mung 3 1293 1 This is to certify that the thesis entitled RIPENING BEHAVIOR OF WILD TOMATO SPECIES presented by RE BECCA GRUMET has been accepted towards fulfillment of the requirements for M.S. degree in Horticulture / Wm WM Date April 28, 1980 0-7639 LIBRARY Michigan State ”JN‘fl‘L‘ - . “(fill \ _, \--‘n ’1',” . ‘ 4 4; CM mom W OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS \: Place in book return to remove charge from circulation records RIPENING BEHAVIOR OF WILD TOMATO SPECIES By Rebecca Grumet A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE 1980 Gags/7c ABSTRACT RIPENING BEHAVIOR IN WILD TOMATO SPECIES By Rebecca Grumet Nine wild tomato species were surveyed for variability in ripening habit. Fruits harvested at different ages were examined for ethylene production, respiratory behavior, and external ripening changes. Ethylene production and 002 evolution were monitored using an ethylene-free flow-through system and gas chromatography. The three color-fruited species all exhibited typical climacteric behavior in correspondence with visible ripening changes. Variability was observed for timing and magnitude of the climacteric response. Two of the green- fruited species did not ripen prior to abscission. They differed, however, for timing of endogenous ethylene produc— tion relative to abscission. The other green-fruited species ripened on the vine. They varied with respect to ethylene production, rate of ripening changes, and whether or not ripening occurred off the vine. The diversity exhibited for ripening habit by the wild tomato species implies differences in regulatory mechanisms. ACKNOWLEDGMENTS I would especially like to acknowledge the help and support of my major professors, Dr. Jon Fobes and Dr. Robert C. Herner who were always ready to assist in any way possible. I would also like to acknowledge the other members of my guidance committee, Dr. Andrew Hanson and Dr. David Dilley for their assistance and helpful suggestions. I would like to thank Jim O'Connel for growing and caring for my plants and Marcia Browers for helping me to make crosses. A special note goes to Beverly Burley for helping me to prepare the manuscript. Finally, I would like to thank Peter Irwin for coming to the rescue so many times when the G.C.'s weren't working. ii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . . . . . . . . . V LIST OF FIGURES . . . . . . . . . . . . . . . . . . . vi INTRODUCTION . . . . . . . . . . . . . . . . . . . . . 1 LITERATURE CITED . . . . . . . . . . . . . . . . . . . 5 CHAPTER I RIPENING BEHAVIOR OF WILD, COLOR-FRUITED TOMATO SPECIES Abstract . . . . . . . . . . . . . . . . . . . . . . . 8 Introduction . . . . . . . . . . . . . . . . . . . . . 8 Materials and Methods . . . . . . . . . . . . .~. . . 11 Results . . . . . . . . . . . . . . . . . . . . . . . 15 Discussion . . . . . . . . . . . . . . . . . . . . . . 26 Literature Cited . . . . . . . . . . . .-. . . . . . . 32 CHAPTER II RIPENING BEHAVIOR OF WILD, GREEN-FRUITED TOMATO SPECIES: FRUITS THAT ABSCISE PRIOR TO RIPENING Abstract . . . . . . . . . . . . . . . . . . . . . . . 36 Introduction . . . . . . . . . . . . . . . . . . . . . 36 Materials and Methods . . . . . . . . . . . . . . . . 38 Results . . . . . . . . . . . . . . . . . . . . . . . 41 iii Page Discussion . . . . . . . .‘. . . . . . . . . . . . . . 55 Literature Cited . . . . . . . . . . . . . . . . . . . 62 CHAPTER III RIPENING BEHAVIOR OF WILD, GREEN-FRUITED TOMATO SPECIES: FRUITS THAT RIPEN ON THE VINE Abstract . . . . . . . . . . . . . . . . . . . . . . . 65 Introduction . . . . . . . . . . . . . . . . . . . . . 65 Materials and Methods . . . . . . . . . . . . . . . . 67 Results . . . . . . . . . . . . . . . . . . . . . . . 67 Discussion . . . . . . . . . . . . . . . . . . . . . . 86 Literature Cited . . . . . . . . . . . . . . . . . . . 90 iv Table II III II II III LIST OF TABLES CHAPTER I Tomato germplasm used for assay of ripening characteristics . . . Fruit weight and approximate age of maturity: color-fruited tomato species . . . . . Comparison of peak ethylene production and 002 evolution during ripening in the color- fruited tomato Species—summary of several eXperiments . . . . . . . . CHAPTER II Levels of endogenous ethylene production in L. chilense fruit at various stages of development - summary of several experiments. Peak ethylene production in four categories of L. peruvianum fruit CHAPTER III Tomato germplasm used for assay of ripening characteristics . . . . Age of maturity and days to soften for detached L. chmielewskii and L. parviflorum fruits Comparison of peak rates of ethylene production and C02 evolution in L. hirsutum fruit with those of climacteric, color fruited tomatoes. Page In 17 25 46 50 68 77 85 Figure LIST OF FIGURES Page CHAPTER I Wild, color-fruited tomatoes. Left to right: Lycopersicon esculentum var. cerasiforme, L. pimpinellifolium, L. cheesmanii. . . . . . . . 12 Days to ripen vs. age at harvest for the wild, color-fruited tomatoes . . . . . . . . . 18 Ethylene production (a) and respiratory behavior (b) during ripening in the wild, color- fruited tomatoes: L. esculentum var. cerasiforme, L. pimpinellifolium and L. cheesmanii. Full color development was attained 3-5 days after peak ethylene production . . . . . . . . . . . . . . . . . . 21 Ethylene production and respiratory activity for three L. pimpinellifolium fruit per jar. . 23 The effect of propylene treatment on ethylene production (a) respiration (b) and color development of L. pimpinellifolium fruit . . . 27 CHAPTER II Green-fruited tomatoes that abscise prior to ripening. Left, Lycopersicon chilense; right, L. peruvianum . . . . . . . . . . . . . 39 Ethylene production of younger (a) and older (b) L. chilense fruit with, and without, propylene. Both sets of fruit had already lost their chlorophyll . . . . . . . . . . . 42 Ethylene production in abscised L. chilense fruit . . . . . . . . . . . . . . . . . . . . 44 Ethylene production in softening and non- softening, hand— detached L. pgruvianum fruits. Softening occurred in the high ethylene producing group at 66 days . . . . . . . . . . 48 vi Figure Page Natural abscission zone and point of calyx attachment in L. Peruvianum fruit . . . . . . . 52 Ethylene production in abscised L. peruvianum fruits, and fruits about to fall. Measurements made daily until softening occurred . . . . . . 53 Time-line of developmental processes in L. chilense fruit . . . . . . . . . . . . . . . . 57 Time-line of developmental processes in L. peruvianum fruit . . . . . . . . . . . . . . . 60 CHAPTER III Green-fruited tomatoes that ripen on the vine. Left to right: Solanum pennellLL, Lycopersicon hirsutum, L. chmielewskii, L. parviflorum . . . 69 Days to ripen vs. age at harvest for S. pennellii fruit 72 Ethylene production (a), respiratory behavior (b), and response to propylene in S. pennellii fruit. Softening occurred at 70 days in treated and untreated fruits . . . . . . . . . 74 Initial levels of ethylene production vs. fruit age at harvest in L. chmielewskii fruit . . . . 78 Ethylene production in L. hirsutum fruit harvested at different ages . . . . . . . . . . 81 Respiration in L. hirsutum fruit harvested at different ages . . . . . . . . . . . . . . . 83 vii Readers: The paper format utilized in this thesis meets the requirments stipulated by the Horticulture Department and the University. The thesis body was separated into three sections. They were prepared for PLant Physiology and follow the manuscript style of that journal. viii INTRODUCTION The cultivated tomato, L. esculentum, has long been a choice eXperimental unit for investigations into the ripening process. Much progress has been made toward understanding the highly regulated series of biochemical and physiological changes that mark transition of an immature green tomato fruit, to a ripe tomato. These changes include bursts in ethylene production and respiratory activity, chlorophyll degradation, biosynthesis of flavor components and carotenoid pigments, breakdown of starches and fruit softening (9). Early studies of respiratory activity in ripening fruits led to the discovery of the respiratory climacteric, an increase in respiration accompanying visible ripening changes (10). At approximately the same time, exogenous ethylene was reported to hasten ripening in a wide variety of fruits (1). Investigations into the role of endogenous ethylene production were thus stimulated (7). With the advent of gas chromatography, a rise in endogenous ethylene production was found to shortly preceed the rise in respiratory activity (3). Thus, a causal relationship between ethylene production and subsequent fruit ripening 2 with its associated respiratory climateric was hypothesized. Other supportive evidence included the inhibition of ripening by removal of ethylene from fruit storage contain- ers (1,4). Endogenous ethylene production by ripening fruit is not a universal phenomenon, however. Other fruits were studied which produce very little ethylene and do not undergo a respiratory climacteric (13). These fruits have been termed non-climacteric. In addition, aspects of ripening in climacteric fruit suggest ethylene is not the sole regulator of ripening. There are examples of increased respiration occurring without increased ethylene production (20), and increased ethylene production occurring without heightened respiration (1). Perhaps one of the strongest arguments for the limited role of ethylene in tomato is that immature green fruits cannot be induced to ripen normally by exposure to exogenous ethylene (9,12,15). Thus, some other changes must occur in the maturing fruit that enable it to respond to ethylene and subsequently ripen. Even clearer evidence for this comes from the gin and n9; non-ripening tomato mutants. They remain green and hard for months, produce little ethylene and do not undergo a respiratory climacteric (8,11). In response to exogenous ethylene, they appear typically non-climacteric and are not induced to ripen (8,11). Thus, their block to ripening must lie at some point prior to the ability to respond to ethylene. 3 This provides many questions of physiological and genetic import. How do gene products differ between mutant and normal fruits? How are developmental processes regulated? What roles do selective gene expression, environment, and physiological conditions play? Perhaps the most fundamental question facing biologists today is how gene eXpression is regulated. The tomato system may offer an effective approach to answer this question. Many models for ripening changes in tomato fruit have been proposed. The presence of genetically altered forms have enabled investigators to test these models more fully. Changes in hormone levels (2,5,6), membrane permeability (17,19), and various enzyme activities (15,16,18) have been assayed in normal and mutant fruits. Genetic diversity is therefore a potentially powerful tool to investigate physio- logical processes and understand the integration of genetics and physiology in the regulation of development. If diversity exists for ripening behavior, it may be possible to manipulate the process genetically. This study was initiated to survey ripening habit among wild tomato species. Wild tomato populations exhibit variability for a host of characteristics, thereby enabling breeders to increase yields, incorporate disease and insect resistance, increase stress tolerances, and heighten nutritional values (14). Observation in natural habitats suggests that vari- ability for ripening behavior was likely: wild tomato 4 fruits differ with respect to external ripening changes including softening, pigmentation, and abscission. Accord- ingly, the characterization of ripening undertaken for these fruits included determination of indices of ripening, age at maturity, ethylene production and respiratory behavior. The information obtained has led to hypotheses for ripening control mechanisms and has suggested numerous additional exPeriments. 10. 11. 12. LITERATURE CITED Abeles, F. B. 1973. Ethylene in plant biology. Academic Press, New York. Abdel-Rahman, M., T. H. Thomas, G. J. Doss and L. Howell. 1975. Changes in endogenous plant hormones in cherry tomato fruits during development and maturation. Physiol. Plant. 34:39-43. Burg, S. P. and E. A. Burg. 1968. Ethylene, plant senescence and abscission. Plant Physiol. 43:1503-1511. Burg, S. P. and E. A. Burg. 1966. Fruit storage at subatmosperic pressures. Science. 153:31A-315. Dilley, D. R. 1969. Hormonal control of fruit ripen— ing. HortSci. 4:111-114. Frenkel, C. and S. A. Garrison. 1976. Initiation of lycopene synthesis in the tomato mutant rin as influenced by oxygen and ethylene interactions. HortSci. 11:20—21. Gane, R. 1935. The formation of ethylene by plant tissue and its significance in the ripening of fruits. J. Pomol. 18:351. Herner, R. C. and K. C. Sink. 1973. Ethylene production and reSpiratory behavior of the rin tomato mutant. Plant Physiol. 52:38-42. Hobson, G. E. and J. N. Davies. 1971. The tomato. Lg A. C. Hulme (ed.) Biochemistry of fruits and their products. Vol. 2. Academic Press, New York. Kidd, F. and C. West. 1925. Gt. Brit., Dep. Sci. Ind. Res. Food Invest. Bd. Rep. 192A:27. Ng, T. J. and E. G. Tigchelaar. 1977. Action of the non-ripening (nor) mutant on fruit ripening of tomato. J. Amer. Soc. Hort. Sci. 102:505-509. Pratt, H. K. and J. D. Goeschl. 1969. Physiological role of ethylene in plants. Ann. Rev. Plant Physiol. 20:541-584. 13. 14. 15. 16. 17. 18. 19. 20. 6 Rhodes, M. J. C. 1970. The climacteric and ripening of fruits. Ln A. C. Hulme (ed.) Biochemistry of fruits and their products. Vol. 1. Academic Press, New York. Rick, C. M. 1973. Potential genetic resources in tomato species: clued from observations in native habitats. Ln A. M. Syrb (ed.) Genes, enzymes and populations. Plenum Press, New York. Sacher, J. A. 1973. Senescence and post-harvest biology. Ann. Rev. Plant Physiol. 24:197-224. Sawamura, M., E. Knegt, and J. Bruinsma. 1978. Levels of endogenous ethylene, C02 and soluable pectin and activities of pectin methylesterase and polygalacturonase in ripening tomato fruits. Plant Cell Physiol. 19: 1061-1069. Simon, E. W. 1974. Phospholipids and plant membrane permeability. New Phytol. 73:377-420. Tigchelaar. E. C., W. B. McGlasson and R. W. Buescher. 1978. Genetic regulation of tomato fruit ripening. HortSci. 13:508-513. Vickery, R. S. and J. Bruinsma. 1973. Compartments and permeability for potassium in developing fruits of tomato. J. EXp. Bot. 24:1261-1270. Watada, A. and L. L. Morris. 1967. Growth and respiration patterns of snap bean fruits. Plant Physiol. 42:751-761. CHAPTER I: RIPENING BEHAVIOR OF WILD, COLOR-FRUITED TOMATO SPECIES ABSTRACT Wild, color-fruited tomatoes, L. esculentum var. cerasiforme, L. pimpinellifolium, and L. cheesemanii, were surveyed for variability in ripening habit. Ethylene production and respiration of ripening fruits were monitored using an ethylene-free flow-through system and gas chromo- tography. The color-fruited tomatoes all exhibited climac- teric ethylene production and respiratory behavior similar to the cultivated tomato. There were differences, however, for the timing and magnitude of climacteric response. Levels of ethylene production and CO evolution were correlated with 2 age at maturity over a three-fold range from the earliest maturing L. pimpinellofolium, to the latest maturing, L. cheesemanii, fruit. INTRODUCTION Ripening in the cultivated tomato, Lycopersicon esculentum, comprises a series of biochemical and physio- logical events, including the following: softening, pigment changes, development of flavor components, autocatalytic ethylene production and climacteric respiratory behavior (8). Many investigations have been made to elucidate the 9 mechanisms initiating and integrating these processes, but much is unresolved. Most workers agree that ethylene plays a central role in triggering climacteric fruit ripening (1,10,15,16,24). Mature green tomato fruits can be induced to ripen with exogenous ethylene: once ripening is initiated, there is an autocatalytic burst in ethylene production resulting in quantities far in excess of the threshhold level (8). Ethylene, however, is not the sole regulator of ripening. Immature tomato fruits cannot be induced to ripen normally I by treatment with ethylene (8,15,24). Thus, changes must first occur within the fruit before normal ripening can take place. Various possibilities that have been investigated include: changes in membrane permeability (26,29), changes in levels of endogenous hormones (2,3,4), and changes in the activities of various enzymes (24,25,28). There is not sufficient evidence, however, to establish any one of these as the key to ripening. There must be some as yet unresolved change, or series of changes, in the developing fruit which eventually enables response to ethylene and subsequent ripening. 'The most basic resource that can be used for plant improvement is the genetic diversity available in natural populations of the cultigens and related species. Modern tomato breeding provides an excellent example of the exploitation of wild species by introgression to improve cultivated materials; disease- and pest-resistant cultivars 10 are prime examples of breeders' successes (17). Within the gene pools of tomato species, much variability exists for various morphological (17) and biochemical characters (18, 20-23) and observations in wild habitats have suggested that considerable variation for ripening behavior is readily available for use in physiological studies and crop improve- ment. The nine known tomato species fall into two categories, color-fruited Species and green-fruited Species. The color- fruited species include Lycgpersicon esculentum (the cultivated tomato), L. esculentum var. cerasiforme (the wild cherry tomato), L. pimpinellifolium, and L. cheesmanLL. The latter three are readily crossable with the cultivated tomato. L. esculentum var. cerasiformg is a pantropic weed native to the Andean region, and also found in Brazil, Columbia, Central America, and Mexico. L. pimanellifolium represents a diminutive esculentum, and is indigenous to the coastal zones of Peru and Ecuador: L. cheesmanii is a wild species endemic to the Galapagos Islands. Genetic manipulation is only possible if there is variability among the materials. This study is a survey to characterize the wild tomato fruits on the basis of: 1) the type of visible ripening changes that occur, 2) age at maturity, 3) ethylene production, and 4) respiratory behavior. 11 MATERIALS AND METHODS Plant materials: Wild, color-fruited tomato species were studied for ripening behavior and compared to the cultivated tomato. They are pictured by Figure 1 and the source of wild germplasm is listed in Table I. Only one accession of each species was tested. Flowers were selfed by hand so exact fruit age could be determined. The plants were grown in an air-conditioned greenhouse (25/17OC day/night temperature), with 16 hours/day of supplemental lighting provided by 1000 watt metal halide lamps. Standard Michigan greenhouse cultural practices were followed. Measurement of ethylene and carbon dioxide: To determine age at maturity for each species, 2-3 fruits were harvested every two or three days from the time they reached full size, until they began to change color. The resultant set of 10 of 12 fruits was monitored for ethylene production and C02 evolution. Once age at maturity was determined, another set of 10 to 12 fruits was harvested just prior to the onset of ripening and monitored for respiration and ethylene pro- duction. Detached fruits were weighed, placed individually into 50 ml. jars containing a layer of glass beads, and held in a 200C storage room with 16 hours of mixed incandescent and flourescent light per day. The jars were connected to a calibrated, humidified, ethylene-free flow-through system. 1‘ . . (I, ,,.o..... . , .e l a . .\ ..:,.Al. , l , ,7 ,. .. ..|.u . . I . '11,} .. 11,1. , I... .I ., .,, , . . 4 \ C V I atnt, z.l.¢~l|:vu:’.l: .4 .‘2 pun-eta 33:114.. ...suav .. 5.33;:3!....:.-.:’|:.'.po.ul.01 LiQta «[11:11 .SIIiladt’tl‘allIIl... I1} I o.... 12 Figure 1: Wild, color-fruited tomatoes. Left to right: Lycopersicon esculentum var. cerasiforme, L. pimanellifolium, L. cheesmanii. 14 Loomsom..mucmHmH mowmmmamw .mHHmnmmH HflCmEmoono .m oozfi shod .ROH .mocflso ashaooflaamcfldsfim .4 mmofi .spmm .mEHA .ohMomaomno mesomflmmsmo .pm> HSpCoH30mm .4 momfi omm> .>o .ESPCmasomw CocamLMQOOhm om: Cowpmooq mmfloomm .oz . agendasomm .4 0: 03A wmm> .>o ESpCoHSOmo Cocampoaooa4 Acoflpmcflaaom pmpmm mzmov mw< Hawv szflms smoum mofloogm copfispmupoaoo .wmflommm opmsoe "SeahSQmE pm ow< mwmsflxopma< ccm pzmfloz pflssm .HH manme 18 Figure 2: Days to ripen vs. age at harvest for the wild, color-fruited tomatoes. $29 omhmm>m3 Eo:§:=od 3.3 «3.3 :2. .0 can as 1 ..- . mm . ..n 3 3 2. 2. 3 2. 2. an an .5. . . c... s... ....4 . .... k =0 / 4:2: 8 / . 8. / I ...... I W a... I In 0 3. .I o 3 . $8“ 7. Z .. I fl. 2: H ... a . 6. b. o t \ x o. A u- :‘N 6 .. J H h. . . D. a .. . m 0. o o. o 3'.“ c .- ..fi 0 o o l L .. 2...... 1. 2:. .. .. 8... ....L Li. L...” 9.»: u I ... .. 3N Y. o .25; u 0'. ... . con 3 .. .. J no. ”aid”... .4 .0000. .0000. :NH .. .... .3... it... If... \ I 0'6 con .31..“ y C! ' '0 23 Figure 4: Ethylene production and respiratory activity for three L. pimpinellifolium fruit per jar. 20 a- 0.0m.“ ‘100 ‘120 m ‘ q 75. .-.... c: «o 1 P 1 14“)- D 1 .-5. .-.... «co 9.. 1300* son- 5m- SM- 300-- dayo am: pollination 25 0003-0000 002-000 440m2mmmno .4 OOONIOOOH OWNIOON 0m m> .>o Espcmasomm .4 ooom-oooH onN-oom mesomflmmpmo .pm> ESP:04:omm .4 0004-000 004-05 esflaocflaamcflmsfim coonmsmmoosq AH-zmp nmx way A ->m0 H-mx 41v soapsam>m moo coawoswopm mamahcpm moflommm .mpcmeflsmmxw Hmpo>mm mo hpmeesm .mowoomm 090809 cmpfispm-hoaoo .0443 639 :4 mCHComflm wcflnsu Soapsao>m moo 02m coaposoosm ocmaznpm xmom mo :omflsmmEoo .HHH oHQme 26 , while L. cheesmanii exhibited peak 002 evolution of 2 kg-1 day-1. day-1 3000-4000 mg. CO Response togpropylene treatment: Another characteristic of climacteric fruit behavior is hastened ethylene production, increased respiration, and earlier ripening in response to propylene treatment (13). Since the respiratory trend for L. pimpinellifolium was unclear, fruits of this species were treated with propylene. The resultant ethylene production is illustrated by Figure 5a. Propylene treatment hastens endogenous ethylene production, and color development occurred three days earlier for propylene treated fruit than for control fruit. Respiration of treated and control fruits stored individually declined to low, sustained levels (Figure 5b). There was no discernable difference between the two groups. DISCUSSION Variability for ripening characteristics among the wild tomato species represents a genetic pool for future physiological investigations and for possible incorporation of specific control mechanisms into the ripening process of cultivated tomatoes. The color-fruited species are pre- dicatably, quite close in ripening behavior to the cultivated tomato. In addition to chlorophyll degradation, carotenoid biosynthesis, and softening, the three color-fruited species 27 Figure 5: The effect of propylene treatment on ethylene production (a), respiration (b) and color development of L. pimpinellifolium fruit. 1Data are the mean of 3 replicates : S.E. 8:05:05 8:0 mama . «n c.- 3. on 2. Na 3 an I can L Sn L 89. ... 8: u o a a . 2.0.306! I .0 o .3 III . . 08. a . l.Iltzp #5“ 30:) Du: 3.3 to. 0302.0: 1...: 23.9.. 2525 .— co:ac=.oa .23 1.3 N... 3 on ma 3” an am am . d J- 11 1 .0 ’ 'd d .. , ss 0 ' X a; o o H o 4 ON I III C - C I I O ' xi ssh... 5 -H . \ .0! Ii. ‘— .r ‘ ,. O Q _ _ . a . LI 0 . o. . - 0m 7 . * 7 i 0 a .. . EoEQEoso—c 5.00 . - . o . H . a s 0' .s 1 cm A L .r 03 * 02 29.50.33 5.8 0:230... : .. I I WNW/RH 3:) M 29 all Show fairly typical climacteric behavior. They all exhibit a peak in ethylene production that is correlated with other ripening phenomena such as softening, color change, and climacteric respiration (Figure 3). Peak ethylene production preceeds full color development in these fruits by three to five days, as is characteristic of the cultivated tomato (16). ‘ The wild color-fruited species have different magnitudes of ethylene production directly correlated to their age at maturity. The older fruits produce higher amounts of ethylene. Those fruits that mature later also have higher base levels of ethylene production prior to the climacteric burst. A poorly understood phase in fruit ripening is the transition from the immature pre-climacteric fruit to the mature pre-climacteric fruit. In tomatoes the basic ethylene forming mechanism exists at a low level throughout develop- ment, but it is not fully activated until a critical physiological age (13). If fruit are harvested too young, they are never capable of being activated (15). Thus, some sort of change must occur during the transition from an immature to mature fruit. The change might be a gradual increase in sensitivity to endogenous ethylene, a gradual increase in endogenous ethylene to a threshhold concentra- tion (11), a loss of feedback inhibition (15), or a combination of these possibilities. 3O McGlasson g3 gL. (11) studied the response of immature tomato fruits to propylene treatment. Their results show that the ability of immature fruits to respond to propylene increases with fruit age. They suggested that some relatively slow change must preceed the burst in ethylene production and that the development of some cellular component during aging may result in a system capable of autocatalytic ethylene production. Our results support this hypothesis. Since the base level of ethylene production prior to the climacteric is lower in earlier maturing fruits, these fruits may have a lower threshhold level for response. If the threshhold barrier for autocatalytic ethylene production and ripening is broken sooner, perhaps the cellular component developed during fruit aging that enables autocatalytic ethylene pro- duction is not developed to the same extent as in later maturing fruits. Thus the capacity for autocatalytic ethylene production would be lower in earlier maturing fruits. An interesting next eXperiment would be to test for threshhold level of response to propylene treatment in fruits of the different species. One would hypothesize that L. pimpinellifolium would require a lower threshhold concen- tration than L. cheesmanii. Although the same kind of comparison relating age at maturity and levels of ethylene production cannot be made for different cultivars within L. esculentum (7,14,28), it is not contradictory to this Speculation. Through centuries 31 of domestication man has manipulated the cultivated tomato to suit his needs, including selection for uniform, early ripening (19). Thus it is not unreasonable to presume that the development of early and late cultivars has altered the .relationship between age at maturity and capacity for ethylene produCtion. The negative linear relationship between age at harvest, and days to ripen, for the color-fruited species is consis- tent with ripening behavior in the cultivated tomato (9). Once of sufficient maturity, the machinery is present, and ripening will proceed on or off the vine at approximately the same rate (12). This is not the case for several of the green-fruited tomatoes (5,6). As is the case of the cultivated tomato, it is possible to harvest the wild, color- fruited tomatoes while still immature (16). These fruits never ripen fully after being removed from the vine. Thus, ripening behavior in the color-fruited tomato Species is closely related to ripening in the cultivated tomato. These fruits exhibit typical climacteric ethylene production and respiratory behavior, followed by softening and pigment changes.‘ There is a difference among the Species for timing of the ripening process and magnitude of the climacteric response. The direct relationship between fruit age at maturity, and magnitude of autocatalytic ethylene production suggests that some factor which promotes ripening by enabling autocatalytic production, increases with fruit age. 10. 11. 32 LITERATURE CITED Abeles, F. B. 1973. Ethylene in plant biology. Academic Press, New York. Abdel-Rahman, M., T. H. Thomas, G. J. Doss and L. Howell. 1975. Changes in endogenous plant hormones in cherry tomato fruits during development and maturation. Physiol. Plant. 34:39-43. Dilley, D. R. 1969. Hormonal control of fruit ripening. HortSci. 4:111-114. Frenkel, C. and S. A. Garrison. 1976. Initiation of lycopene synthesis in the tomato mutant rin as influenced by oxygen and ethylene interactions. HortSci. 11:20-21. Grumet, R., J. F. Fobes and R. C. Herner. Ripening behavior in wild, green fruited tomato species: fruits that abscise prior to ripening. (in preparation). Grumet, R.,J. F. Fobes and R. C. Herner. Ripening behavior in wild, green fruited tomato species: fruits that ripen on the vine. (master's thesis). Herner, R. C. and K. C. Sink. 1973. Ethylene production and respiratory behavior of the rin tomato mutant. Plant Physiol. 52:38-42. Hobson, G. E. and J. N. Davies. 1971. The tomato. Ln A. C. Hulme (ed.) Biochemistry of fruits and their products, Vol. 2. Academic Press, New York. Kader, A. A., M. A. Stevens, M. Albright-Holton, L. L. Morris and M. Algazi. 1977. Effect of fruit ripeness when picked on flavor and composition in fresh market tomatoes. J. Amer. Soc. Hort. Sci. 102:724-731. McGlasson, W. B. 1970. The ethylene factor. Ln A. C. Hulme (ed.) Biochemistry of fruits and their products, Vol. 2. Academic Press, New York. McGlasson, W. B., H. C. Dostal, and E. C. Tigchelaar. 1975. Comparison of propylene induced responses of immature normal andqug mutant tomatoes. Plant Physiol. 55:218-222. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 33 McGlasson, W. B. and I. Adato. 1977. Relationship between the capacity to ripen and ontogeny in tomato fruits. Aust. J. Plant. Physiol. 4:451- 58. McMurchie, E. J., W. B. McGlasson and I. L. Eaks. 1972. Treatment of fruit with propylene gives information about the biogenesis of ethylene. Nature 237:235-236. Ng, T. J. and E. G. Tigchelaar. 1977. Action of the non-ripening (nor) mutant on fruit ripening of tomato. J. Amer. Soc. Hort. Sci. 102:505-509. Pratt, H. K. and J. D. Geoschl. 1969. Physiological role of ethylene in plants. Ann. Rev. Plant Physiol. 20:541-584. Rhodes, M. J. C. 1970. The climacteric and ripening of fruits. Lg A. C. Hulme (ed.) Biochemistry of fruits and their products, Vol. 1. Academic Press, New York. Rick, C. M. 1973. Potential genetic resources in tomato species: clued from observation in native habitats. Lg A. M. Srb (ed.) Genes enzymes, and populations. Plenum Press, New York. Rick, C. M. 1975. The tomato. Ln R. C. King (ed.) Handbook of genetics, Vol. 2. Plenum Press, New York. Rick, C. M. 1978. The tomato. Sci. Amer. 239:76-87. Rick, C. M. and J. F. Fobes. 1975. Allozymes of Galapagos tomatoes: polymorphism, geographic distri- bution and affinities. Evolution. 29:443-457. Rick C. M. and J. F. Fobes. 1975. Allozyme variation in the cultivated tomato and closely related species. Bull. Torrey Bot. Club 102:376-384. Rick, C. M., J. F. Fobes and M. Holle. 1977. Genetic variation in Lygopersicon pimpinellifolium: evidence of evolutionary change in mating systems. Plant System. and Evol. 127:139-170. Rick, C. M. and E. Kesicki, J. F. Fobes and M. Holle. 1976. Genetic and biosystematic studies on two new sibling species of Lycopersicon from interandean Peru. Sacher, J. A. 1973. Senescence and ost-harvest biology. Ann. Rev. Plant Physiol. 2 :197-224. Sawamura, M., E. Knegt and J. Bruinsma. 1978. Levels of endogenous ethylene, C02 and soluable pectin and activities of pectin methylesterase and polygalacturonase inéripening tomato fruits. Plant Cell Physiol. 19:1061- 10 9. 26. 27. 28. 29. 34 Simon, E. W. 1974. Phospholipids and plant membrane permeability. New Phytol. 73:337-420. Tigchelaar. E. C., W. B. McGlasson and R. W. Buescher. 1978. Genetic regulation of tomato fruit ripening. HortSci. 13:508-513. Tigchelaar. E. C., W. B. McGlasson and M. J. Franklin. 1978. Natural and ethephon stimulated ripening of F1 hybrids of the ripening inhibitor (gin) and non-ripening (nor) mutants of tomato. Aust. J. Plant Physiol. 5W9456. Vickery, R. S. and Bruinsma. J. 1973. Compartments and permeability for potassium in developing fruits of tomato. J. EXp. Bot. 24:1261-1270. CHAPTER II: RIPENING BEHAVIOR OF WILD, GREEN-FRUITED TOMATO SPECIES: FRUITS THAT ABSCISE PRIOR TO RIPENING. 35 ABSTRACT Fruit of two wild, green-fruited tomato species, L. chilense and L. peruvianum were studied for ripening habit. These species are'different from other tomatoes because their fruit abscise prior to ripening. Ethylene production and external ripening changes were studied in fruits collected prior to and following abscission. Ethylene production was monitored using an ethylene-free flow-through system and gas chromatography. The ability of fruits of the two Species to produce ethylene and ripen varied with maturity. The two species differed from each other for timing of endogenous ethylene production relative to abscission. L. peruvianum fruit have elevated ethylene production preceding abscission. In L. chilense fruit, high levels of endogenous ethylene production only occurs after fruit drop. This suggests differences in control mechanisms regulating ripening in the two types of fruit. 'INTRODUCTION Many investigations have been made in recent years to elucidate the mechanisms initiating and integrating changes associated with tomato fruit ripening. Much, however, is unresolved. Most workers agree that ethylene plays a key 36 37 role in triggering the process. In cultivated tomatoes and the closely allied color—fruited species ethylene production shortly precedes changes in color, texture, and flavor (7,10). Mature green fruits can be induced to ripen normally by exposure to exogenous ethylene (1,7). Furthermore, removal of ethylene by scrubbing the atmOSphere or reduced atmospheric pressures inhibits ripening (1). Ethylene, however, is not the only factor involved. Immature tomato fruits cannot be induced to ripen normally by treatment with ethylene (9,12,14). Thus, changes must occur within the fruit before ripening can take place. Various possibilities that have been investigated include: changes in membrane permeability (16,19), changes in levels of endogenous hormones (3,4,5), and changes in the activities if various enzymes (14,15,17). There is not sufficient evidence, however, to establish any one of these as the key to ripening. There must be some as yet unresolved change, or series of changes, in the developing fruit which eventually enables reSponse to ethylene and subsequent ripening. This study characterizes ripening in two wild, green- fruited tomato species, L. chilense and L. peruviangm. L. chilense is commonly found in the slopes and bottoms of guebradas that disect the desert of southern Peru and northern Chile; L. pgruvianum inhabits multifarious niches in the coastal river valleys of Peru and northern Chile. Fruit from both species do not ripen until they have abscised from the plant. The relationship between ethylene production and the 38 transition from immature, non-ripening, to mature, ripening fruit was investigated in these two Species: the results may lead to a better understanding of control mechanisms underlying this transition. MATERIALS AND METHODS Two green-fruited, wild tomato species, L. peruvianum and L. chilense, were examined for ripening behavior. They are pictured by Figure 1. L. peruvianum was LA.1351 from Rope, Cajamarca, Peru; L. chilense was LA 455 from Tambo, Ariquipa, Peru. Only one accession of each species was tested. Since both Species are self-incompatable, cross- pollinationswere made by hand; pollinated flowers were tagged so that exact fruit age could be determined. Growing conditions were the same as for Grumet, et al. (7). Fruits harvested at various stages prior to abscission as well as fruits that naturally abscised from the plant were monitored for ethylene production. Ten to twelve fruits were used to characterize any one stage. For L. chilense, the stages delineated were: 1) prior to and during chlorophyll loss, 2) subsequent to chlorophyll loss, but prior to abscission, and 3) after abscission. For L. p333- vianum, the stages studied were prior to and following abscission. Ethylene measurements were done as in Grumet, g3 gL. (7). Propylene studies also followed the procedure of Grumet, g3 gL. (7). 39 Figure 1: Green-fruited tomatoes that abscise prior to ripening: Left, Lycopersicon chilense; right L. peruvianum. 41 RESULTS Indices of ripening in the wild tomato species include color change, softening and abscission (7,8). This paper describes two green-fruited Species for which abscission is the first external indicator of ripening, L. chilense and L. peruvianum. L. chilense: L. chilense fruits showed various patterns of ethylene production (Figures 2,3), depending on when they were collected. Natural abscission generally takes place after the fruits are 100 days old. One of the normal developmental stages in L. chilense fruit is a loss of chlorophyll beginning approximately 55 days after pollination. Fruits change from green to whitish; there is neither concomitant nor subsequent carotenoid biosynthesis. The loss of chlorophyll takes several days, as opposed to rapid degradation characteristic of the climacteric, color-fruited tomato species (7). It is also not accompanied by ethylene production (Table I). Older fruits that had already lost chlorophyll, fell into two groups. Up until approximately eighty days, they produce almost no ethylene and do not respond to propylene by endogenous ethylene production (Figure 2a). When harvested more than eighty days after pollination, they produce small amounts of ethylene, up to 40 pl kg-1 day"1 (Figure 2b). If these fruits are treated with 42 Figure 2: Ethylene production of younger (a) and older (b) L. chilense fruit with, and without, propylene. Both sets of fruit had already lost their chlorophyll f"?‘l\"‘ * t "“‘ '120 100 ‘ _ L 30- ii . J so 60- 3’ S -. PYOF-‘Yiene 40- :- ' '- ,, ’x 1 40 l u A I { ‘ or” Aq‘f-‘r ’ ta" . I :3! ’ I . . lo in. 7'1 is 10 1:. days aiier pollination Figure 1: Ethylene production of younger (a) and older (b) L. chilense fruit with, and without, propylene. Both sets of'fruit had already lost their chlorophyll. pl (22 Ha kg" tiny" 44 Figure 3: Ethylene production in abscised L. chilense fruit. l 1 l L 1 l I 2 3 4 5 6 days after abscission 300 200 100 ' pl 02 H4 kg-l day-1 46 com o: 04 conflomnm scammfloQO-opm coapmpspme mmoa Hah£9040430 WCHMSU +004 004-00 ow-mo 00-00 A -sae 4-m4 4:0 COHPomooum oCoHSLPm xmom undemoao>om mo ommpm ACOHHmsflaaom popmm mhmcv ow< .mpcoefluomxm 4m4o>mm mo humessm I anoemoao>oo mo mommpm m3044m> Pm pflsum mmsoawco .4 :4 coflposcohm osmflhnpm msocomoccm go mao>oA .4 040ae 47 propylene, they are stimulated to produce as much as 1 300 pl 02H“ kg- day.1 (Figure 2b). Finally, fruits that naturally abscise exhibit a climacteric rise in ethylene 1 production to 300 pl kg- day'1 (Figure 3). L. peruvianum: The first external Sign of ripeness in L. peruvianum fruit is abscission from the vine approxi- mately 70 days after pollination. Fruits remain green and hard while on the vine and soften after they fall. There is no loss of chlorophyll corresponding to degreening of L. chilense fruit. L. peruvianum fruit remain green even after they abscise and soften. Fruits harvested prior to natural abscission fall into two categories: a minority that softens, and the majority that does not. Fruits that do not soften have lower ethylene production that those that do (Figure 4). lknuipicked.fruits that do not soften can be further divided into two groups based on ease of detachment from the plant by breaking at the abscission zone. There is a difference in the type of break that occurs in the two groups of fruits. Those that are difficult to break leave a rough interface at the abscission zone. Those that are not as difficult to break leave a smooth interface. Fruits that break off relatively easily also have higher levels of ethylene production than those that are diffi- cult to break (Table II). Both have lower ethylene production than fruits that soften. 48 Figure 4: Ethylene production in softening and non- softening, hand-detached L. peruvianum fruits. Softening occurred in the high ethylene producing group at 66 days. 500 " . i - \ l 450' \ l l i 400 ‘ l \ 350- l l '7 l 3.. .g l '0 x \ o v ll 1 \ I ‘ harvested- 5‘ 250. \ I \ softened 3- \ ’ l I \ l I \ o 150' 100- harvested-didn’i soften 50- days after pollination 50 was 000 0044a0 we» own 44mm 04 ozone 0: 004 compmc 64 445044440 402 o: 00 00m4m0 04 0pm; -zm0 4-wx H: canopMom scape: 64m ozoakswm xmom pcoemoao>oa mo owmpm .4454m Escmfl>sowm .4 mo moflpowoumo 430m :4 :04poscopm ocoahnpm xmom .HH oHQme 51 Another eXperiment was done to eliminate the possi- bility that wounding is responsible for high ethylene production in the fruits that soften. Since the most feasible source of wounding during hand—detachment is removal of the calyx, fruits were harvested and monitored with their calyxes attached. Figure 5 shows relative positions of the natural abscission zone and point of calyx attachment. After three days in storage, calyxes were removed from fruit with low initial ethylene produc- tion. The results Show that calyx removal does not increase ethylene production or induce softening (unpublished data). Fruits having high initial levels of ethylene production were not included in the eXperiment because an increase in production due to calyx removal would not be detected. These fruits softened with their calyzes attached, as do fruits that naturally drop from the vine. Fruits that fell from the vine were also studied and found to have high levels of ethylene production (Figure 6). They softened within a few days after abscission (Table I). Also studied were fruits that fell off the vine when they were touched. These fruits were estimated to be one day prior to abscission. They also soften quickly and have high levels of ethylene production (Figure 6). \ natural abscission zone point of calyx attachment Natural abscission zone and point of calyx attachment in h; Deruvianum fruit. 53 Figure 6: Ethylene production in abscised L. peruvianum fruits and fruits about to fall. Measurements made daily until softening occurred. pl (:2 ii.- in" day" 500 400 350 300 250 200 150 100 _ taHen l . \ l - l - I i / l / r -\ l l l / X P ./ ./ X l . I about todmp / l ’1 P v i .i'oé'z'ste days after fruit droo 55 DISCUSSION Both L. chilense and L. peruvianum have fruits that do not ripen, as indicated by softening, until they fall from the vine. Thus the arrival of maturity in these Species can be recognized by abscission. Although ripening is accompanied by endogenous ethylene production in both types of fruit, the timing of ethylene production rela- tive to abscission is not the same. In L. chilense, ethylene production rises after fruit drop (Figure 3). In L. peruvianum, elevated ethylene production precedes abscission (Table II). This implies that the control mechanisms regulating ripening might be different for the two Species. L. chilense: The different patterns of ethylene produc- tion in L. chilense fruits probably correspond to stages of maturity. Fruits in the first stage, (which includes loss of chlorophyll), produce little ethylene, even when treated with propylene (Figure 2a). The slow loss of chlorophyll, lasting approximately ten days, suggests that active degradation, comparable to the climacteric color-fruited species is not occurring (7). Slow degreen- ing is characteristic of non-climacteric citrus fruits which also do not produce much ethylene (18). This would tempt one to classify L. chilense as non-climacteric. Studies of older fruit, however, show that L. chilense is capable of natural ethylene production (Figure 2b). 56 Treating these fruits with propylene induces a burst of ethylene production. Finally, abscised fruits undergo climacteric ethylene production (Figure 3), and soften in a few days. Maturation processes in L. chilense fruit are dia- gramatically summarized by Figure 7. Immature fruits do not naturally produce ethylene, whereas mature fruits do. The final stage in maturation prior to ethylene production and softening is abscission. This behavior is similar to avocado fruit which undergo climacteric ripening after harvest (6). In avocado, ripening is presumably due to the removal of some restraining factor previously supplied by mother plant (6). L. peruvianum: L. pgruvianum fruit are similar to L. chilense: they do not soften until they fall from the vine. Unlike L. chilense, however, the majority of L. peruvianum fruit harvested prior to natural abscission do not soften during storage. Thus detachment itself is not the stimulus for ripening. It is not simply removal of the fruit from the source of inhibitory substance that enables abscised L. peruviangm fruit to ripen. Wounding is also not responsible for softening that occurs in a minority of hand-detached fruits. If wounding occurred during harvest, it would probably be a result of calyx removal; but some frits have high levels of ethylene production and soften while their calyxes are still attached. .ungu om:o__zo am :4 mommmooca paucosoopm>mo $0 mcwp-os_h "0.0L304d 00440000g0 0:044:40 ? """"""" :0_40:_—_00 swabs 0400 om4 oo4 pm pm cw, hm“ aw ' """" ‘ , """""""""""" ' ’ """""""""" + 1? lllllllllllllllllllll + 1 d S m 3 I: 0 o D. U: J IL I, a... IL I... n IL 1. n O C L. L. a J S N ..+ u u D— S D. .u L. ”- J Lo 5 O on O O U .I. M U l. 1 U- 58 In other fruits, calyx removal does not induce an increase in ethylene production, or cause softening. Fruits that are hand-removed just before they drop soften and produce high levels of ethylene, 300 pl kg"l day”1 (Figure 6). Thus physically falling from the plant to the ground and possible subsequent wounding, is also not responsible for ethylene production and softening. Finally, fungal infection is probably not responsible for softening as there was no external Sign of infection even when the fruits were held for weeks after becoming soft. In fruits that have fallen, and those about to drop, an abscission layer must already be formed. It is not detachment or wounding, but probably formation of an abscission layer and concomitant development of ethylene production machinery which stimulates ripening. One would postulate that the minority of hand-detached fruits which soften have already begun abscission zone formation at the time of harvest. This hypothesis is consistent with models for abscission zone development in leaves (1,13). Abeles g3 gL. (2), working with beans exPlants, describe a three part process of abscission zone formation as follows: 1) a period of cell aging when ethylene does not promote abscission, 2) the induction of wall-degrading enzymes and synthesis of ethylene production machinery (at this time ethylene enhances abscission) and 3) a loss of break strength due to cell separation. 59 Perhaps high levels of ethylene are required, either directly or indirectly, to induce softening; and the development of sufficient ethylene production machinery is, in turn, dependent upon cell aging. Thus, in the fruits which are about to fall, or have fallen, ethylene production machinery is well developed. Possibly, hand-detached fruits which soften also have adequate ethylene production machinery and are in stage 3 of abscission zone formation. In these fruits, however, there has not yet been sufficient cellwall.degradation to cause fruit drop. In contrast, those fruits which do not soften have not yet produced sufficient ethylene synthesis machinery, and are in stage 1 or 2 of abscission zone formation. The difference in levels of ethylene production between the two groups of non-softening fruits that were either difficult, or not so difficult, to break at the abscission zone may be because one group is in stage 1 of abscission zone formation (prior to ethylene production), and the other group in stage 2 (synthesis of ethylene production machinery). A possible next eXperiment would be to take fruits of each category and test for response to propylene. One would predict group 2 fruits to respond by enhanced ethylene production and/or fruit softening and group 1 fruits to be unaffected. These stages of develop- ment are summarized diagramatically by Figure 8. A possible indirect effect of ethylene is to initiate synthesis of a product that can break down a hypothesized Q) C O .C N 4.: c. a 3 as . ... 'r- 2 °P U C C H m In (B 0r- w- 6 W E U! C C H 0!- Ul Q 0!- 0!- U o 'P H :— 3 U) ‘0- U ‘0- r- S- .D in O a q. M .3 a: fi ............ ‘- ---------------- 2. -------- fi .- ..... _‘ ...... - -4 ban-ad 0 20’ 40 60 85 days after pollination ......... C----d heightened ethylene production Figure 7: Time-line of developmental processes in L, peruvianum fruit. 61 inhibitory substance transmitted from the mother plant. This suggestion is consistent with our data. While on the plant there is a continual source of restraining factor, but once the fruit naturally abscise, the factor is degraded. Fruits harvested too young have not yet developed the product necessary for degration of the inhibitory substance, and thus do not soften during storage. It may be concluded that although fruit of L. chilense and L. peruvianum both abscise from the plant before ripening, the control mechanisms underlying their behavior are differ- ent. Both, however, have mature and immature developmental stages when ripening does or does not occur, respectively. If there is some inhibitory substance involved in the transition of an immature tomato fruit to a mature fruit, further studies with these Species may help to characterize the factor and processes leading to its degradation. U:- 10. 11. LITERATURE CITED Abeles, F. B. 1973. Ethylene in plant biology. Academic Press, New York. Abeles, F. B., L. E. Cracker and G. R. Leather. 1971. Abscission: the phytogerontological effects of ethylene. Plant Physiol. 47:7-9. Abdel-Rahman M., T. H. Thomas, G. J. Doss and L. Howell. 1975. Changes in endogenous plant hormones in cherry tomato fruits during development and maturation. Physiol. Plant. 34:39-43. Dilley, D. R. 1969. Hormonal control of fruit ripening. HortSci. 4:111-114. Frenkel, C. and S. A. Garrison. 1976. Initiation of lycopene synthesis in the tomato mutant gig as influenced by oxygen and ethylene interactions. HortSci. 11:20-21. Gazit, S. and A. Blumenfeld. 1970. ReSponse of mature avacado fruits to ethylene treatments before and after harvest. J. Am. Soc. HortSci. 95:229-231. Grumet, R., J. F. Fobes and R. C. Herner. Ripening behavior in wild, color-fruited tomato species. (in preparation). Grumet, R., J. F. Fobes and R. C. Herner. Ripening behavior in wild, green-fruited tomato Species: fruits ”that ripen on the vine. (in preparation). Hobson, G. E. and J. N. Davies. 1971. The tomato. Lg A. C. Hulme (ed.) Biochemistry of fruits and their products, Vol. 2. Academic Press, New York. McGlasson, W. B. 1970. The ethylene factor. Ln A. C. Hulme (ed.) Biochemistry of fruits and their products, Vol. 1. Academic Press. New York. McMurchie, E. J., W. B. McGlasson and I. L. Eaks. 1972. Treatment of fruit with propylene gives information about the biogenesis of ethylene. Nature 4:451-458. 62 12. 13. 14. 15. 16. 17. 18. 19. 63 Pratt, H. K. and J. D. Goeschl. 1969. Physiological role of ethylene in plants. Ann. Rev. Plant Physiol. 20:541-584. Rubenstien, B. and A. C. Leopold. 1963. Analysis of the auxin control of bean leaf abscission. Plant Physiol. 38:262-267. Sacher, J. A. 1973. Senescence and ost-harvest biology. Ann. Rev. Plant Physiol. 2 :197-224. Sawamura,jM" E. Knegt and J. Bruinsma. 1978. Levels of endogenous ethylene, C02 and soluable pectin and activities of pectin methylesterase and polygalac- turonase in ripening tomato fruits. Plant Cell Physiol. 19:1061-1069. Simon, E. W. 1974. Phospholipids and plant membrane permeability. New Phytol. 73:337-420. Tigchelaar, E. C., W. B. McGlasson and R. W. Buescher. 1978. Genetic regulation of tomato fruit ripening. HortSci. 13:508-513. Ting, S. V. and J. A. Attaway. 1971. Citrus fruits. Lg A. C. Hulme (ed.) Biochemistry of fruits and their products, Vol. 2. Academic Press, New York. Vickery, R. S. and J. Bruinsma. 1973. Compartments and permeability for potassium in developing fruits of tomato. J. EXP. Biol. 24:1261-1270. CHAPTER III: RIPENING BEHAVIOR OF WILD- GREEN-FRUITED TOMATO SPECIES: FRUITS THAT RIPEN ON THE VINE 61+ ABSTRACT Fruit of four wild, green-fruited tomato species, L. hirsutum, L. chmielewskii, L. parviflorum and S. pennellii were surveyed for variability in ripening habit. Ripening changes were studied in fruits harvested at different ages; ethylene production and CO2 evolution were monitored using an ethylene-free flow through system and gas chromatography. Results indicate differences in ripening behavior among the Species with respect to ethylene production and respira- tory patterns, rate of ripening changes, and whether or not ripening occurs off the vine. Ethylene production in S. pgnnellii and L. hirsutum was not correlated with external ripening changes, making questionable the role of ethylene as the ripening hormone in these fruits. INTRODUCTION Although ripening in the cultivated tomato, Lycgpersicon esculentum, has been a topic of much research in recent years, control mechanisms underlying various ripening phenomena are still poorly understood. Most workers agree that ethylene has a central role in triggering the process (1,6,10,11,18), but the inability of ethylene to induce immature tomato fruits to ripen normally indicates that other changes must occur as well. (4,5,7,9). 65 66 An uneXplored approach to this problem is the use of wild germplasm to study the ripening process. Within the tomato species, a tremendous amount of variability exists for myriad morphological (12) and biochemical characters (12,14-17). Observations in wild habitats have suggested possible inter-generic variation for ripening behavior. The green-fruited tomato species fall into two categories, those that abscise prior to ripening and those that ripen on the vine. The latter category includes L. hirsutum, Solanum pgnnellii, L. chmielewskii and L. parviflorum. All the wild Species can be hybridized with the cultivated tomato L. esculentum. L. hirsutum is naturally distributed from south-central Peru to northern Equador, having an altitudinal range from 500-300 m. This robust plant is particularly known for its hairy plant parts and resistance to a variety of insect pests. §. pgnnellii is indigenous to the exceedingly dry, lower slopes of the central Peruvian Andes. It is most outstanding for its ability to withstand drought conditions (17). The two sibling Species, L. chmielewskLL and L. parviflorum (17), occupy a restricted area in the interandean region of central Peru; their native distribution is bounded by the Ecuadorian frontier in the north, and the Departamento Cuzco in the south. L. parviflorum is distinguished from L. chmielewskii by its greatly reduced plant parts and flower size. 67 MATERIALS AND METHODS Plant materials: Four wild green-fruited species were characterized for ripening behavior: L. chmielewskii, L. pgrviflorum, L. hirsutum and S. pennellii. They are pictured in Figure 1 and the source of germplasm is listed on Table I. Only one accession of each species was tested. L. chmielewskii, L. parviflorum and S. pgnnellii and F1 flowers were selfed by hand so exact fruit age could be determined L. hirsutum is self-incompatible, so outcrosses were made for this Species. Growing conditions were the same as for Grumet, g3 gL. (2). To determine age at maturity for each species, fruits were harvested every two to three days from the time they reached full size until they indicated some sort of ripening change. The resultant set of 10-12 fruits were monitored for respiration and ethylene production as in Grumet, g3 gL. (2). Propylene studies also followed the procedure of Grumet, g3 gL.(2). RESULTS The four green-fruited species in this study L. chmielewskii, L. parviflorum, L. hirsutum and S. pennellii "ripen" while attached to the vine. This may be contrasted with L. chilense and L. peruvianum which do not ripen prior abscission (3). For L. chmielewskii, L. parviflorum, and S. pennellii, ripening is indicated by fruit softening. 68 zoom .mm4swo4< .0044< 4444mcsom escm4om 045 Show .2mdoc< .mEmmo 04v ammmmmmm .4 o mmm4 Shem l .ooscocmx .monoo 6444cmwmco 8340444>4mm .4 now spam .om84:hm< .mss4noc4mmo 44xm3o4o4eno :004m4omoom4 mmo4 :04pmoo4 mo4oomm .0: <4 .mo4pm44owom4mno w:4com4m mo zmmm¢ pom pom: Ewm4QEHoU censoe «H o4nme 69 Figure 1: Green-fruited tomatoes that ripen on the vine. Left to right: Solanum pgnnelliL, Lycopersicon hirsutum, L. chmielewskii, L. pgrviflorum. 71 L. hirsutum has no external sign of ripening, but ethylene and respiratory data show this species to have highly regulated climacteric behavior. S. pennellii: The external index of ripening in S. pgnnelliL fruit is softening occurring both on and off the vine 70-80 days after pollination. Fruit on the vine become extremely soft and eventually abscise from the plant. Time to soften in hand-picked fruits is directly correlated with fruit age, once full size has been attained (Figure 2). Once begun, softening occurs quickly. In a period of 2-3 days fruit change from firm to extremely soft, and split open with the slightest pressure. If fruits are kept after they soften, they are susceptible to molds. S. pennellii fruit are also very sensitive to water; both immature and mature fruit will split open if washed. Ethylene production in S. pennellii fruit is very erratic. Many observations have shown individual fruits to have several production peaks (Figure 3a); however, there is no peak in production directly corresponding to the onset of fruit softening. Respiratory activity is also erratic and does not correspond to peaks in ethylene production, or to the onset of fruit ripening (Figure 3b). Propylene treatment does not affect ripening in these fruits. Fruit softening, ethylene production, and CO2 evolution are not altered in the treated group with respect to controls (Figure 3). 72 Figure 2: Days to ripen vs. age at harvest for S. pennelliL fruit. 7 35 days 38 days 41 days 48 days 49 days i h m m m 7th 7°! VINO .1 m days aitor pollination 74 Figure 3: Ethylene production (a), respiratory behavior (b), and response to propylene in S. pennellii fruit.1 Softening occurred at 70 days in treated and untreated fruits. 1. data are mean of 3 replicates I S.E. . ... 40 0 45:55.2..23 240.. ..o....:=.c:. ... > .0 0K 4: 3 45 S. as 4:. so . 00 S. .llllllll-lqtl-l-iIII-l . I! so». - - cm - 4x: . .0 L 0004 .4 o. -- 0:0 >12: r ~ _ . I 0.... N 003 w 5 00 x - o . 000 .m. A 4 - X- b.“ _ 4 .. coon m % .4 saw I... _ (no . t I A 053.3%. -Ill :0 1 can - comm r Lfi l 328 coo—ago... :0 («‘39 (~53 7H 30 id 76 L. chmielewskii and L.‘parviflorum: Ripening, as indicated. by fruit softening in the two sibling species, L. chmielew- SkLL and L. parviflorum occurs in mature fruit both on and off the vine. If abscission takes place, it occurs much later than fruit softening. It is possible, however, to harvest these fruits before they are capable of ripening. If harvested too young, they remain hard for more than 30 days in storage; if sufficiently mature when harvested, they soften in 6-12 days (Table II). L. chmielewskii and L. pagnylorum do not flower readily under Michigan conditions. As a result, ethylene and respiratory studies are not sufficiently replicated to be conclusive. From the information obtained to date, however, climacteric behavior is implicated. The magnitude of ethylene production immediately following harvest for L. chmielewskLL fruit is dependent on fruit age (Figure 3). The youngest fruits produce less than 20 pl/kg/day whereas the oldest fruit produce more than 150 pl kg‘1 '1. day A set of fruits harvested during the transitional period for ability to soften in storage, 37—48 days after pollination, was studied. They softened 10—12 days after harvest, thereby corresponding with ripening on the vine. Ethylene production declined following harvest and then rose again to what might be considered a climacteric peak. Ethylene production in L. pgrviflorum differs in the younger non-softening fruits from the older softening 77 .0w04040 44 404400 40: 00 0M0 04:4 04 40444 00400>40£ 44:44 4 04-04 00-00 0040444>nam .4 0-0 00-00 . 440030404000 .4 440440444404 40440 0400v 40440m 04 040m 44443402 40 0w< 004004m .044344 8340444>40m .4 0:0 444030404850 .4 00:00409 404 40440m 04 0400 040 44444340: 40 0m< .44 04909 78 Figure 4: Initial levels of ethylene production vs. fruit age at harvest in L. chmielewskii fruit. 79 80 fruits. Non—softening fruits harvested prior to 40 days old, have erratic production ranging from 50 to 200 pl day-1. Fruits harvested at 41 days are mixed; although some fruits softened, ethylene production is relatively low and erratic. Older, softening fruits have climacteric production with peak rates of 400-500 ul kg'1 day-1. L. hirsutum: L. hirsutum fruits have no readily distinguish- able, external ripening changes. They remain hard, green and attached to the plant, long after they have undergone climacteric bursts in ethylene production and respiration. These fruits have an interesting timing mechanism regulating their ethylene production and respiratory behavior. If the fruit are harvested too early, i.e. 40-50 days after pollination, they have steady low levels of ethylene production and low CO2 evolution, (Figures 5,6). Eventually the fruits wither. If harvested somewhat later, i.e. at 55 days, there is again low ethylene production. This is followed by a small rise that might be termed climacteric. Respiration in these fruits declines throughout storage (Figure 6). At 58 and 60 days the ethylene peak is increasingly pronounced and respiration also rises. Finally at 65 days the ethylene peak is extraordinary, 2000-4000 ul kg"1 '1. day This is ten times peak ethylene production in the cultivated L. esculentum (Table III). Peak respiration in L. hirsutum is three times that of L. esculentum. Fruit harvested at 70 days begin with high ethylene production and decline 81 Figure 5: Ethylene production in L. hirsutum fruit harvested at different ages. pl 02H4 kg-1 day-1 2000 1500' 1000' 1 500 65 days 40 days { W5 days 4.5....- .......:::. ...... 50 5'6 70 ab DAYS AFTER POLLINATION 83 Figure 6: ReSpiration in L. hirsutum fruit harvested at different ages. =o=0£=3 3:: is: i 83. i coco 1 8A: LJep V5" 203 5w 85 400.40.40 405040 5040 0400 0044:44-40400 4 0004-0000 0000-0000 44440444 .4 0000-0004 000-000 4400500000 .4 ooom-0004 omm-oom 0mm> .>0 5344043000 .4 ooom-0004 omm-oom 05404400400 .40> 5344043000 .4 0004-000 044-04 5044004440040540 .4 4044340>m moo 4044030044 0404444m 4-400 4-mx we 4-400 4-04 4: 0040040 N00 040 4044030044 0404444m 40 0040m 4004 40 4004404500 ”444 04409 .00040509 0044344 40400 .04404005440 40 000:4 044; 44:44 5040044; .4 :4 0044:40>m 86 rapidly. Fruit harvested even later have low levels of ethylene production and respiration. Figures 5 and 6 show that harvesting too early not only diminishes climacteric response, but delays it as well. Fruits harvested later exhibit peak production sooner than young fruits. These patterns for ethylene production and reSpiratory behavior have been replicated three times. For fruits grown in the winter the timing is shifted later by a few days, but the pattern reamins the same. DISCUSSION In addition to exhibiting diversity for visible ripening changes, L. parviflorum, L. chmielewskii, L. hirsutum and §. pennellii also exhibit variability for other physiological traits associated with the ripening process. Such traits include ethylene evolution, internal timing mechanisms, and whether or not fruit remain incapable of ripening for some time after attaining full size. g. pennellii: Based on ethylene, respiratory and propylene data, it is difficult to conclude that ethylene plays a key regulatory role in ripening of §. pennellii fruit. Ethylene is produced by detached fruit, but there is no single climac- teric peak, production levels do not correspond to softening and subsequent abscission of mature §. pennellii fruit, however, as well as the negative linear relationship between fruit age at harvest and time to ripen, indicates that some 87 control mechanisms must be involved. Ethylene may play a role in regulation of softening and abscission for fruits attached to the vine, but this cannot be determined from our studies. L. chmielewskii and L. parviflorum: It is possible to harvest L. chmielewskii and L. parviflorum fruits after they have attained full size, but before they are capable of ripening. The failure of younger fruits to ripen after harvest suggests that some maturation process must occur while attached to the plant that cannot take place off the plant. This is similar to behavior of the cultivated tomato (5). There, too, it is possible to harvest a fruit before it is fully capable of ripening, although it may have already attained full size (5). Preliminary ethylene data for L. chmielewskLL fruit show the magnitude of production following detachment to be correlated with fruit age (Figure 4), thus one might expect ethylene production in harvested fruit to continue to increase as the fruit ages. In fruit harvested 37-41 days after pollination, however, ethylene production declines following harvest and the subsequent climacteric peak remains within the range of initial ethylene production levels. For L. parviflorum, it is interesting to note, that although younger fruit are capable of producing quite large amounts of ethylene, they do not soften. Either those 88 amounts are not sufficient for this species, or some factor other than ethylene is induced. For both Species, additional replication is needed before conclusions can be drawn. L. hirsutum: L. hirsutum fruit are very unusual. Their ethylene production and respiratory behavior are typically climacteric, but show extremely precise timing. There is a very Specific age when detached fruits produce large quantities of ethylene and have corresponding heightened respiration (Figures 5,6). If fruits are harvested too young, they show no, or minimal response. If harvested too old, they also do not exhibit climacteric behavior. The time difference between too young and too old for peak production is only a few days. An interesting experiment might be to harvest fruits daily, or to monitor ethylene and CO2 evolution of fruits attached to the vine. These patterns of ethylene and 002 evolution are also outstanding because the peak rates of evolution of both gasses are much greater than for any other tomato Species (Table III). Additionally, the peak rates do not coincide with any external ripening changes. Apparantly, in this case ethylene does not serve as a trigger for ripening. Whether or not the ethylene production is correlated with seed maturation is unknown. To conclude, although L. chmielewskii, L. parviflorum, L. hirsutum and g. pennellii all "ripen" while attached to the vine, there are differences in the regulatory mechanisms involved. Softening in S. pennellii fruit occurs very 89 quickly and is Shortly followed by abscission. This closely synchronized behavior suggests some sort of regulatory' mechanism. Whether ethylene plays a key role is questionable because peaks in ethylene production are not directly correlated to fruit softening. In contrast to S. pennellii, L. chmielewskii, and L. parviflorum fruit take a relatively long time to soften. If abscission occurs at all, it takes place much later than fruit softening. Thus ripening processes are not as tightly integrated in these fruits. However, the inability of young detached fruits to soften suggest that there are control mechanisms responsible for fruit maturation on the vine. Finally, L. hirsutum has an extremely Specific timing mechanism regulating its climacteric behavior despite the absence of resultant external ripening change, As in §. pennellii the physiological significance of ethylene production in these fruits is a mystery. 10. ll. LITERATURE CITED Abeles, F. B. 1973. Ethylene in plant biology. Academic Press. New York. Grumet, R., J. F. Fobes and R. C. Herner. Ripening behavior in wild, color-fruited tomato species. (in preparation). Grumet, R., J. F. Fobes and R. C. Herner. Ripening behavior in wild, green fruited tomato Species: fruits that abscise prior to ripening. (in preparation). Herner, R. C. and K. C. Sink. 1973. Ethylene production and respiratory behavior of the rin tomato mutant. Plant Physiol. 52:38-42. Kader, A. A., M. A. Stevens, M. Albright-Holton, L. L. Morris and M. Algazi. 1977. Effect of fruit ripeness when picked on flavor and composition in fresh market tomatoes. J. Amer. Soc. Hort. Sci. 102:720-731. McGlasson, W. B. 1970. The ethylene factor. Lg A. C. Hulme (ed.) Biochemistry of fruits and their products, Vol. 1. Academic Press, New York. McGlasson, W. B. and I. Adato. 1977. Relationship between the capacity to ripen and ontogeny in tomato fruits. Aust. J. Plant Physiol. 4:451~h58. McMurchie, E. J., W. B. McGlasson and I. L. Eaks. 1972. Treatment of fruit with propylene gives information about the biogenesis of ethylene. Nature 237:235-236. Ng, T. J. and E. G. Tigchelaar. 1977. Action of the non-ripening (nor) mutant on fruit ripening of tomato. J. Amer. Soc. Hort. Sci. 102:505-509. Pratt, H. K. and J. D. Geoschl. 1969. Physiological role of ethylene in plants. Ann. Rev. Plant Physiol. 20:541-584. Rhodes, M. J. C. 1970. The climacteric and ripening of fruits. Ln A. C. Hulme (ed.) Biochemistry of fruits and their products, Vol. 1.‘ Academic Press, New York. 90 12. 13. 14. 15. 16. 17. 18. 91 Rick, C. M. 1973. Potential genetic resources in tomato species: clued from observations in native habitats. Ln A. M. Serb (ed.) Genes, enzymes and populations. Plenum Press, New York. Rick, C. M. 1975. The tomato. Ln R. C. King (ed.) Handbook of genetics, Vol. 2. Plenum Press, New York. Rick, 0. M. and J. F. Fobes. 1975. Allozymes of Galapagos tomatoes: polymorphism, geogra hic dis- tribution and affinities. Evolution 29: 43-457. Rick, C. M. and J. F. Fobes. Allozyme variation in the cultivated tomato and closely related species. Bull. Torrey Bot. Club 102:376-384. Rick, C. M., J. F. Fobes and M. Holle. 1977. Genetic variation in Lycopersicon pimpinellifolium: evidence of evolutionary change in mating systems. Plant System. and Evol. 127:139-170. Rick, C. M. and E. Kesicki, J. F. Fobes, and M. Holle. 1976. Genetic and biosystematic studies on two new sibling species of Lycopersicon from interandean Peru. Theoret. Appl. Genet. 47:55-68. Sacher, J. A. 1973. Senescence and post-harvest biology. Ann. Rev. Plant Physiol. 24:197-224.