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Cellular image profile analysis of apples exhibiting corking disorders as related to calcium and potassium
Scientia Horticulturae, 16 (1982) 217--231 Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
217
CELLULAR IMAGE PROFILE ANALYSIS OF APPLES EXHIBITING CORKING DISORDERS AS RELATED TO CALCIUM AND POTASSIUM
R.K. SIMONS and M.C. CHU
Department of Horticulture, University of illinois, Urbana, IL (U.S.A.) (Accepted for publication 16 June 1981)
ABSTRACT Simons, R.K. and Chu, M.C., 1982. Cellular image profile analysis of apples exhibiting corking disorders as related to calcium and potassium. Scientia Hortic., 16: 217--231. Cellular characterization and electron microprobe studies of apple fruit cultivars 'Starking Delicious' and 'Spigold' were made in relation to corking as the fruit approached maturation. Profiles of tissue development and subsequent breakdown indicate that this physiological disorder has developed in formative and developmental stages of growth, with extensive tissue breakdown. Cultivar differences were noted, with 'Spigold' having larger cortical cells than 'Starking', although both had similar anomalies in cellular development. These were characterized by extensive cell proliferation throughout the cortex and extending between the hypodermis and the core-line bundle. Numerous large lacunae were found, with smaller ones being interspersed contiguous to small, supporting, senescent vascular bundles. Both potassium and calcium were low in all affected tissues, although the potassium level was higher than calcium in most of the sampled areas.
INTRODUCTION
The physiological disorders of apples, variously termed "bitter pit" and "cork spot", are related to the inability of calcium (Ca) to be absorbed into the developing tissues. These disorders develop over a long period of time and are influenced b y cultural and environmental factors, including cultivar, nutrition and weather (Perring, 1979). Although descriptive terms for corking disorders vary in relation to the maturity of the apple for macroscopic observation, microscopically they are virtually indistinguishable from each other (except in severity) as the interrelated symptoms typical of corking disorders. In Netherlands orchard trees after mid-July, the intake of Ca into the fruit was counter-balanced b y Ca leaving the fruit in the water flowing back at the high water stresses occurring with the tree during the day (Tromp, 1979). In the Central United States this would be comparable to the stage of growth when the terminal bud has " s e t " and, at the same time, fruit enlargement occurs at a rapid rate. Wiersum (1979) found that it may be related to the accumulation of Ca whieh occurred to a very large extent in the pedicels, while
0304-4238/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishing Company
218 only a limited amount reached the fruit. There were also Ca-containing crystals in phloem parenchyma, ray parenchyma and pith of one-year-old shoots (Terblanche et al., 1979). Other studies (Terblanche et al., 1980) showed that orchard nutrition is the most important scientifically-based tool b y which the incidence of bitter pit can be manipulated. Hanger (1979) found Ca movement in plants to be unidirectional, moving up from the roots and generally routed to meristematic zones and young tissue. Once deposited in a leaf, it is not recycled, even under conditions of Ca-stress. Thus, it is the most immobile macronutrient in the plant. Schumacher et al. (1978) found that fruits susceptible to bitter pit come from the most vigorous parts of the trees. Apical dominance influences the deposition of assimilates and nutrient elements into the fruits. This disorder has a positive correlation with K/Ca and the K content of the fruit, as well as the average fruit size. A negative correlation exists between the disorder and the Ca content. Simons et al. (1971, 1980) found that corking development of apples at the mid-season stage of development was expressed b y extreme cell-wall thickening in the hypodermis. The fruit was incapable of further growth expansion in the affected area as a result of cell collapse. MacArthur (1940) reported that cellular development in relation to internal cork produced numerous "reactivated" cells external to the phellem. The walls of such cells were definitely thinner. The nuclei were interspersed among the reactivated cells, often filling the intercellular spaces as a second t y p e of abnormal cell. The abnormal tissue was composed of attenuated cells that were initiated by: (a) division of cortical cells; (b) the linear growth of phellem cells. Miller (1980) found similar development concerning the ontogeny and cytogenesis of cork spot in 'York Imperial' apple fruit. Sharples and Johnson (1977) have d o c u m e n t e d that Ca influences permeability, maintains membrane integrity and lowers respiration rates. Apple fruits of low Ca content showed earlier and more severe s y m p t o m s of cell membrane breakdown than fruits of high Ca content (Fuller, 1976). A negative correlation exists between fruit size and Ca concentration with Ca increasing in very small fruits (Perring and Preston, 1974). An erratic climatic regime has been shown to have an impact upon texture and cell-wall thickening, with some cultivars being more susceptible than others. Tree parts with high evaporation and development of hormones seemed to influence the deposition of nutrient-elements in the fruits during early development and thus also their susceptibility to bitter pit (Schumacher et al., 1978). Since corking development has been difficult to correct under different environmental stress conditions, the purpose of our research was (a) to examine the developmental aspects of corking tissues, and (b) to record the presence or absence of K and Ca in specific anomalous areas in relation to tissue breakdown. It is a cell-by-cell analysis of Ca-content in corky tissues.
219 MA TER I ALS AND METHODS
Fruit samples, approaching maturation and exhibiting corking indentations, of 'Starking Delicious' and 'Spigold' were obtained for cellular characterization by scanning electron microscopy (SEM) and for electron microprobe studies. Under mid-west U.S.A. growing-conditions, fruit were nearing maturity before the corking disorders were visible macroscopically. Although corking development occurred on both cultivars, the 'Spigold' trees had a previous history of severe corking. Surface necrosis was more prevalent on both cultivars between the equatorial axis and the calyx region. Ten samples of fresh fruit of each cultivar were prepared for SEM studies and for electron microprobe analysis (only on a qualitative basis) of nutrient elements, particularly K and Ca. The samples were placed in 50% ETOH, and carried through a 4-step series of ethanol--water before utilizing critical-point procedures (Anderson, 1951; Horridge and Tamm, 1969). SEM studies were made with a Cambridge Mark II microscope. The electron microprobe X-ray analysis was made on an ORTEC scanner with a JSM-U 3 scanning electron microscope. Samples for the microprobe analysis were critical-point dried and then coated with chrome. RESULTS AND DISCUSSION
'Starking Delicious' Cellular breakdown.-- The profile of tissue development and subsequent breakdown (Figs. 1 and 2) illustrates that this physiological disorder had developed in the formative and developmental stages of growth, with extensive tissue breakdown. Cross references (small letters on plates) indicate sampling areas of specific supporting vascular tissue in the outer cortex which have been analyzed by the electron microprobe and presented in Figs. 3--5. Extensive cell proliferation was found throughout the cortex, extending between the hypodermis and the core-line bundle (Fig. 1A). Abnormal growth of the fruit, as a precursor to corking, occurred over a succession of developmental growth periods. There were numerous large lacunae, with smaller ones being interspersed contiguous to small supporting vascular bundles. Isolated breakdown was also found, and was the end result of the collapse of large cortical cells. Intact cells surrounded large lacunae (Fig. 1A). A profile outline in a transverse section showed tissue degradation throughout the fruit, including the cuticle and hypodermis, which continued into the inner cortex (arrow, Fig. 1). This area showed cell necrosis, lacunae formation, cell division and proliferation within cells of the outer cortex, and disintegration of cells having abnormally thick walls near the hypodermal cell layers. There was a progression of chain-like cell proliferation, contiguous to large lacunae, deep within the cortex which had thick cell walls (bottom, Fig. 1). Cellular development was shown in relation to vascular tissues of the outer
220 cortex. The hypodermis and epidermis remained intact, although the cuticle had disintegrated at the edge of corking. A break in the fruit surface, with visual corking development, was also evident (at the right of Fig. 1). Cuticle thickness was variable, being approximately 1/3 as thick at the edge of the corking as in the areas of normal cuticle. Supportive vascular tissue of the outer fruit cortex (arrow, Fig. 1A; enlarged, from arrow, Fig. 1) showed disintegration of cells having a different size and shape adjacent to the bundle. Number and size of regenerative cells indicated progressive development involved in tissue breakdown. Fruit tissue did not regenerate normally when mature (Simons and Lott, 1964). However, under abnormal development, regeneration was accentuated during these later developmental stages. Starch-grain accumulation was not abundant in the small, proliferated cells, but did accumulate in cells adjacent to the minute vascular bundles in the cortex, which appeared to contribute to abnormal maturation in localized tissues. Cells were thick-walled, with pectic protuberances appearing on the cell-wall surface. This was previously verified by light microscopy (Simons, 1962). The apex of supporting vascular tissues in the outer cortex (z, Fig. 2) is shown in relation to adjacent vascular tissues (d, Fig. 1A). The apical part of this vascular bundle was adjacent to large lacunae, which were formed from small proliferated cells, abnormally thick-walled and collapsing. Thick cell walls of the vascular tissue appeared incapable of normal translocation (Fig. 2e,f). Vascular branching occurred deep within the cortex adjacent to cell proliferation where there were necrotic areas, with the eventual formation of large lacunae that persisted throughout the middle-outer cortex to the hypodermal region (Fig. 2c,d). A growth pattern, combining senescence with a resumption of meristematic activity directly associated with irregular growing regimes, has been observed in watercored 'Starking' apples (R.K. Simons, 1981, unpublished data.) E l e c t r o n m i c r o p r o b e a n a l y s e s . - - These showed variations in K and Ca through-
out the sampled areas (Figs. 3--5). Parts of the fruit that were devoid of corking symptoms contained different levels of K and Ca (Fig. 3A--F). This included the hypodermis (3A), beginning of cortical cells (3B), lacunae (3C) and other areas of cortical cells (3D--F). These electron microprobe evaluations revealed that the K content was greater than Ca in all instances. The cortical tissues contained less of both elements than the hypodermis and epidermis. However, they were in the same ratio and all were at low levels. Electron microprobe analyses of vascular tissue (Fig. 4A--G) were made from the areas indicated in Fig. la--g. K and Ca trends occurred in the same ratio for these specific tissues as for those illustrated in Fig. 3. K and Ca content of the tissues in the general area leading to disintegration (see Fig. l a --d) decreased in the hypodermis and throughout the vascular tissues supporting this region (area enlarged in Fig. 2d--g). There was a slight accumulation of K and Ca within the e,f~g portion of the vascular tissue at the mid-section
221
of the fruit cortex. Variations in silicon (Si), K and Ca were found by electron microprobe recordings between the equatorial axis and the calyx region. Ca had accumulated as opposed to a decrease in K. This region is usually susceptible to apparent corking symptoms. The cuticle and epidermis not showing corking (Fig. 5A) m a y be compared with the edge of the corked area (Fig. 5B) in which Si had increased. This trend continued within the cuticle and epidermis of the corked area as Ca increased, K decreased, and Si remained high (Fig. 5C,D). 'Spigold' Cellular b r e a k d o w n . -- A cellular profile through the cuticle and continuing
into the cortex is shown in a transverse section, with the specific areas indicated where the analysis was made (Fig. 6A--H). The cellular structure of 'Spigold' was different from that of 'Starking' in that the cortical cells were larger. Breakdown of entire areas extended beyond and included cortical cells away from the point of corking, although the cuticle had remained intact (Fig. 6). These cells collapsed and formed lacunae (Figs. 6 and 7). This mass degeneration of large numbers of cortical cells and lack o f regenerative tissues (which included cell division and proliferation) contrasted to the 'Starking' samples, where similar development occurred throughout the same tissues on a smaller scale, although abundant cell proliferation was present (illustrated in I, Fig. 6, and further enlarged in Fig. 7). Thick cortical cells were found throughout the affected fruit in relation to corking. The entire outer cortex contained cells with thick walls. Disintegration was specific between the epidermis, hypodermis and the pitted area (Figs. 6A, 7, arrow in 8A,B). However, as in other studies (Simons, 1962), the cortical cells were affected between the epidermis and core-line bundles in the fruit that exhibited corking. Large lacunae were found contiguous to vascular bundles, and cells were subsequently breaking down in this area. The cuticle disintegrated markedly at the edge of the affected area, and metabolic products of breakdown (Fuller, 1976) were apparent in this tissue (Fig. 8B). The vascular bundle shown in Fig. 7(vb) has been enlarged in Fig. 8C to show the large contiguous cell which was filled with starch grains (s) and which was adjacent to the lacunae, indicated by I, Figs. 6 and 7. E l e c t r o n m i c r o p r o b e analyses. - - The areas designated in Fig. 6 are analyzed
in Fig. 9A--H. Figure 9A (corresponding to the area in Fig. 6A) indicates Si accumulation in the epidermis at the pit edge. The hypodermis (Fig. 9B) (directly adjacent to the epidermis in Fig. 9A) was also high in Si, which declined in the outer cortical cells (Figs. 6C and 9C). There was also proportionately less K and Ca in these respective areas. K and Ca were found in minute amounts at the edge of cellular breakdown (Figs. 6D and 9D) where Si had also decreased.
222 The epidermis of tissues not in the immediate breakdown area (Figs. 6E and 9E) was higher in Si, b u t not as high as that in the corked area {Fig. 9A), while K and Ca were at low levels in this area. This corresponds to the findings (Bramlage et al., 1979) in which Ca was found to decline in the outer flesh during maturation and reached a minimum at, or shortly following, harvest. The remaining tissues studied in Fig. 6F--H were analyzed by the electron microprobe (Fig. 9F--H). These areas include lacunae in the corked area, dividing cells at the edge of the broken surface, and the cuticle from the area not exhibiting s y m p t o m s of breakdown. In these cases, K and Ca were at levels t o o low to be of significance. In both cultivars, the surface corking areas may have appeared macroscopically to be very small. However, this was always accompanied b y large areas o f abnormal breakdown deep within the fruit with premature senescence. These growth combinations initiated abnormally early fruit maturation. K and Ca were at low levels in the fruit that exhibited corking. CONCLUSION The incidence of corking
223
Fig. 1. Transverse section of 'Starking Delicious' showing extent of cell proliferation in relation to vascular tissues in the fruit cortex, as enlarged from Fig. 1A (upper right) with the continuous arrow (Fig. 1 ) extending through the cortical tissue to the left side of the vascular bundle shown in Fig. 2. Figure 1, a---g is shown in Figs. 3 and 4 with electronmicroprobe analysis of each area. Fig. 1, x 62; Fig. 1A, x 12.5.
224
Fig. 2. Transverse section of 'Starking Delicious' fruit 4 weeks before maturation, with areas enlarged contiguous to the vascular bundle. This corresponds to areas designated c--g in Fig. 1A. These specific areas were recorded by electron-microprobe analysis, see Figs. 3 and 4. z indicates the apex of the supporting vascular tissues in the outer cortex and corresponds to d, Fig. 1A. X 61.
225
Fig. 3. A - - F . Microprobe analysis of 'Starking Delicious' indicating areas of tissue sampled in Fig. 1A. A, hypodermis; B, beginning of cortical cells; C, lacuna (asterisk, Fig. 1A); D,E,F, cortical cells. X 5000; 5 K. Fig. 4. A--G. Microprobe analysis of 'Starking Delicious' showing vascular bundle supporting the fruit cortex from near the core-line bundle to the outer extremities of the cortex adjacent to the hypodermis. Specific areas in Fig. 4, A--G correspond to Fig. 1, A--G. × 5000; 5 K.
226
si "
kca
si
kca
Fig. 5. A--D. Microprobe analysis o f mature 'Starking Delicious' showing corking located between the equatorial axis and calyx region. A, cuticle avd epidermis not in pit area; B, cuticle and epidermis at edge of pit; C,D, cuticle and epidermis in pit area. × 5000; 5K
Fig. 6. Transverse section of mature 'Spigold' fruit showing tissue disintegration in the outer cortex at the periphery of tissue breakdown. A--H indicate electron-microprobe analysis references in Fig. 9 A--H. × 24.
227
Fig. 7. Transverse section in mature 'Spigold' fruit to illustrate corking as related to c u t i c l e epidermis, h y p o d e r m i s and o u t e r c o r t e x with supporting vascular tissues, x 40.
228
Fig. 8.
229
Fig. 8. Transverse section of mature 'Spigold' fruit showing (A) cuticle, epidermis, hypo_ dermis and outer cortical cells; (B) cuticle degradation at edge of tissue breakdown; (C) vascular tissue in the outer cortex with starch grain accumulation contiguous to the bundle A, C, × 240; B, x 1220.
230
Fig. 9. Electron-microprobe analysis of mature 'Spigold' fruit with corking. A, epidermis at edge of tissue breakdown; B, hypodermis contiguous to A; C, hypodermal cells adjacent to cortex; D, the beginning of cortical cells; E, area contiguous to breakdown with epidermis; F, cavity in corked area contiguous to epidermis; G, dividing cells at the edge of corking; H, cuticle from area not exhibiting corking symptoms, x 5000; 5K. REFERENCES Anderson, T.F., 1951. Critical point method. Trans. N.Y. Acad. Sci. Ser. I.I, 13: 130. Bramlage, W.J., Drake, M. and Baker, J.H., 1979. Changes in calcium level in apple cortex tissue shortly before harvest and during postharvest storage. Commun. Soil Sci. Plant Anal. 10: 417--426. Fuller, M.M., 1976. The ultrastructure of the outer tissues of cold-stored apple fruits of high and low calcium content in relation to cell breakdown. Ann. Appl. Biol., 83: 2 9 9 304. Hanger, B.C., 1979. The movement of calcium in plants. Commun. Soil Sei. Plant Anal., 10: 171--193. Horridge, G.A. and Tamm, S.L., 1969. Critical point drying for scanning electron microscopic study of ciliary motion. Science, 163: 817--818.
231 MacArthur, M., 1940. Histology of some physiological disorders of the apple fruit. Can. J. Res., C18: 26--34. Miller, R.H., 1980. The ontogeny and cytogenesis of cork spot in 'York Imperial' apple fruit. J. Am. Soc. Hortic. Sci., 105: 355--364. Perring, M.A., 1979. The effects of environment and cultural practices on calcium concentration in the apple fruit. Commun. Soil Sci. Plant Anal., 10: 279--293. Perring, M.A. and Preston, A.P., 1974. The effect of orchard factors on the chemical composition of apples. III. Some effects of pruning and nitrogen application in Cox's Orange Pippin fruit. J. Hortic. Sci., 49: 85--93. Schumacher, R., Fankhauser, F. and Stadler, W., 1978. Mineralstoffgehalte und Stippeanf~illigkeit yon Apfelm in Abh~/ngigkeit yon Ihrer Ansatzstelle in der Baumkrone. Schweiz. Z. Obst-Weinbau, 114(87): 295--303. Sharpies, R.O. and Johnson, D.S., 1977. The influence of calcium on senescence change in apple. Ann. Appl. Biol., 85: 450--453. Simons, R.K., 1962. Anatomical studies of the bitter pit area of apples. Proc. Am. Soc. Hortic. Sci., 81: 41--50. Simons, R.K. and Lott, R.V., 1964. The morphological and anatomical development of apples injured by early season hail. Proc. Am. Soc. Hortic. Sci., 85: 60--73. Sirnons, R.K., Hewetson, F.N. and Chu, M.C., 1971. Sequential development of the 'York Imperial' apple as related to tissue variances leading to corking disorders. J. Am. Soc. Hortic. Sci., 96: 247--252. Simons R.K., Chu, M.C. and Doll, C.C., 1980. Physiological disorders of the apple at midseason stage of development. Scientia Hortic., 13 : 227--233. Terblanche, J.H., Wooldridge, L.G., Hesebeck, I. and Joubert, M., 1979. The redistribution and immobilization of calcium in apple trees with special reference to bitter pit. Commun. Soil Sei. Plant Anal., 10: 195--215. Terblanche, J.H., Gurgen, K.H. and Hesebeck, I., 1980. An integrated approach to orchard nutrition and bitter pit control. In: Mineral Nutrition of Fruit Trees. Chap. 8. Butterworths, London, 435 pp. Tromp, J., 1979. The intake curve for calcium into apple fruits under various environmental conditions. Commun. Soil Sci. Plant. Anal., 10: 323--335. Wiersum, L.K., 1979. Effects of environmental and cultural practices on calcium nutrition. Commun. Soil Sci. Plant Anal. 10: 259--278.
217
CELLULAR IMAGE PROFILE ANALYSIS OF APPLES EXHIBITING CORKING DISORDERS AS RELATED TO CALCIUM AND POTASSIUM
R.K. SIMONS and M.C. CHU
Department of Horticulture, University of illinois, Urbana, IL (U.S.A.) (Accepted for publication 16 June 1981)
ABSTRACT Simons, R.K. and Chu, M.C., 1982. Cellular image profile analysis of apples exhibiting corking disorders as related to calcium and potassium. Scientia Hortic., 16: 217--231. Cellular characterization and electron microprobe studies of apple fruit cultivars 'Starking Delicious' and 'Spigold' were made in relation to corking as the fruit approached maturation. Profiles of tissue development and subsequent breakdown indicate that this physiological disorder has developed in formative and developmental stages of growth, with extensive tissue breakdown. Cultivar differences were noted, with 'Spigold' having larger cortical cells than 'Starking', although both had similar anomalies in cellular development. These were characterized by extensive cell proliferation throughout the cortex and extending between the hypodermis and the core-line bundle. Numerous large lacunae were found, with smaller ones being interspersed contiguous to small, supporting, senescent vascular bundles. Both potassium and calcium were low in all affected tissues, although the potassium level was higher than calcium in most of the sampled areas.
INTRODUCTION
The physiological disorders of apples, variously termed "bitter pit" and "cork spot", are related to the inability of calcium (Ca) to be absorbed into the developing tissues. These disorders develop over a long period of time and are influenced b y cultural and environmental factors, including cultivar, nutrition and weather (Perring, 1979). Although descriptive terms for corking disorders vary in relation to the maturity of the apple for macroscopic observation, microscopically they are virtually indistinguishable from each other (except in severity) as the interrelated symptoms typical of corking disorders. In Netherlands orchard trees after mid-July, the intake of Ca into the fruit was counter-balanced b y Ca leaving the fruit in the water flowing back at the high water stresses occurring with the tree during the day (Tromp, 1979). In the Central United States this would be comparable to the stage of growth when the terminal bud has " s e t " and, at the same time, fruit enlargement occurs at a rapid rate. Wiersum (1979) found that it may be related to the accumulation of Ca whieh occurred to a very large extent in the pedicels, while
0304-4238/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishing Company
218 only a limited amount reached the fruit. There were also Ca-containing crystals in phloem parenchyma, ray parenchyma and pith of one-year-old shoots (Terblanche et al., 1979). Other studies (Terblanche et al., 1980) showed that orchard nutrition is the most important scientifically-based tool b y which the incidence of bitter pit can be manipulated. Hanger (1979) found Ca movement in plants to be unidirectional, moving up from the roots and generally routed to meristematic zones and young tissue. Once deposited in a leaf, it is not recycled, even under conditions of Ca-stress. Thus, it is the most immobile macronutrient in the plant. Schumacher et al. (1978) found that fruits susceptible to bitter pit come from the most vigorous parts of the trees. Apical dominance influences the deposition of assimilates and nutrient elements into the fruits. This disorder has a positive correlation with K/Ca and the K content of the fruit, as well as the average fruit size. A negative correlation exists between the disorder and the Ca content. Simons et al. (1971, 1980) found that corking development of apples at the mid-season stage of development was expressed b y extreme cell-wall thickening in the hypodermis. The fruit was incapable of further growth expansion in the affected area as a result of cell collapse. MacArthur (1940) reported that cellular development in relation to internal cork produced numerous "reactivated" cells external to the phellem. The walls of such cells were definitely thinner. The nuclei were interspersed among the reactivated cells, often filling the intercellular spaces as a second t y p e of abnormal cell. The abnormal tissue was composed of attenuated cells that were initiated by: (a) division of cortical cells; (b) the linear growth of phellem cells. Miller (1980) found similar development concerning the ontogeny and cytogenesis of cork spot in 'York Imperial' apple fruit. Sharples and Johnson (1977) have d o c u m e n t e d that Ca influences permeability, maintains membrane integrity and lowers respiration rates. Apple fruits of low Ca content showed earlier and more severe s y m p t o m s of cell membrane breakdown than fruits of high Ca content (Fuller, 1976). A negative correlation exists between fruit size and Ca concentration with Ca increasing in very small fruits (Perring and Preston, 1974). An erratic climatic regime has been shown to have an impact upon texture and cell-wall thickening, with some cultivars being more susceptible than others. Tree parts with high evaporation and development of hormones seemed to influence the deposition of nutrient-elements in the fruits during early development and thus also their susceptibility to bitter pit (Schumacher et al., 1978). Since corking development has been difficult to correct under different environmental stress conditions, the purpose of our research was (a) to examine the developmental aspects of corking tissues, and (b) to record the presence or absence of K and Ca in specific anomalous areas in relation to tissue breakdown. It is a cell-by-cell analysis of Ca-content in corky tissues.
219 MA TER I ALS AND METHODS
Fruit samples, approaching maturation and exhibiting corking indentations, of 'Starking Delicious' and 'Spigold' were obtained for cellular characterization by scanning electron microscopy (SEM) and for electron microprobe studies. Under mid-west U.S.A. growing-conditions, fruit were nearing maturity before the corking disorders were visible macroscopically. Although corking development occurred on both cultivars, the 'Spigold' trees had a previous history of severe corking. Surface necrosis was more prevalent on both cultivars between the equatorial axis and the calyx region. Ten samples of fresh fruit of each cultivar were prepared for SEM studies and for electron microprobe analysis (only on a qualitative basis) of nutrient elements, particularly K and Ca. The samples were placed in 50% ETOH, and carried through a 4-step series of ethanol--water before utilizing critical-point procedures (Anderson, 1951; Horridge and Tamm, 1969). SEM studies were made with a Cambridge Mark II microscope. The electron microprobe X-ray analysis was made on an ORTEC scanner with a JSM-U 3 scanning electron microscope. Samples for the microprobe analysis were critical-point dried and then coated with chrome. RESULTS AND DISCUSSION
'Starking Delicious' Cellular breakdown.-- The profile of tissue development and subsequent breakdown (Figs. 1 and 2) illustrates that this physiological disorder had developed in the formative and developmental stages of growth, with extensive tissue breakdown. Cross references (small letters on plates) indicate sampling areas of specific supporting vascular tissue in the outer cortex which have been analyzed by the electron microprobe and presented in Figs. 3--5. Extensive cell proliferation was found throughout the cortex, extending between the hypodermis and the core-line bundle (Fig. 1A). Abnormal growth of the fruit, as a precursor to corking, occurred over a succession of developmental growth periods. There were numerous large lacunae, with smaller ones being interspersed contiguous to small supporting vascular bundles. Isolated breakdown was also found, and was the end result of the collapse of large cortical cells. Intact cells surrounded large lacunae (Fig. 1A). A profile outline in a transverse section showed tissue degradation throughout the fruit, including the cuticle and hypodermis, which continued into the inner cortex (arrow, Fig. 1). This area showed cell necrosis, lacunae formation, cell division and proliferation within cells of the outer cortex, and disintegration of cells having abnormally thick walls near the hypodermal cell layers. There was a progression of chain-like cell proliferation, contiguous to large lacunae, deep within the cortex which had thick cell walls (bottom, Fig. 1). Cellular development was shown in relation to vascular tissues of the outer
220 cortex. The hypodermis and epidermis remained intact, although the cuticle had disintegrated at the edge of corking. A break in the fruit surface, with visual corking development, was also evident (at the right of Fig. 1). Cuticle thickness was variable, being approximately 1/3 as thick at the edge of the corking as in the areas of normal cuticle. Supportive vascular tissue of the outer fruit cortex (arrow, Fig. 1A; enlarged, from arrow, Fig. 1) showed disintegration of cells having a different size and shape adjacent to the bundle. Number and size of regenerative cells indicated progressive development involved in tissue breakdown. Fruit tissue did not regenerate normally when mature (Simons and Lott, 1964). However, under abnormal development, regeneration was accentuated during these later developmental stages. Starch-grain accumulation was not abundant in the small, proliferated cells, but did accumulate in cells adjacent to the minute vascular bundles in the cortex, which appeared to contribute to abnormal maturation in localized tissues. Cells were thick-walled, with pectic protuberances appearing on the cell-wall surface. This was previously verified by light microscopy (Simons, 1962). The apex of supporting vascular tissues in the outer cortex (z, Fig. 2) is shown in relation to adjacent vascular tissues (d, Fig. 1A). The apical part of this vascular bundle was adjacent to large lacunae, which were formed from small proliferated cells, abnormally thick-walled and collapsing. Thick cell walls of the vascular tissue appeared incapable of normal translocation (Fig. 2e,f). Vascular branching occurred deep within the cortex adjacent to cell proliferation where there were necrotic areas, with the eventual formation of large lacunae that persisted throughout the middle-outer cortex to the hypodermal region (Fig. 2c,d). A growth pattern, combining senescence with a resumption of meristematic activity directly associated with irregular growing regimes, has been observed in watercored 'Starking' apples (R.K. Simons, 1981, unpublished data.) E l e c t r o n m i c r o p r o b e a n a l y s e s . - - These showed variations in K and Ca through-
out the sampled areas (Figs. 3--5). Parts of the fruit that were devoid of corking symptoms contained different levels of K and Ca (Fig. 3A--F). This included the hypodermis (3A), beginning of cortical cells (3B), lacunae (3C) and other areas of cortical cells (3D--F). These electron microprobe evaluations revealed that the K content was greater than Ca in all instances. The cortical tissues contained less of both elements than the hypodermis and epidermis. However, they were in the same ratio and all were at low levels. Electron microprobe analyses of vascular tissue (Fig. 4A--G) were made from the areas indicated in Fig. la--g. K and Ca trends occurred in the same ratio for these specific tissues as for those illustrated in Fig. 3. K and Ca content of the tissues in the general area leading to disintegration (see Fig. l a --d) decreased in the hypodermis and throughout the vascular tissues supporting this region (area enlarged in Fig. 2d--g). There was a slight accumulation of K and Ca within the e,f~g portion of the vascular tissue at the mid-section
221
of the fruit cortex. Variations in silicon (Si), K and Ca were found by electron microprobe recordings between the equatorial axis and the calyx region. Ca had accumulated as opposed to a decrease in K. This region is usually susceptible to apparent corking symptoms. The cuticle and epidermis not showing corking (Fig. 5A) m a y be compared with the edge of the corked area (Fig. 5B) in which Si had increased. This trend continued within the cuticle and epidermis of the corked area as Ca increased, K decreased, and Si remained high (Fig. 5C,D). 'Spigold' Cellular b r e a k d o w n . -- A cellular profile through the cuticle and continuing
into the cortex is shown in a transverse section, with the specific areas indicated where the analysis was made (Fig. 6A--H). The cellular structure of 'Spigold' was different from that of 'Starking' in that the cortical cells were larger. Breakdown of entire areas extended beyond and included cortical cells away from the point of corking, although the cuticle had remained intact (Fig. 6). These cells collapsed and formed lacunae (Figs. 6 and 7). This mass degeneration of large numbers of cortical cells and lack o f regenerative tissues (which included cell division and proliferation) contrasted to the 'Starking' samples, where similar development occurred throughout the same tissues on a smaller scale, although abundant cell proliferation was present (illustrated in I, Fig. 6, and further enlarged in Fig. 7). Thick cortical cells were found throughout the affected fruit in relation to corking. The entire outer cortex contained cells with thick walls. Disintegration was specific between the epidermis, hypodermis and the pitted area (Figs. 6A, 7, arrow in 8A,B). However, as in other studies (Simons, 1962), the cortical cells were affected between the epidermis and core-line bundles in the fruit that exhibited corking. Large lacunae were found contiguous to vascular bundles, and cells were subsequently breaking down in this area. The cuticle disintegrated markedly at the edge of the affected area, and metabolic products of breakdown (Fuller, 1976) were apparent in this tissue (Fig. 8B). The vascular bundle shown in Fig. 7(vb) has been enlarged in Fig. 8C to show the large contiguous cell which was filled with starch grains (s) and which was adjacent to the lacunae, indicated by I, Figs. 6 and 7. E l e c t r o n m i c r o p r o b e analyses. - - The areas designated in Fig. 6 are analyzed
in Fig. 9A--H. Figure 9A (corresponding to the area in Fig. 6A) indicates Si accumulation in the epidermis at the pit edge. The hypodermis (Fig. 9B) (directly adjacent to the epidermis in Fig. 9A) was also high in Si, which declined in the outer cortical cells (Figs. 6C and 9C). There was also proportionately less K and Ca in these respective areas. K and Ca were found in minute amounts at the edge of cellular breakdown (Figs. 6D and 9D) where Si had also decreased.
222 The epidermis of tissues not in the immediate breakdown area (Figs. 6E and 9E) was higher in Si, b u t not as high as that in the corked area {Fig. 9A), while K and Ca were at low levels in this area. This corresponds to the findings (Bramlage et al., 1979) in which Ca was found to decline in the outer flesh during maturation and reached a minimum at, or shortly following, harvest. The remaining tissues studied in Fig. 6F--H were analyzed by the electron microprobe (Fig. 9F--H). These areas include lacunae in the corked area, dividing cells at the edge of the broken surface, and the cuticle from the area not exhibiting s y m p t o m s of breakdown. In these cases, K and Ca were at levels t o o low to be of significance. In both cultivars, the surface corking areas may have appeared macroscopically to be very small. However, this was always accompanied b y large areas o f abnormal breakdown deep within the fruit with premature senescence. These growth combinations initiated abnormally early fruit maturation. K and Ca were at low levels in the fruit that exhibited corking. CONCLUSION The incidence of corking
223
Fig. 1. Transverse section of 'Starking Delicious' showing extent of cell proliferation in relation to vascular tissues in the fruit cortex, as enlarged from Fig. 1A (upper right) with the continuous arrow (Fig. 1 ) extending through the cortical tissue to the left side of the vascular bundle shown in Fig. 2. Figure 1, a---g is shown in Figs. 3 and 4 with electronmicroprobe analysis of each area. Fig. 1, x 62; Fig. 1A, x 12.5.
224
Fig. 2. Transverse section of 'Starking Delicious' fruit 4 weeks before maturation, with areas enlarged contiguous to the vascular bundle. This corresponds to areas designated c--g in Fig. 1A. These specific areas were recorded by electron-microprobe analysis, see Figs. 3 and 4. z indicates the apex of the supporting vascular tissues in the outer cortex and corresponds to d, Fig. 1A. X 61.
225
Fig. 3. A - - F . Microprobe analysis of 'Starking Delicious' indicating areas of tissue sampled in Fig. 1A. A, hypodermis; B, beginning of cortical cells; C, lacuna (asterisk, Fig. 1A); D,E,F, cortical cells. X 5000; 5 K. Fig. 4. A--G. Microprobe analysis of 'Starking Delicious' showing vascular bundle supporting the fruit cortex from near the core-line bundle to the outer extremities of the cortex adjacent to the hypodermis. Specific areas in Fig. 4, A--G correspond to Fig. 1, A--G. × 5000; 5 K.
226
si "
kca
si
kca
Fig. 5. A--D. Microprobe analysis o f mature 'Starking Delicious' showing corking located between the equatorial axis and calyx region. A, cuticle avd epidermis not in pit area; B, cuticle and epidermis at edge of pit; C,D, cuticle and epidermis in pit area. × 5000; 5K
Fig. 6. Transverse section of mature 'Spigold' fruit showing tissue disintegration in the outer cortex at the periphery of tissue breakdown. A--H indicate electron-microprobe analysis references in Fig. 9 A--H. × 24.
227
Fig. 7. Transverse section in mature 'Spigold' fruit to illustrate corking as related to c u t i c l e epidermis, h y p o d e r m i s and o u t e r c o r t e x with supporting vascular tissues, x 40.
228
Fig. 8.
229
Fig. 8. Transverse section of mature 'Spigold' fruit showing (A) cuticle, epidermis, hypo_ dermis and outer cortical cells; (B) cuticle degradation at edge of tissue breakdown; (C) vascular tissue in the outer cortex with starch grain accumulation contiguous to the bundle A, C, × 240; B, x 1220.
230
Fig. 9. Electron-microprobe analysis of mature 'Spigold' fruit with corking. A, epidermis at edge of tissue breakdown; B, hypodermis contiguous to A; C, hypodermal cells adjacent to cortex; D, the beginning of cortical cells; E, area contiguous to breakdown with epidermis; F, cavity in corked area contiguous to epidermis; G, dividing cells at the edge of corking; H, cuticle from area not exhibiting corking symptoms, x 5000; 5K. REFERENCES Anderson, T.F., 1951. Critical point method. Trans. N.Y. Acad. Sci. Ser. I.I, 13: 130. Bramlage, W.J., Drake, M. and Baker, J.H., 1979. Changes in calcium level in apple cortex tissue shortly before harvest and during postharvest storage. Commun. Soil Sci. Plant Anal. 10: 417--426. Fuller, M.M., 1976. The ultrastructure of the outer tissues of cold-stored apple fruits of high and low calcium content in relation to cell breakdown. Ann. Appl. Biol., 83: 2 9 9 304. Hanger, B.C., 1979. The movement of calcium in plants. Commun. Soil Sei. Plant Anal., 10: 171--193. Horridge, G.A. and Tamm, S.L., 1969. Critical point drying for scanning electron microscopic study of ciliary motion. Science, 163: 817--818.
231 MacArthur, M., 1940. Histology of some physiological disorders of the apple fruit. Can. J. Res., C18: 26--34. Miller, R.H., 1980. The ontogeny and cytogenesis of cork spot in 'York Imperial' apple fruit. J. Am. Soc. Hortic. Sci., 105: 355--364. Perring, M.A., 1979. The effects of environment and cultural practices on calcium concentration in the apple fruit. Commun. Soil Sci. Plant Anal., 10: 279--293. Perring, M.A. and Preston, A.P., 1974. The effect of orchard factors on the chemical composition of apples. III. Some effects of pruning and nitrogen application in Cox's Orange Pippin fruit. J. Hortic. Sci., 49: 85--93. Schumacher, R., Fankhauser, F. and Stadler, W., 1978. Mineralstoffgehalte und Stippeanf~illigkeit yon Apfelm in Abh~/ngigkeit yon Ihrer Ansatzstelle in der Baumkrone. Schweiz. Z. Obst-Weinbau, 114(87): 295--303. Sharpies, R.O. and Johnson, D.S., 1977. The influence of calcium on senescence change in apple. Ann. Appl. Biol., 85: 450--453. Simons, R.K., 1962. Anatomical studies of the bitter pit area of apples. Proc. Am. Soc. Hortic. Sci., 81: 41--50. Simons, R.K. and Lott, R.V., 1964. The morphological and anatomical development of apples injured by early season hail. Proc. Am. Soc. Hortic. Sci., 85: 60--73. Sirnons, R.K., Hewetson, F.N. and Chu, M.C., 1971. Sequential development of the 'York Imperial' apple as related to tissue variances leading to corking disorders. J. Am. Soc. Hortic. Sci., 96: 247--252. Simons R.K., Chu, M.C. and Doll, C.C., 1980. Physiological disorders of the apple at midseason stage of development. Scientia Hortic., 13 : 227--233. Terblanche, J.H., Wooldridge, L.G., Hesebeck, I. and Joubert, M., 1979. The redistribution and immobilization of calcium in apple trees with special reference to bitter pit. Commun. Soil Sei. Plant Anal., 10: 195--215. Terblanche, J.H., Gurgen, K.H. and Hesebeck, I., 1980. An integrated approach to orchard nutrition and bitter pit control. In: Mineral Nutrition of Fruit Trees. Chap. 8. Butterworths, London, 435 pp. Tromp, J., 1979. The intake curve for calcium into apple fruits under various environmental conditions. Commun. Soil Sci. Plant. Anal., 10: 323--335. Wiersum, L.K., 1979. Effects of environmental and cultural practices on calcium nutrition. Commun. Soil Sci. Plant Anal. 10: 259--278.