas mixture over the fruit. preventing the accumu- lation of any apple volatiles. The results of this experiment (Table IX), showing no difference between the treatments and the control. may be interpreted as an indication that 002 is not the factor which in- hibited spotting. However. the experiment was started late in January. when some apples had already developed Jonathan spot. According to Clerx (1960). who experimented on the effects of various combinations of storage periods of controlled atmosphere and regular cold storage on Jonathan spot development. once the fruit had attained a certain stage of development in ventilated storage. spotting occurred even when the fruit was subsequently transferred to C.A. storage. Therefore no con- clusion could be drawn from this last experiment and the question whether it was CO2 or some other apple volatile that retarded spot development remains open. Whatever the case may be. the possible practical applica- tion of this true or pseudo CO2 effect is quite obvious: it should be rather technically simple to increase the 002 level (and concomitantly the concentration of other’volatiles) in regular or common storages to a level of. or associated with. approximately 1% 002. One possible method would be plastic lining of bulk containers (Dewey'gt,al, 1959). Using a suitable material. establishment of the slight atmospheric modification needed to control Jonathan spot should present no difficult problem. Such a modification may be also established with a CO2 generator. Another finding that carries practical ramifications is the rela- tively low amount of spotting - 32.2% - that was observed in the control fruit examined as late as September ? (Table VIII). almost a.year followb ing harvest. This may reflect a residual effect of controlled atmosphere storage. where the apples were held since harvest. prior to the beginning 56 of the experiment on May 10. The sensitivity of a factor in the pathway of Jonathan spot forma- tion to C02 or some apple volatile may offer a clue to the absence of spot development in certain high temperatures. The higher the tempera- ture. the higher the rate of respiration and other metabolic processes which result in an increased gaseous production by the fruit. modifying the internal atmosphere. Apple fruits. however. are known to greatly vary in their degree of permeability. this degree being a function of the skin anatomy. number and size of open lenticels and other factors. If high temperature inhibited spot formation by altering the internal atmosphere. than the less permeable the fruit. the lower must be the inhibiting temperature and vice versa. This is suggested as a possible explanation for the experimentally observed shift in optimal temperature for spotting that was mentioned previously. An important aspect of this study was the insight it provided into the buffering mechanism of plant cells. Jacobson and Ordin (1954). in- vestigating organic acid metabolism associated with ion absorbtion in excised wheat roots concluded that"when excess cation absorbtion occurs. exchange for previously absorbed cations and the production of malate are the most important means of ion compensation.“ Ulrich (l9hl) from simi- lar studies summarized that when excised roots absorbed cations in excess of anions. and provided that an ample supply of sugar was available in the cells. organic acids were formed as a response to the tendency to- wards an increase in the pH of the root sap. Significantly. the results reported here for Jonathan spot. a disorder in which the affected cells are apparently stimulated to maintain ionic balance. support the conclu- sions derived from studies that were conducted with excised plant organs. 57 The doubling of buffering capacity seen in the titration curves of the spotted tissue (Fig. 3) is understandable in view of the organic acid and cation content of this tissue. The two-fold increase in or- ganic acid and three-fold increase in cation equivalents would both double the buffering capacity and raise the pH. Assuming that the negative charges of the phosphorous compounds in the cell approximately equal the positive charges contributed by the calcium and magnesium present. the pH of the tissue homogenate can be explained stoichiometrically with the analytical data obtained for potassium and total acid. Employing the Henderson-Hasselbalch equation. pH ‘5 pK + log ( (Salt; / [acid] ). and considering 7.0 and 3.5 meq/100g fresh wt of total acid (as malate) in the spotted and normal tissues. respectively (Fig. 4); and 2.4 and 5.8 meq of K (Table XV) respectively. the following approximations held: In the normal homogenate. there are-' 1.7 meq of each carboxyl of malic (3.5/2). At pH of 4.7 (Fig. 3) the first carboxyl of the acid (pK = 3.“) is close to being completely dissociated,in equilibrium withr»1.7 meq of K. Thus~0.7 meq of K’ (2.1+ - 1.7) are left for the titration of the second carboxyl (pK.-:-: 5). With 0.7 meq of this carboxyl dissociated. the pH becomes: pH 5'5 + log %&%.(tota1 malic and malate" g‘1.7 meq). pH -‘-’ n.85; high ray-0.15 pH unit from the observed pH of n.7o. At pH 5.2 of the spotted tissue. (Fig. 3) again the first carboxyl is fully dissociated. 1eaving~3. 5 meq (7.0 - 3.5) to react with 2.3 meq or K (5.8 '3 3.5). The pH becomes: pH 5 5 + log %L3.(total malate‘ and malate= g3.5 meq). 58 pH 2’ 5.28; high by0.08 pH units from the observed pH. However. such a good stoichiometric agreement did not always exist. The pH of apple tissue homogenates varies greatly after storage. being affected by such factors as the date of harvest. temperature in the storage. and the length of the storage period (Clerx. 1960). Thus the pH analysis in 1962 showed the usual pH of the normal and spotted tissue homogenates to be 3.? to 3.9 and “.5 to 4.7. respectively. Such pH values can not be explained as satisfactorily as above on the basis of the analytical data. pointing to a basic difficulty encountered through- out this study that stemmed from the extreme variability and complexity of the material. Nevertheless. even with the above frequently observed low pH. certain analytical relations hold: Using the former assumptions and formula and starting with the acid concentration that the Henderson. Hasselbalch equation would demand for the observed pH and potassium cone tent found in the normal tissue. the rise in pH of the spotted tissue can be explained as fellows: Initially. in the normal tissue: pH of 3.7 a 3.“ + log X; X = 2 Ratio of salt/acid is thus 2/1 and total malic and malate’ = 3 units. In the spotted tissue. where the total acidity is twice as high. total malic and malate = 6 units (3.x 2).) If K content would have only doubled. the same ratio of salt to acid as in the normal homogenate would have been maintained. doubling the buffering capacity only (as observed. Fig. 3) but leaving the pH unchanged. Potassium. however. had more than doubled in the spotted homogenate (Table XV). The pH thus rises. (usually by 0.7 units). and becomes 4.4/(3.7 + .7). Thus: N “on = 3.“ + log X: X :3 10 59 Ratio of salt/acid became ca. 5.4§/.55. which involved a 2.? fold in- crease in K (from 2.0 to 5.46). which is not very far from the 2.4 fold increase reported in Table XV. However. at pH 4.4. some 25% of the second carboxyl (pKl,§'5.0) is also dissociated. Also, ca. 25% of the total acidity in the spotted tissue is comprised of citric acid. which at pH 4.4 is nearly fully dissociated at one car-bowl (p195 3.1) and some 35% dissociated at another carboxyl (pK;=4.75). These factors would further increase the demand on K needed to bring the pH to the observed level. Therefore. in the spotted tissue. K can not be assumed to be the only important cation associated with the buffering mechanism of the cell. The four-fold increase in Ca and Mg found in the spotted tissue must be considered here. Very probably. a certain part of these cations react with.malic acid forming salts or complexes of low solubility products which do not participate in the buffering mechanism of the cell. This would decrease the concentration of free acid in the cell sap and thus decrease the amount of K needed to bring the pH of the spotted homogenate to that ob- served. thus diminishing the above mentioned discrepancy between the empirically found and theoretically needed amounts of K for the estab- lishment of the observed pH. The fact that the acid profile in the spotted tissue changed to include a higher percentage of citrate deserves special attention. Accepting Ulrich's interpretation of acid synthesis accompanying cation uptake as a mechanism to preserve the pH in the cell. a tricarboxylic acid such as citrate will be more effective than malic for this purpose. Normal tissue also contains some citric acid. thus the rising pH could be thought as merely changing the balance in the synthesis of malate and 60 citrate. The following mechanism. suggested by Biale (1960) for lemon fruits. may be considered: aspartate __..._a oxaloacetate -—-—-——-3 citrate an malate Biale stated that the enzymes required for these reactions - malic dehydrogenase. transaminase and condensing enzyme were widely encountered in organisms. These may be present in apple fruits also. Tentatively adapting such or similar mechanism for apple peel. it is possible to envisage a change in pH bringing about a modification in the equilibrium involving the synthesis of the acids. The observed increase in cation equivalents was one of the major subjects investigated. One question that was posed concerned the origin of the transported elements. The fact that the extent of the increase in K. Mg, P and Cu was modified when whole peel sections containing spots were analyzed while Mn and Ca did not show an increase in such material may be significant. It may suggest that the latter two elements reach the spotted tissue from the peel itself. causing the overall Mn and Ca content of the peel to remain the same. The former 4 elements may be translocated to the spotted tissue from the cortex. thus showing an in- crease. even though modified. in whole spotted peel sections. Another question concerned the cation accumulation itself. According to the above discussion. cation accumulation is suggested to be responsi- ble for the rise in pH which presumeably triggers the eventual formation of the spot. To provide evidence for this proposed sequential relation between cation accumulation and spot development. 05137 was introduced into the fruit as an analog of K. The initial migration of the label 61 from the spotted tissue was not anticipated. but it may be explained from the afore mentioned conclusions of Jacobson and Ordin that cation uptake involved an exchange of Mg and Ca for absorbed K. Accordingly; Cs may be thought as being initially excreted from the spotted tissue as a compensatory measure for the cation influx that must occur in that tissue. However. acceptance of this explanation for the migration of Cs from the spotted tissue carries the conclusion that Cs cannot be regarded as an analog of K without some reservations. contrary to the current idea adapted in cation uptake studies. Since later autoradiographs (Fig. 6) indicated in many cases a Cs accumulation in the spotted peel tissue. there may be a reabsorbtion of Cs in later stages of spot development. If so. Cs is reabsorbed followb ing the assumed continuous cation influx into the affected cells. Thus. autoradiographs taken later. after spots'had developed for some time, should show three basic patterns of Cs distribution. namely: White areas. corresponding to young spots: dark areas corresponding to old spots: and no definite pattern corresponding to the inébetween stages of spot de- velopment. All these patterns may be observed in Fig. 6. where the autoradiographs were prepared when spotting of the peel was well estab- lished. Accepting that the first stage in cation influx is accompanied by an.outward migration of Cs. two possibilities arise. One possibility is that cation accumulation takes place after the initial formation of the spots and thus the white areas on the autoradiograph must correspond only to a fully recognizable spots. This did not occur. The second possibility is that the accumulation of cations proceeds the formation of the brown pigment. Then in some very early stages of spot develop- ment. Cs would have already migrated from the susceptible to spotting 62 areas before the spot can be observed. This offers an explanation for some white areas which may be seen in the autoradiographs presented in Fig. 8 which show no correspondence to observable spots in the photos. The anthocyanin content of the spotted peel was determined to offer a possible clue to the origin of the black-brown color of the spot. Since the anthocyanin content of spotted peel was between 30 to 60% of normal. some anthocyanin destruction must have occurred. There is evidence that degradated anthocyanin.may serve as a starting material for the observed brown pigment which typifies Jonathan spots. According to Joslyn and Ponting (1951). Nagai (1921) reported that certain antho- cyanins were completely decolorized by the action of‘oxidizing enzymes. He showed that purified preparations of many plant pigments yielded brownish colors by the action of freshly prepared plant Juices contain» ing oxidizing enzymes. Markakis gt’gl, (1957) proposed a type of antho- cyanin hydrolysis that involved the opening of the pyrilium ring at position 1-2. with formation of a ketone: 0 H O l ” v I O "H /’ oc6 +4.. 05 Markakis gt al. also stated that "further degradation of this ketone would eventually lead to the brown precipitation which has constantly been observed as an end product of the degradation of the pigment." They further reported that when pelargonidin-B-monoglucoside Clu'was stored in citrate buffer. 85% of the original radioactivity of the pig- ment was recovered in the brown precipitate. 63 There are mamr additional reports concerning brown pigment synthe- sis. Hodge (1953) stated that naturally occurring compounds from which browning originates contain usually a multiplicity of potential carbonyl groups such as catechols. which are easily oxidized to quinones. Joslyn and Ponting (1951) concluded that some of the reactions of quinones may conceivably be enzyme-catalyzed but that nonenzymatic browning is be- lieved to predominate. It is therefore apparent from the literature that anthocyanin may be degradated enzymatically and that the reactions leading to the polymerization of the brown pigment are usually catalyzed mnenzymatically. Although Jonathan spots develop earlier in the red in color areas of the apple skin. the eventual occurrence of spots on the non-red colored areas indicates that anthocyanin is not essential for the for- mation of this disorder. Considering the multiple substrates from which . the brown pignent may be formed. it would have been surprising if de- gradated anthocyanin was to serve a sole possible substrate for the various reactions involved in brown pigment synthesis. Thus Siegelman (1955) isolated l-epicatechol from peel tissues of Grimes Golden and Golden Delicious apples and found it to be the principal browning sub- strate for polyphenoloxidase in apple peel tissue. Likewise. it is possible that quinic and shikimic acids. which occur in relative abun- dance in apple peel tissue (Fig. 4). are instrumental in the biogenesis of the brown pigment. Davis (1951) identified shikimic acid as a pre- cursor of phemrlalanine and tyrosine in certain strains of bacteria and fungi. Oxidative metabolism of tyrosine may then result in the forma- tion of melanin. a brown pigment which is believed to be a polymer of indo1-5.6.quinone (Cromartie gt 3.1..” 1957). A sumary of these pathways 64 is given by Fruton and Simmonds (1958). Accordingly, tyrosine may be oxidized to phenylalanine-3.4—quinone. subsequently forming a dihydroxy- indol. which upon oxidation becomes a dihydroindol quinone. An alter- nate pathway is proposed for this compound: At pH 1.3 to 2.0. it is converted to a carboxy dihydroindole. whereas at pH 5.6 to 6.8, it eventually'becomes indol-5.6-quinone. the polymerization of which yield- ing melanin. According to the above proposed pathway. the pH of the system governs the eventual formation of melanin. Noteworthy. the pH of scraped spotted peel was found in one analysis to be as high as 5.45. It is certainly possible that the pH of the 4—5 outer layers of cells. where the brown pigment was observed. is 5.6 or even higher. The actual pH of these cells was impossible to obtain.with the methods employed here. There are still other possible pathways for the genesis of the brown pigment. Haas and Stadtman (1949) reported that quinones. among other compounds. will undergo browning in the absence of enzymatic catalysis. and that in most foods. these reactions occur in the presence of accelerators such as carboxylic acids and their salts. phosphates and metallic ions. As already discussed. all these accelerators increased by two or three-fold in the spotted tissue. The malate decarboxylation studies were conducted to determine if accelerated decarboxylation of malic acid in the spotted tissue would account for the rise in pH accompanying the development of the spots. The observed decrease in malate decarboxylation in that tissue does not lend support to such a theory. The very similar endogenous 02 uptake that newly spotted and normal discs exhibited in early experiments (1962) was not substantiated later 65 (1963). when a 50% decrease in endogenous 02 was observed in the spotted discs. Possibly. this discrepancy occurred because the 1963 experiments were conducted some two months later than in the previous year or be- cause citrate-phosphate instead of phosphate buffer was used. Since an identical 02 uptake resulted from the introduction of ascorbic acid and catechol to spotted and normal discs. the amounts of ascorbic acid and pclyphenol oxidases per unit dry wt of these tissues is similar. Polyphenol oxidase is known to be an important terminal oxidase in mature apple fruits (Rubin gt,al.. 1952). and thus the re- duction in endogenous 02 uptake that the spotted tissue exhibited pro- bably reflected a decrease in substrate in that tissue. The phase in the ontogeny of the fruit at which Jonathan spot is initiated is not known. Some findings suggest that inception occurs prior to harvest of the fruit. The observations on the anatomy of the skin indicate a higher cell number per unit area in the spotted skin. Early inception is therefore indicated since cell division is apparently completed within the first 25 days after full bloom (Tukey and Young. 1942). This is substantiated by reports concerning the positive effect of kinetin applied at bloom and relating the seed number to the incidence of Jonathan spot (C.S.I.R.0. 1958. Martin gt,§l,. 1961). Kinetin is known as a regulator of cell division (Das gt,al,. 1956) and apple ovules are known to excrete indolaceticacid (IAA) shortly after bloom (Luckwill. 1957). It is possible that some factor that is excreted more abundantly by the ovules of Jonathan and related cultivars affects certain cells at the periphery of the fruit at some early stage of cell division. This could result in certain areas in the hypodermis under- going different rates of cell division and possibly some permanent 66 modifications in the Donan free space. In time. after harvest. when the fruits would be exposed to suitable external conditions. these pre- disposed areas of the skin may become Spotted. 0n the basis of various findings and pertinent literature that were reported in this thesis. the following hypothesis is suggested: 1. 2. 3. Some factor. perhaps associated with the seeds. brings about a certain modification in some cells in the peripherial tissue of the young fruit. After the harvest. there exists a preparatory stage in the path of Spot formation which is biphasic in regard to temperature. the second phase having a lower optimal temperature than the first one. During this preparatory stage cations and phosphorus start to accumulate in the spot susceptible cells. The cation influx into these cells triggers a higher rate of acid production or affects a lower rate of acid destruction. As a result of this cation influx. the pH of these cells rises. The rise in pH is suggested as the initial causal factor pro- moting anthocyanin breakdown and possibly channeling carbonylic compounds such as indolquinonevand polyphenols into the synthe- sis of the brown pigment that visually characterizes Jonathan spot. CONCLUSIONS AND SUMMARY The genesis and physiology of a spot disorder affecting the epider- mal tissue of matured Jonathan apple fruit was investigated in an attempt to postulate a theory concerning the causal sequence involved in its formation. It was initially determined from morphological observations that the disorder commonly termed as Jonathan spot consists of two types. one of which occurs in association with an aperture in the epidermis. particularly the lenticels. and has therefore been referred to in this study as lenticel spot. The other. or "true" Jonathan spot occurs with- out relation to lenticels or other breaks or openings in the epidermis. Differing anatomical modifications of the skin tissue were observed for lenticel and Jonathan Spots. Lenticel spot occurs in conjunction with a break in the epidermal tissue. whereas Jonathan spot occurs with- out relation to any apertures and is characterized by a higher number of compressed cells per unit cross sectional area of epidermal tissue. Temperature had a dissimilar effect upon the development of the two spot types. Lenticel spot was often accelerated by high temperature (70°F). whereas Jonathan spot was inhibited by high temperature. The optimal temperature for Jonathan spot development was below 60°. It is evident that much of the confusing and contradictory data of the literature is due to a lack of a distinction between the two dis- orders. Having once resolved that there was a definite distinction between these two spot types. this study was devoted exclusively to the 67 68 genesis and physiology of Jonathan spot. Jonathan spot seemed biphasic in respect to the influence of tem- perature in that its incidence was accelerated when fruits were initially exposed to a high temperature after harvest. but lower temperature was required for the formation of the visible symptoms that characterize the spots. It is thus possible that Jonathan spot should be classified as a low temperature disorder. Noteworthy. prolonged exposures of apples to temperatures near 60°F substantially decreased the disorder. Relative humidity was an irrelevant factor in Jonathan spot de- velopment. This finding would seem contrary to observations made by other workers. who were probably concerned with lenticel spot rather than with Jonathan Spot in their studies. Above normal levels of CO2 surrounding the fruit. as low'as 0.7% inhibited Jonathan spot. The possibilities that other fruit volatiles were involved with this apparent C02 effect. however. were not resolved. Similar to the effects of high temperature. prolonged storage in con- trolled atmospheres (3% 02. 5% 002) tended to delay and to reduce the incidence of Jonathan spot. Various analytical determinations were made in an attempt to ex- plain the rise in pH of the spotted tissue. Although the pH of the spotted tissue homogenate varied considerably from 4.4 to 5.2. it was consistently 0.5 to 0.7 pH units higher than the pH of normal tissue but had an approximately double the normal buffering capacity all along the titration curve. Therefore. total titratable acidity of spotted and normal tissues was very similar. Mineral accumulation in spotted tissue was confirmed. amounting to a three-fold increase in the major cations (K. Ca. Mg) and P. 69 03137 was employed in an attempt to ascertain a possible sequential relation between the actual formation of the spots and the accumulation of K. It was applied to apple fruits about 3 weeks before harvest through the leaves or twigs. Autoradiographs showed that the label was uniformly distributed in the peel right after harvest of the fruits. challenging the idea that an early. pro-harvest accumulation of cations is responsible for Jonathan spot. Autoradiographs taken when spots were first observed indicated the absence of the tracer at the spots. Other autoradiographs taken later in the season were less conclusive. but re- vealed in several cases an accumulation of C3137 in the spots. Thus C3137 proved of little value in ascertaining if cation accumulation occurred prior. concomitantly or after the actual appearance of Jonathan spots. Nevertheless. one possible interpretation of the autoradiographs is that cation accumulation occurred prior to the visual symptoms of Jonathan spots. Acid extraction and fractionation showed an approximate two-fold increase in total acidity in the spotted tissue. as well as an increase in the percentage of citrate. It is postulated that the changes in cations and acidity were re- flected in the raised pH and in the titrational characteristics of the spotted homogenate. Jonathan spot characteristically appeared earlier and to a higher extent on the red in color areas of the apple skin. Newly spotted tissue showed a marked loss of anthocyanin. which.became greater with time. Although Jonathan spot has long been considered a disorder af- fecting anthocyaninébearing cells. anthocyanin in itself. however. can not be considered as a mandatory precursor for the formation of Jonathan 70 spot since mature green apples eventually developed extensive spotting. It is speculated that degradated anthocyanin served as a precursor for the synthesis of the brown pigment which is typical to the disorder. but that it was only one possible precursor for this pigment. others being quinic and shikimic acids and l-epicatechol that were found in relative abundance in apple peel. The respiratory measurements of spotted peel discs in a warburg respirometer revealed that endogenous respiration of spotted peel discs varied greatly. In 1962. newly spotted discs suspended in phosphate buffer showed a similar to normal endogenous 02 uptake. refuting the idea that Jonathan spot involved an immediate breakdown of the affected tissue. Old spotted tissue. however. exhibited a 50% decrease in 02 uptake. In 1963. when respiration measurements were conducted some two months later than in the previous year and citrate-phosphate buffer was used. the discs exhibited a 50% decrease of the normal endogenous 02 uptake. Addition of malate showed that the spotted discs partially lost the capacity to decarboxylate malate. weakening a tenative assumption that Jonathan spot resulted from an accelerated decarboxylation of malic acid. Addition of catechol and ascorbic acid resulted in identical 02 uptake for both the spotted and normal discs. indicating a similar con- tent of ascorbic acid and polyphenol oxidases per unit dry wt. It was suggested that the reduction in endogenous 02 uptake that the spotted tissue exhibited may be due to a lack of substrate in that tissue. A hypothesis of the causal sequence involved in Jonathan spot de- velopment was proposed. It included two major assumptions. namely. (1) that some cells in the peripherial tissues of the receptacle undergo 71 a certain modification early in the ontogeny of the fruit. and (2),that cation and phosphorus accumulation which later occurs in that tissue. triggers the eventual formation of Jonathan spot. It is evident that a satisfactory comprehension of the genesis and development of Jonathan spot can be accomplished only with a better basic understnading of the gross effects of the seeds in young fruits and of the processes involved in mineral transport through cellular membranes. LITERATURE CITED Australia. Commonwealth Sci. Ind. Research Organization. 1958. Fruit investigations. Tenth Annual Report: 20-29. Biale. J. B. 1960. The post harvest biochemistry of tropical and subtropical fruits. Advances in Food Research 10:293-354. Brooks. 0.. and J. S. Cooley. 1917. Effects of temperature aeration and humidity on Jonathan Spot and scald of apples in storage. J. Agr. Research 9:287-318. . and D. F. Fisher. 1918. Irrigation experiments on apple spot diseases. J. Agr. Research 12:109-137. Bunemann. 0. 1958. The relation of the nutrient element content in the leaves and fruits to the storage quality of Jonathan apples in regular and controlled atmospheres. Unpublished Ph.D. Thesis. 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