THE ROLE OF ABSCISLC ACID IN PEACH (Prunus persica L.) SEED DORMAN‘CY Dissertation for the Degree of Ph. D. MLCHLGAN STATE UNIVERSLTY PRINCE ALBERT BONAMY 1976 This is to certify that the thesis entitled The Role of Abscisic Acid in Peach (Prunus pers'ica Ll.) Seed Dormancy presented by Prince Albert Bonamy has been accepted towards fulfillment of the requirements for Ph. D. Horticulture degree in Major professor Dammy 16 . 1976 0-7 639 m 800K BINDERY INC. leRARY BINOEHS mulmm. mung ."/ l r | ' ".lllll V“- Fg‘i \L, ABSTRACT THE ROLE OF ABSCISIC ACID IN PEACH (Prunus persica L.) SEED DORMANCY By Prince Albert Bonamy Abscisic acid (ABA) was identified in a methanol extract of dormant peach seed by combined gas liquid chromatographydmass spectro— metry (CC-MS). ABA levels were measured during seed maturation using electron capture-gas liquid chromatography (EC-CC). Recovery of 14C-ABA indicated that more than 50% of the ABA present in the extract should normally have been recovered by the procedure used for fractionation. ABA levels in the embryonic axes could not be related to the germination potential of embryos excised from maturing seeds. Drying and storage of seeds did not significantly affect total ABA content, although an increase in both free and bound ABA occurred in the embryonic axes. On imbibition, levels of both free and bound ABA dropped except for free ABA in the seed coat and bound ABA in the embryonic axes, neither of which changed significantly. Both free and bound ABA declined in all seed portions during stratification at both 5° and 20°C, yet only the 5°C treatment broke dormancy. Interruption of low temperature (5°C) stratification by 10 days at 27°C promoted final germination after 12 weeks, and increased ABA slightly, but not significantly. Extracts of chilled seeds were less inhibitory to germination of non-dormant seeds than were extracts Prince Albert Bonamy of non-chilled seeds, but ABA content of the extracts, as measured by GLC, was sufficient to account for only a small part of their biological activity. I therefore conclude that the level of ABA is not the major factor controlling the dormancy of peach seeds. THE ROLE OF ABSCISIC ACID IN PEACH (Prunus persica L.) SEED DORMANCY BY Prince Albert Bonamy A DISSERTATION Submitted to Michigan State university in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1976 D E D I C A T I 0 N TO MY MOTHER, LOUISE ii ACKNOWLEDGEMENT I am deeply appreciative of my Major Advisor, Dr. F. G. Dennis, for his constant encouragement, patience, assistance, and meticulous care in the preparation of this thesis. Gratitude is expressed to Drs. M. J. Bukovac, R. F. Carlson, R. C. Herner, and C. J. Pollard for their guidance and encouragement. Thanks are also due to Dr. C. C. Sweeley for making the mass-spectrometer facilities available and to Mr. J. E. Harten for his technical assistance for performing sample analyses. iii TABLE OF CONTENTS Page LIST OF TABLES.................................................. vi LIST OF FIGURES................................................. vii ABBREVIATIONS................................................... ix INTRODUCTION.................................................... 1 LITERATURE REVIEWOOOOOO00......OOOOIOOOOOOOOOOO00.000.000.000... 3 Definitions................................................ 3 Causes of Dormancy......................................... 4 Exogenous Control of Dormancy in Prunus Seed............... 5 Seed Coat............................................. 5 Temperature........................................... 5 Light................................................. 7 Gases................................................. 7 Chemicals............................................. 7 Endogenous Control of Dormancy in Prunus Seed.............. 9 EffeCt 0f the Seed coatooooooooooaooo0.000000000000000 10 Respiratory Changes................................... 10 Auxins................................................ ll Gibberellins.......................................... ll Cytokinins............................................ ll Inhibitors............................................ ll Interaction of Promoters and Inhibitors............... 14 Summary.................................................... 15 iv Page SECTION I: THE ROLE OF ABSCISIC ACID IN PEACH (Prunus persica L.) SEED DORMANCY. I. INDUCTION 0F DORMANCY.................... l8 Abstract..................................................... 19 Introduction................................................. 19 Materials and Methods........................................ 21 Results...................................................... 26 Discussion................................................... 37 Literature Cited............................................. 41 SECTION II: THE ROLE OF ABSCISIC ACID IN PEACH (Prunus persica L.) SEED DORMANCY. II. EFFECTS OF CHILLING 44 Abstract..................................................... 45 Introduction................................................. 45 Materials and Methods........................................ 47 Results...................................................... 43 58 DiscuSSionOOOOOOOOOOO00.0.0.000.000000.00000000000000.0000... Literature Cited...nono00000000000000.0-ooooooooooooooooooooo 63 S WY MD CON CLUS IONS . . . . O . O . . . O . . . . . . . . . . . . . . . . . . C . C . . . . . . . . O . . 65 BIBLIOGWHYOOOOOOOOOOOOOOO.00...OOOOOOOOOOOOOOOOOOOOOOOOOOOIOOOO. 67 Table LIST OF TABLES SECTION ONE Retention times (min) of cis, trans and trans, trans isomers of Me-ABA, and Me-DPA, and of ABArlike component in methylated extract from peach seed following gas chromato- graphy on two column supports............................... Recovery of 2—14C-ABA during fractionation of extracts of non-stratified and stratified peach seeds................... Fruit diameter, seed weight, and germination of excised embryos during maturation of 'Redhaven' peach in 1974 and 1975. Means for 10 fruits, 10 seeds (wt), or 4 x 10 seeds (germination)............................................... Effect of time of sampling on concentration of free (F) and bound (B) ABA (ng/g) in 'Redhaven' seed..................... Effect of time of sampling on total free (F) and bound (B) ABA (ng/seed) in developing 'Redhaven' peach seed........... Effect of drying and storage on ABA content. Total free and bound ABA content (ng/seed) of peach seed analysed fresh, after 1 year of dry storage, and after imbibtion............ SECTION TWO Free (F) and bound (B) ABA in peach seeds as affected by duration and temperature of stratification.................. Free (F) and bound (B) ABA (ng/g) in peach seed as affected by removal from 5°C to 27°C....OIOC00......OOOOCOOOCOOICOOOO vi Page 27 31 32 33 34 38 52 56 Effects of extracts of non-stratified and stratified peach seed on the germination (%) of stratified seed............ Biological activity (ng ABA-eq per seed) of extracts of stratified and non-stratified peach seed as determined by assay with stratified peach seed z§_ABA content (ng/seed) as determined in EC-GLC................................... vii Page 57 61 Figure LIST OF FIGURES SECTION ONE Procedure for fractionation of methanolic extracts of peach seed tissues to yield free and bound ABA fraCtionSIOOOIOOOOOOOOOOIOOIOI.OOOOOOOOOOOIIOOOOOOOOOOO Mass spectra of authentic cis, trans-ABA and of presumed ABA in extract of peach seeds. Intensities of major ions as percent of base peak.................. Concentrations of free and bound ABA in extracts of embryonic axes during maturation 2s, germination of EXCised embryOSoooocoo-00000000000000.-0000000000000... SECTION TWO Free and bound ABA in peach seed as affected by time and temperature of stratification. ABA concentration (ng/g) in embryonic axes 2s. germination............... Germination and free and bound ABA (ng/g) in embryonic axes of peach seed as affected by removal from 5°C to 27°:1°C.0..0.000IOOOIOIOIIOOIOIOOOOOOIOOOOOOOOOOOOOOO Effect of peach seed extract and ABA on seed germina- tionIOOOOOOOOIIOOOOOO0.00IOOOOOOOOOOOOOOO0.000.000.0000 viii Page 23 3O 36 50 55 6O ABBREVIATIONS The following abbreviations will be used in this dissertation. ABA BA DPA EC-GC GA GLC GC-MS IAA PPO abscisic acid N6-benzyladenine dihydrophaseic acid electron capture gas-liquid chromatography denotes the series of gibberellins and use of a subscript denotes a specific gibberellin, as GAl gas-liquid chromatography combined-gas liquid chromatography and mass spectrometry indole-3—acetic acid 2, S-diphenyl oxazole ix GUIDANCE COMMITTEE: The Paper-Format was adopted for this dissertation in accordance with Departmental and University regulations. The dissertation body was separated into two sections, both of which were prepared for publication in the Journal of the American Society for Horticultural Science. INTRODUCTION INTRODUCTION Viable seeds which fail to germinate on exposure to favorable environmental conditions of temperature, light, moisture and oxygen supply, are said to be in a state of dormancy (36). Such seed require special pretreatment to induce germination (43). Dormancy in many grains, e.g., Vicland oats (61) disappears during storage at room temperature, while dormancy of Rosaceous seeds may be broken by cold treatment (43). Leopold (38) indicated that internal mechanisms regulating germination may be those involving (a) the enlargement and restriction of the embryo, and/or (b) growth substances in the seed. Agents that can terminate dormancy under certain conditions are (a) mechanical treat- ment such as scarification, (b) light, (c) temperature, and (d) certain chemicals. Inhibitors have been implicated in the dormancy of seed of many temperate zone fruits. Abscisic acid (ABA) has been identified in peaCh (78), and measured in plum (39), and is thought to be one of the factors in the control of dormancy (35). The study of ABA and its role in dormancy may lead to a better understanding of this important phenomenon. ABA can be measured by GLC (62) which eliminates the need for extensive purification of extracts prior to bioassay, as well as interference from other inhibitors. This study was designed to measure the level and distribution of ABA in peach seed during entrance into dormancy, and during the breaking of dormancy by chilling: and (b) to determine the effect of increasing the temperature prematurely during chilling, and thereby inducing secondary dormancy, on the levels of abscisic acid. Preliminary experi- ments were also conducted to determine the effects of exogenous growth regulators and extracts of non-stratified and stratified peach seed on germination of stratified seed. LITERATURE REVIEW LITERATURE REVIEW Definitions This review is concerned primarily with the literature relating to dormancy in Prunus seed. Dormancy is often separated into endogenous dormancy (also called rest, true dormancy, or constitutive dormancy) and exogenous dormancy (also called quiescence, external dormancy, or imposed dormancy). Endogenous dormancy is a condition in the seed that delays or prevents germination under favorable environmental conditions and may be attributable to metabolic blocks, or the presence of inhibitors. (67). Exogenous dormancy involves external controls, for germination will occur if the seeds are placed under suitable environmental conditions (36) . Stratification is the practice of holding imbibed seeds in a moist environment, regardless of temperature (31), while low temperature (0° - 10°C) stratification is referred to as after-ripening (43). The chilling requirement prevents germination during unfavorable climatic conditions, and is viewed as a survival mechanism (74). Increasing the temperature prematurely may re-introduce dormancy, and then a second low temperature exposure may be required. Nicholaeva (47) and Janick 35 El. (31) both refer to this as secondary dormancy. 0n the other hand, the effects of several separated periods at low temperature may be cumulative (63). Causes of Dormancy in Prunus Seeds Crocker (l3, l4) classified the various causes of dormancy as follows: a. embryo immaturity b. seed coat impermeability to water or gases c. seed coat restriction to embryo growth d. metabolic block within the embryo e. a combination of the above f. secondary dormancy Recently, Kozlowski (36) re-classified these as follows: a. morphologically mature butphysiologically dormant embryos b. rudimentary embryos c. immature embryos d. mechanically resistant seed coat e. impermeable seed coat Morphologically, the peach seed consists of an embryo surrounded by testa. Within the testa are thin layers of nucellar and endosperm tissue, but the cotyledons of the embryo make up the bulk of the seed, and contain the food reserves used by the developing seedling. The embryos of Prunus seeds are fully developed, but germination does not occur unless the seed coat (testa) is removed, or the seed after-ripened. If a Inechanically resistant seed coat were responsible for dormancy, then low temperature stratification should reduce this resistance. However, Wong (78) found no difference in the resistance of seed coats of non-stratified .Xi' stratified peach seed. Thus, chilling does not appear to affect germination by weakening the seed coat in peach. 0n soaking in water, peach seeds become fully imbibed within 24 to 48 hours, indicating that the seed coat is not a barrier to the penetration of water. Prunus embryos are morphologically mature but are physio— logically dormant. Low temperature stratification of seed results in normal seedlings, while excised embryos germinated without low temperature exposure give rise to dwarfed seedlings (20). Exogenous Control of Dormancy in Prunus Seeds Seed Coat. As noted above, removal of the seed coat in Prunus results in embryo germination. For example, Chao and Walker (10) obtained 40 to 80% germination of non-chilled excised embryos of apricot and 50 to 70% germination of similarly treated peach embryos. However, the seedlings generally have short internodes and various abnormalities, and are not as vigorous as seedlings from stratified embryos (10, 20). Thus, in effect, the seed coat imposes dormancy upon the embryo, and can be thought of as an exogenous factor. Once the chilling requirement is completed, the seed coat is much less effective in restricting germination. The fact that seed coat resistance does not change with chilling (78) suggests that either inhibitors present in the seed coat prevent germination (see below), or that the non-chilled embryo is not sufficiently vigorous to break through the mechanical barrier provided by the testa. Temperature. Pillay, g£_§l, (49) working with cherry seeds, Obtained consistently higher germination in seeds held at 7.2°C than in those held at 3.3°C. Chao and Walker (10) stratified apricot seed, with Inaricarp removed, for O to 4 weeks at 0°, 3.3°, 7.2°, 10°, or 22.2°C. After 2 weeks highest germination occurred at 7.2°C, but after 4 weeks 95% germination was observed in seeds held at 3.3°C. Their data suggest an interaction between length of storage and temperature. However, since germination was recorded at the temperature of stratification, the results obtained may not reflect true germination potential. Suszka (68, 69, 70) held P, azigm_and P, EEEEE seeds at 20°C for 2 to 4 weeks prior to after-ripening at 3°C, and observed that subsequent germination was greater than that of seeds held continuously at 3°C. Suszka (70) reasoned that in nature Prunus seeds first go through a period of warm moist stratification after the fruits ripen and drop from the tree. This is followed by a longer period of cold, moist stratification during which the seed undergo physiological changes that prepare them for germination. Joley (32) and Tehrani (71) confirmed Suszka's observations. Pollock (5) tested the effect of temperature upon the growth of embryos excised from non-stratified peach seeds. Embryos germinated at 19°C. showed no abnormalities, while those germinated at 23° or 27°C were dwarfed. He concluded that dwarfing symptoms were controlled by the temperature of germination. When partially after-ripened peach embryos were transferred to 25°C in a moist environment, secondary dormancy was induced (33). However, if the embryos were dried at 5°C or 25°C for two days, and then stored at 26°C before re-imbibition, chilling was cumulative. Thus drying prevented the induction of secondary dormancy. These results differ from those of Flemion (19) and Coston (12) both of whom reported no inhibitory effect On germination of peach seed when chilling was interrupted by a period of time at 20°C to 25°C. Supplementary light accelerated the germination of Ligh . excised embryos of peach, cherry, apricot, and plum seeds (56), but stratified seeds germinated in either light or darkness, suggesting that light plays no active role in Prunus seed dormancy under natural conditions. Gases. Tissaoui (72) showed that exposure to N2 broke dormancy in apple embryos even in the absence of chilling. Thus 02 was not necessary for release from dormancy. No data are known from similar studies with Prunus seed. Coston (12) observed that germination of peach seed was stimu- lated when either ethylene or ethephon was applied either before or after stratification at 0°C. However, abnormalities were observed following ethephon treatment. Chemicals. Certain chemicals can partially substitute for chilling in Prunus seed. Tukey and Carlson (73) treated non-chilled sseed of 23 cultivars of peach with thiourea, using concentrations of 0.005% t:o 0.5% and exposure periods of 2 to 16 hours. Thiourea at 0.25% resulted :Ln.100% germination of 'Lovell' seeds. Dormancy of 6 additional cultivars fives not broken, and all others showed less response than 'Lovell'. Seedlings firom the treated seeds were dwarfed, and had shortened internodes and aIIomalous leaves, typical of seedlings from non—stratified excised embryos. Later, Carlson and Badizadegan (9) reported that thiourea was not as Effective as BA or GA3 in stimulating germination of seeds of several Lipe (40) reported °t1162r peach cultivars after one month of stratification. thaltboth thiourea and GA3 were able to break dormancy in peach seed. He “Otlead that GA3-treated seeds germinated slowly, and the resulting seedlings were not dwarfed, whilst thiourea-treated seeds germinated readily,but the seedlings showed typical symptoms of insufficient chilling. Gray (23) was among the first to note that GA3 could substitute for chilling in peach seed. Treatment of non—stratified seed with GA3 (10 ppm) in agar medium resulted in 35% germination in 13 days, whereas the control seeds had not germinated after 53 days. However, seedling growth was not discussed. Chao and Walker (10) stimulated the germin- ation of non-stratified peach and apricot seed with high concentrations (4,000 to 20,000 ppm) of GA3, but only the apricot seedlings grew normally. However, application of 20 to 1,000 ppm GA3 to excised embryos of peach and apricot resulted in normal seedling growth. Fogle (22) soaked P, avium seed in GA3 solution (100 ppm) prior to stratification. After 3 months at 5°C, 64% germination occurred in GA3-soaked seed vs. 28% germination in water-soaked seed. BA at 10 and 20 ppm stimulated germination in seeds of 3 varieties ()f peach after stratification for one month (9). On the other hand, non— astratified embryos or intact seeds of plum did not respond to 1,2,4 or 8 PM BA (39) . The effects of GA and cytokinins may be additive or more than axiditive (synergistic). When 6A3 and BA were applied together to non- St:ratified peach seeds, no germination occurred (16); however, partially chilled seeds responded to treatment. No data were presented for the effects of the two chemicals alone. Lin and Boe (39) applied GA3 and BA to Inon-stratified intact seeds and embryos of plum. Intact seed did not resSpond to treatment, but GA3 (16 ppm) plus BA (8 ppm) resulted in 100% Selrtnination of the embryos compared to 25% for no treatment. However, GA éilone at 32 ppm gave 62.5% germination, which was not significantly different from the effect of the mixture. The dataare, however, suggestive of additive effects of GA and BA on germination. Effects on seedling growth were not discussed. Naringenin, a growth inhibitor isolated by Hendershott and Walker (26) from peach flower buds, had no effect on germination of stratified peach seed or on seedling development (21). Lipe (40) applied ABA (5.0 ppm) to excised embryos of peach and reduced germination from 83% (water control) to 5.6%. When Diaz and Martin (16) applied ABA to excised peach embryos and then stratified them for 2, 10, or 12 weeks, germination was also inhibited. However, the effect decreased with stratification, suggesting either that the inhibitory effect of ABA.was being nullified by promoters produced at the low temperature, or that chilling increased the embryo's capacity to inactivate ABA. Similar results were reported by Rudnicki and Pieniazek (59) with apple (Pyrus malus L.) seeds. Lipe (40) applied various combinations of IAA, GA, and ABA to ciormant and non-dormant peach seed. ABA reduced the germination of non- ciormant seed, as well as the response of dormant seed to GA. Endogenous Control of Dormancy in Prunus Seeds Three phases of seed dormancy are recognized by Amen (3), namely, induction, maintenance, and release. Relatively little information is aVailable on induction in Prunus seeds. Weaver and Rough (76) noted that in ‘the early-ripening peach cv. 'Raritan Rose', germination of excised emIDGryos following 8 weeks at 4°C, was greater in samples taken 71 to 91 day's; after full bloom than those taken at 99 and 107 days. There was no appc'ilrent increase in inhibitor content (wheat coleoptile bioassay) or lO germination of excised embryos. Pillay (48) using the Avena mesocotyl bioassay noted four growth promoters in methanol extracts of cherry seeds. He suggested that their disappearance at maturity might be related to the induction of dormancy. Bausher (5) used electron capture-GLC to follow ABA levels in seeds of 'Okinawa' peach as the embryo matured. The concen- tration at maturity was 30 ng/g fresh weight, which is probably far too low to have imposed dormancy, judging from the data of Lipe and Crane (41). Studies have dealt with the maintenance and breaking of dormancy in Prunus seeds. Effect of the seed coat. As previously noted, the seed coat prevents the germination of non-chilled Prunus embryos. The reasons for this are not well established. However, one can postulate that the effect is due either to chemicals in the seed cost which inhibit radicle growth, or'to the mechanical resistance of the seed coat. If the latter, then Viong's (78) data suggest that the properties of the embryo, rather than t:hose of the seed coat, change during after-ripening. The possible role c>f inhibitors is discussed below. Dennis (15) suggested the use of reciprocal czrosses between cultivars or species which differed in chilling requirement as a test of the effect of the seed coat. If the seed coat controls d<>rmancy, the chilling requirements of seeds from the two crosses should b€3 similar to those of the female parent. Kester (34) made such crosses in aldnonds and almond/peach hybrids, and observed no maternal effect, suggesting trlat the seed coat has a purely mechanical effect. Respiratory changes, Pollock and Olney (52) found that the rate 0f :respiration increased with stratification in sour cherry (P, cerasus L.) 8953(3. They observed that dinitrophenol (DNP) stimulated respiration in ll dormant seed and that the effect declined with chilling. They suggested that blocks occurred in the electron transport system in dormant seeds, and that these were removed by chilling. Later, LaCroix and Jaswal (37), compared 02 uptake of dormant and non—dormant sour cherry seed in the presence and absence of dinitro— phenol. They observed that DNP increased 02 consumption 91% in dormant embryonic axes gg, only 18% in non-dormant axes, thus confirming the work of Pollock and Olney (52). The 02 uptake of cotyledons was not affected by DNP, implying that the embryonic axis is the site of dormancy. They also noted that the C-6/C—l ratio in the embryonic axis was constant during the 6 weeks of chilling, but decreased sharply at 7 weeks, suggest- ing a shift in respiratory pathway as dormancy was broken. Auxins. Although IAA has been identified in extracts of develop— ing sour cherry seed (28), Biggs (6) could find no marked changes in :indole auxins in peach seeds as stratification time increased. Gibberellins. Until recently, only GA32 had been identified in ]?runus, occurring in developing seeds of apricot (11), and peach (79). IIowever, G.C. Martin (personal communication) has now identified GA29 in immature plum (_P_. domestics L.) seeds and fruits. Although GArlike substances were detected by bioassay of methanol enctracts of mature sweet cherry (P, avium L.) seeds, Proctor and Dennis (EiS) were unable to correlate changes in concentration with changes in SEEIHnination capacity during after-ripening. Mathur final, (42) reported that GA3-like and GA7-like compounds in‘Ztreased 1268% and 553% respectively during chilling of peach seed held at (3°C. Although there was a decrease at the eighth week of stratification, 12 the concentrations increased at the 12th and 16th week. The concentration of GA7-like substance was lower than that of GA3-like substance through- out stratification. Germination increased from 0% for non-stratified seeds to 35% for seeds stratified for 4 weeks. No germination data were given for subsequent samples. However, the authors apparently used a spectrophotofluorometric method which is open to criticism. Lin and Boe (39), also using spectrophotofluorometry, found that dormant plum seed contained 0.125 ug/gm of GAelike substances, and that this increased to 0.17 ug/gm during the 90 day chilling period. They therefore suggested that increases in GA content are associated with the breaking of dormancy. Cytokinins. Webb §£_al, (77) noted that butanol-soluble cytokinins in Acer saccharum seeds reached a maximum concentration after 20 days of stratification, then declined before dormancy was broken. IBorkowska and Rudnicki (8) reported that both rfree and bound (released (an acid hydrolysis) cytokinins increased during stratification of apple aseed with a maximum at 5 weeks. No information is known concerning czytokinin levels in Prunus seed during stratification. Inhibitors. Chao and Walker (10) inhibited the germination of emnbryos excised from.non-chilled peach and apricot seeds with extracts Plfepared from similar seeds, and concluded that substances present in the SEEEd.coat were responsible for activity. However, Flemion and de Silva (231) could not correlate levels of inhibitors extracted from peach seeds W1 th dormancy release. Aitkins (2) identified the phenolic compounds mandelic acid and beclzzoic acid in peach seeds. These products of the hydrolysis of l3 mandelonitrile were detected in non-stratified seed 72 hours after imbibition, but only trace amounts were noted during after-ripening. The concentrations (0.005 to 0.05 ug /g and 0.16 ug /g) of mandelic and benzoic acids, respectively, were not high enough to inhibit seed germination, and Aitkins (2) concluded that these compounds had no direct effect in peach seed dormancy. Lipe and Crane (41) measured the levels of an ABArlike substance in peach seed extracts. Co-chromatography, UV absorption spectra, and bioassay with wheat coleoptile sections indicated that the inhibitor was ABA-like. They correlated the release of dormancy in peach seed with the disappearance of this substance. Although the inhibitor was present throughout the seed, its concentration was greater in the integuments than in the embryo. Application of the inhibitor extracted from dormant :seeds to embryos excised from stratified seeds induced anomalies similar tn: those observed in seedlings from non-chilled embryos. Diaz and Martin (16) investigated a similar compound in peach seaed and showed that one of the components of the extract had the same reatention time as ABA on GLC. They also reported a decline in the concen— tlration of the inhibitor during chilling of two peach cultivars, ’Tetela' and 'Lovell', and this decline was correlated with an increase in the COIIcentration of a similar inhibitor released by base hydrolysis. Milborrow (46) had observed that ABA could be released from its glucose 99t1€3r in this manner. 'Tetela' seeds, which require only a few weeks of Chi--3Lling, contained less inhibitor than 'Lovell' seeds, which must be l4 chilled for 6 weeks, and more inhibitor was present in the embryo than in the seed coat. Wong (78) unequivocally identified ABA in extracts of peach seeds by GC-MS. However, bioassay results indicated that levels in seed coat and cotyledon did not decrease significantly as stratification time at either 3°C or 20°C increased. Although a marked reduction occurred in the embryonic axis, the change was independent of temperature. Thus, although a decrease in ABA in the embryonic axis may be a pre- requisite for germination, other factors, such as gibberellins, must be responsible for the effects of low temperature on germination. Lin and Boe (39), using bioassay, reported a 32% decrease in an ABArlike substance in plum seeds after 30 days of chilling, with a subsequent 10% decline for the remainder of the 90 day chilling period. Interaction of promoters and inhibitors. Dormancy control is often attributed to a balance of endogenous promoters and inhibitors (3) ‘Nith dormancy maintenance being associated with the presence of high levels (of growth inhibitors, and dormancy release with high levels of growth Ipromoters. Amen (3) suggests that exposure to chilling temperature permits garowth promoter production, which then triggers dormancy release. Flemion airid de Silva (21) analysed extracts of peach seed during dormancy release. queir'data indicate no correlation between the breaking of rest and the réiluative levels of promoters and inhibitors. However, Lin and Boe (39) nC>tzed that GAelike substances increased while growth inhibitory substances (ABA-like compounds) decreased in plum seeds exposed to chilling. Khan (35) proposed that germination in some seeds is controlled 15 by interaction between cytokinins, gibberellins, and inhibitors, with cytokinins playing a permissive role. Gibberellins are necessary for germination, but the presence of inhibitors, e.g., ABA, blocks their action. Cytokinins are capable of removing this block. However, Drury (17) states that this model could apply only to discrete data, and that germination of a population of seeds is a continuous function. Further- more, available data on endogenous growth regulators in seeds which require chilling do not support the model. For example, the work of Webb, EEHEA' (77) with A535 seed, suggests that levels of hormones do not determine the state of dormancy in the seed, but rather the sequence .2: changes in growth regulators. They showed that the levels of GA, ABA, and cytokinins were all low following chilling, but germination still occurred. Although data are not available for peach, bioassay results with tapple seeds indicated that GAplike substances reached a maximum after 4 sveeks (64), cytokinins after 5 weeks (8), while ABArlike substances had cleclined to negligible levels after 3 weeks (58). Thus, if Khan's (35) llypothesis were correct, dormancy should have been broken after 4 weeks. Flowever, 6 weeks were required for 50% germination, suggesting that the hy‘pothesis does not hold in this case as well. Summary 1. Removal of the peach seed coat allows germination, but the seeidling grows slowly, and abnormalities occur if temperature of germination is greater than 20°C. l6 2. Holding seeds at 0 to 10°C for a specified period results in normal seedling growth even when the seed coat is intact. 3. The seed coat may either contain inhibitory substances or serve as a mechanical restriction to growth. Some workers (l4, 16) have reported a decline in inhibitory substances in the seed coat, while others (21, 78) have found no such decline. The fact that no maternal (i.e., seed coat) effect is evident in seeds from reciprocal crosses of almonds and peach/almond hybrids which differ in chilling requirement (34) suggests that the seed coat effect is purely mechanical. Chilling does not appear to affect the mechanical resistance of the seed coat of peach (78). Thus germination potential is probably a function of embryo vigor, which is enhanced by chilling. 4. Chilling appears to prepare the embryo for growth. The changes observed in phosphate metabolism and respiration are indicative of growth potential, which perhaps is favored by growth substances produced during chilling. Inhibitors, including ABA, appear to decline, and promoters increase, leading to a favorable balance. However, endogenous regulators are usually present in lower concentrations than are necessary for stimulation or inhibition when applied externally (39) suggesting penetration problems or reduced sensitivity of the target organs. Several aspects of the role of ABA in Prunus seed dormancy require further study. These include: (a) use of physico-chemical methods of measurement, rather than bioassay, (b) relation of ABA levels to the induction of dormancy, as well as the breaking of dormancy, (c) the effect of temperature in the 17 observed decline in ABA content during stratification, (d) the contribution of ABA to the observed inhibition by seed extracts, and (e) the role of esterification of ABA in the disappearance of the free acid during stratification. SECTION ONE THE ROLE OF ABSCISIC ACID IN PEACH (Prunus persica L.) SEED DORMANCY. I. INDUCTION 0F DORMANCY. l8. {-11 ('1 EH all? po THE ROLE OF ABSCISIC ACID IN PEACH (Prunus persica L.) SEED DORMANCY I. INDUCTION OF DORMANCY ABSTRACT A peak observed on gas chromatography of a partially purified methanol extract of mature peach seeds was identified as ABA by combined gas chromatography-mass spectrometry (CC-MS). No germination occurred during seed maturation unless embryos were excised. Germination of excised embryos increased with maturity, and ABA content of embryonic axes and other seed parts did not appear to be related to germination potential. Drying and storage of seeds did not significantly affect total ABA content, although an increase in both free and bound ABA occurred in the embryonic axes. 0n imbibition, levels of both free and bound ABA decreased except for free ABA in the seed coat and bound ABA in the embryonic axis, neither of which changed significantly. In most cases examined, levels of free ABA paralleled those of bound ABA, suggesting that although the former may be converted to the latter, bound ABA is not a major source of free ABA. Growth substances have been measured in developing seeds of peach (22, 21, 11), cherry (15, 9), and apricot (12), but only GA32 (24) and ABA (23) have been unequivocally identified in peach seeds. Using electron capture-gas liquid chromatography, Bausher (2) found that ABA increased in peach seeds as they matured, reaching a maximum concentration (30 mg/g fresh wt) when the embryo had reached full length. Germination potential was not determined but Bausher suggested that the level of ABA present at maturity was sufficient to impose dormancy. 19- 20 Using the Aven§_first internode bioassay, Pillay (15) observed that auxin-like substances decreased as cherry seeds matured, with no activity measurable at maturity. He suggested that this decrease in auxin—like substances might be the cause of rest. Hopping and Bukovac (10) identified IAA in sour cherry seeds, and noted a decrease in total auxin content in the seed as the fruit matured. Bausher (2), found that cytokinin-like substances, as measured by the soybean callus assay, varied from 7.5 to 16.4 ug/g fresh wt. during embryo development of peach. His results suggest that high levels of cytokinin-like substances are associated with embryo growth and that a decline occurs when the embryo matures. Dry storage may reduce the germination potential of seed. Haut (8) reported no significant effect of drying of cherry and peach seed on their germination during subsequent after-ripening. However, Fogle (6), found that dry storage prior to after—ripening reduced germination of cherry (P, azigm_L.) seeds to about 50% of those kept moist. Ross and Bradbeer (18) obtained similar results with hazel seeds. Balboa (1) reported a marked increase in ABA content in the embryonic axes of apple seeds sampled August 24 and subsequently stored for 4 months over CaClz. However, this was not true for mature seed sampled September 21. The present work was undertaken to (a) identify ABA in mature peach seed, (b) relate ABA levels to entrance into dormancy, and (c) determine the effects of drying and storage of the seed on ABA content. 21 MATERIALS AND METHODS Plant material and method of stratification. Mature peach seeds, cv. Halford were used for identification of ABA and for determining recovery of 2-14C-ABA in fractionation. Pits from a California source were purchased from Hilltop Orchards, Hartford, MI, and were stored at 22 i 1°C until used. Dry seeds were removed from the endocarp and soaked in distilled water for 48 hr prior to dissection. For comparison of ABA levels during maturation and storage, seeds, cv. Redhaven, were collected from mature trees at the Horticultural Research Center, East Lansing, MI. from July to September of both 1974 and 1975, and brought to the laboratory for immediate dissection. Extraction of seeds and fractionation of extracts. All seeds were dissected into seed coat, cotyledons, and embryonic axis prior to extraction except where otherwise noted. The tissues were held at 0°C during dissection of 50—seed samples (223 15 min.), then covered with cold methanol and macerated in a Sorval Omni-mixer. Seed coats and cotyledons were ground in 250 ml of solvent, embryonic axes in 100 ml. The macerates were shaken overnight at 2° i 1°C., filtered through cheesecloth and the filtrates were centrifuged. The supernatant was poured off and the tissued was re-suspended in fresh methanol and again centrifuged. The combined supernatant (ca, 500 ml for seed coat and cotyledons, 250 ml for embryonic axis) was evaporated in vagug_at 40° i 1°C. The residue was redissolved in 25 ml of 0.1 M phosphate buffer pH 7.5 and partitioned (Fig. 1). For identification of ABA, an extract 22 Fig. 1. Procedure for fractionation of methanolic extracts of peach seed tissues to yield free and bound ABA fractions. 23 Residue from methanolic extract dissolved in 25 ml phosphate buffer pH 7.5 Wash with 4 x 25 m1 hexane l Hexane (discard) Aqueous Wash with 4 x 25 ml MeClZ Neutral MeC12 (discard) Aqueous Adjust to pH 2.5-3.0 with HCOOH Wash with 4 x 25 ml MeC12 A l Acidic MeClz Aqueous (free ABA) Adjust to pH 11.0 with NH4OH Heat for 1 hr at 60°C Cool. Adjust to pH 2.5 - 3.0 with HCOOH Wash with 4 x 25 ml MeClz [ I MeClz Aqueous (bound ABA) (discard) Fig. l 81 7| 24 of whole seeds (150 gm after imbibition) was prepared in a similar manner, except that larger quantities of solvent were used. Methylation and GLC. Following evaporation of the dichloro- methane, the residues from the free and bound fractions were methylated as described by Schlenk and Gellerman (19) and modified by Powell (16). Each residue was dissolved in 1 ml of ether/methanol (9:1, v/v). Carbitol (1.5 ml), KOH (1.0 ml, 60 g/100 ml H20) and an ethereal solution of diazald (1.5 ml at 115 mg/ml) were added in sequence to a 150 x 15 mm test tube, which was then connected by 'Teflon' tubing through 'Nalgene' stoppers to the tube containing the sample. The diazomethane generated on addition of diazald passed into the sample tube and methylated the organic acids present. The ether/methanol was then evaporated and the residue dissolved in ethyl acetate. For quantitative analysis of ABA, one microliter of sample, representing 5 mg-eq of seed, 30 mg-eq of cotyledon, or 0.1 mg-eq of embryonic axis, was injected into a Packard 7300 gas liquid chromatograph. This was equipped with a 63Ni foil electron capture detector and was operated either at 5 or 7.5 volts. The column (2 mm i.d. x 1.83 m) was packed with 3% SE 30 (methyl silicone) on 80/100 mesh Gaschrom Q. Column temperature was 210°C and inlet and detector temperatures were 26° and 270°C, respectively. The carrier gas was N2 at a flow rate of 40 ml/min at 40 psi. Nitrogen scavenger gas was supplied to the detector at 70 ml/min. Quantitation was based on peak height, using known quantities of synthetic cis, trans ABA (R. J. Reynolds Tobacco Co.) as a reference. 25 Retention time was also determined on a Hewlett-Packard 402B GLC using an electron capture detector and an XE 60 column (25% cyanoethyl, methyl- silicone) at temperature of 200°C. Inlet and detector temperatures were 260° and 270°. Gas chromatography-mass spectrometry. For identification of ABA, the free fraction from“150 gm of whole seed was analysed, following methylation, on an LKB GCéMS interphased with a POP 8/I computer. The glass column (i.d. 2 mm by 1.84 m) contained 3% SP 2100 (methyl silicone) on Supelcoport (acid washed, silanized diatomite 100/120 mesh). Helium was used as carrier gas at a flow rate of 20 ml/min. Column and detector (flame ionization) temperatures were 200° and 290° C, respectively. The mass spectrometer was operated at 70 eV. Recovery of 2-14C-ABA. 2-14C-ABA.(MallinkrodtNuclear) was used to estimate how much ABA was lost during fractionation. In one experiment, methanol extracts were prepared following dissection of 100 non-stratified and 100 stratified (12 weeks at 5 i 1°C) seeds. In a second experiment, a methanol extract of 100 intact, non- stratified seeds was used. Following extraction 1.2 x 10"3 uCi of 2-14C-ABA (specific activity 23 Ci/mole) was added to each sample, and the extracts were partitioned as described above. Ten ml of scintillation fluid (5 gm PPO and 100 gm naphthalene/liter in dioxane) was added to each fraction and the samples were counted for 10 min using a Beckman model LS 100 liquid scintillation counter. Counts were corrected for quenching and efficiency. 26 ABA levels during seed maturation vs._germination. Fruits were collected at weekly intervals from July 31 until fruit maturity in both 1974 and 1975, diameters of 10 fruits and weights of 10 seeds being recorded for each data. Two samples of 50 seeds each were analysed for ABA as previously described. On each sampling date, 4 samples of 10 intact seeds and the same number of excised embryos were placed on 2 layers of moist filter paper in glass Petri dishes. Germination, defined as geotr0pic curvature of the radicle, was recorded after 10 days at 20 i 1°C and 200 ft.-cand1es of fluorescent light. Effects of drying and storage of seed on ABA content. 'Redhaven' seeds from the 1974 harvest were air-dried and stored in the endocarp for 12 months. One lot was dissected and extracted dry, the other imbibed before dissection. ABA.was analysed as previously described. All data on ABA content were analysed by analysis of variance, Duncan's (5) multiple range test being used for comparison of treatment me ans 0 RESULTS The peak assumed to be cis, trans-ABA in the extract had the same retention time as synthetic cis, trans-ABA on both supports (Table 1). None of the other peaks in the extract had retention times identical with trans, trans ABA or with either isomer of DPA. GC-MS of the extract of non-stratified whole seeds showed the presence of ABA at the retention time of synthetic cis, trans-ABA. Major 27 Table l. Retention times (min) of cis, trans and trans, trans isomers of Me-ABA and Me-DPA, and of ABA-like component in methylated extract from peach seed following gas chromatography on two column supports. Column Temp Me-ABA Me-DPA Extract (°C) (c.t) (t.t) (c.t) (t.t) SE 30 210 1.76 2.47 2.35 3.34 1.76 XE 60 200 1.49 -- 2.16 3.00 1.49 28 fragments and intensities were in close agreement with the reference sample (Fig. 2), aside from fragments in the standard at m/e values greater than the molecular ion (MT), which were probably due to impurities. Twenty-seven to 84% of the 2-14-C-ABA added to extracts was recovered in the acidic dichloromethane fraction following fractionation (Table 2), recovery being less than 50% in only one of 9 samples. This represented from 70 to 92% of the total ABA recovered. Only 1 to 4% was recovered in the bound fraction. Both fruit diameter and seed fresh weight increased steadily from mid-July until early September (Table 3). Intact seed failed to germinate, regardless of time of collection. Germination of excised embryos was low in July, increased sharply in early mid-August, and continued to increase until harvest (Table 3). Concentrations of both free and bound ABA in the embryonic axis were 10 to 100—fold that in other portions of the seed throughout maturation (Table 4), while levels in the cotyledons and seed coat were similar. Expression of the data on a per seed basis (Table 5) showed that most of the ABA occurred in the cotyledons, which make up the greatest part of the seed (ca. 67% in mature seeds XE! 32% for the seed coat and less than 1% for the embryonic axis) on a fresh weight basis. Levels of both free and bound ABA increased 3 to 4-fold in the seed coat during maturation in 1974; levels in the embryonic axis and cotyledons exhibited no particular trends (Table 4). In 1975 both free and bound ABA increased significantly during maturation in the embryonic axis but no trends were evident in other tissues. Data for the former are graphed in Fig. 3. No clear relationship between ABA content and 29 Fig. 2. Mass spectra of authentic cis, trans-ABA and of presumed ABA and of presumed ABA in extract of peach seeds. Intensities of major ions as percent of base peak. 30 can can 8!. mbm .zv ”LN ooN 0mm OnN ocm CON DON om. cow now om. oxE on. mm. ho<¢hxw 00. N9 Ohm 00. On Vn. mm. 00.. . 00 ON. ¢n_ uh 00. 00 ms (°lo) AllSNBiNI BALLV'IHU Fig. 2 31 Table 2, Recovery of 2-14C-ABA during fractionation of extracts of non-stratified and stratified peach seeds. Seed Embryonic Intact coat Cotyledon axis seed Imbibed only» Exp. 1 Exp. 2 Total recovery (%) 107 65 60 - 79 % of recovered counts Hexane ll 4 2 - 3 Neutral CHZClZ 8 7 4 - 8 Acidic CH2C12 79 (84)2 84 (55) 92 (55) - 89 (71) Bound CH2C12 2 2 1 - - Aqueous residue 1 3 l - l Imbibed and stratified at 5°C for 8 wk. Total recovery (%) 72 60 31 87 80 % of recovered counts Hexane 8 4 1 2 2 Neutral CHZClz 4 5 4 7 10 Acidic CH2C12 85 (61) 84 (51) 88 (27) 88 (77) 87 (70) Bound CH2C12 1 4 3 l - Aqueous residue 2 2 3 2 l 2Figures in parentheses indicate recovery as a % of total ABA added. 32 Tablei3. Fruit diameter, seed weight, and germination of excised embryos during maturation of 'Redhaven' peach in 1974 and 1975. Means for 10 fruits, 10 seeds (wt.), or 4 x 10 seeds (germination). 1974 1975 Sampling Fruit Seed % Fruit Seed % date diameter weight Germi. diameter weight Germ. (mm) (mg) (mm) (mg) July 16 35 a2 472 a -- 38 a 440 a -— 31 39 b 485 a 15.0 ab 39 a 443 a 20.0 a Aug. 6 48 c 539 b 17.5 b 46 b 489 b 67.5 b 13 53 d 554 b 85.0 c 51 bc 527 bc 80.0 bc 22 58 de 559 b 87.5 cd 53 c 547 c 85.0 c 29 60 e 562 b 90.0 cd 57 c 568 c 90.0 c Sept 7 65 e 573 b 97.5 d -- -- -- zWithin columns, means followed by the same letter are not significantly different from one another at the 5% level 33 Table‘ho Effect of time of sampling on concentration of free (F) and bound (B) ABA (ng/g) in 'Redhaven' seed. Sampling Seed coat Cotyledon Embryonic date axis 1911; F B F B F B July 31 8.8a2 8.1ab 4.8a 3.9a 126a 265ab Aug. 6 7.78 6.0a 9.9b 3.2ab 2113b 323b 13 9.0a 7.3ab 6.5a 3.23 213ab 2073 22 18.9b 15.9abC 8.4ab 7.8b 267b 327b 29 13.9ab 15.2abc 8.43b 8.2b 106a 595c Sept 7 28.4C 22.8bc 9.6ab 8.2b 108a 345b 1975 July 31 10.38 9.43 5.3a 7.48 114a 2318 Aug 6 9.18 10.03 5.98 13.6b 315e 225a 13 11.03 12.2ab 12.1b 6.1b 351c 378b 20 6.9a 10.7ab 11.2b 9.1ab 212b 364b Aug 27 11.08 17.7b 3.9a 6.6a 298c 373b zWithin columns and years, means followed by the same letter are not significantly different from one another at the 5% level. Table 5. ABA (ng/seed) in developing 'Redhaven' peach seed. Effect of time of sampling on total free (F) and bound (B) Sampling Seed coat Cotyledon Embryonic date axis .1213 F B F B F B July 31 2.9082 2.54ab 2.98a 2.42a 0.38a 0.80a Aug 6 2.96a 2.30a 9.48C 3.048 0.76b 1.168 13 3.10a 2.45ab 6.38b 3.158 0.80b 0.788 22 5.45b 4.64b 7.98bc 7.41b 0.95b 1.16b 29 3.328 3.808b 7.31bc 7.11b 0.358 1.31b Sept 7 3.64a 2.82ab 7.32bc 6.25b 0.26a 0.848 1975 July 31 2.55b 2.33ab 2.698 3.79a 0.34a 0.668 Aug 6 1.618 1.75ab 3.198 7.35b 0.67b 0.488 13 1.14a 1.288b 4.82b 2.428 0.558 0.608 20 0.80a 1.24a 6.54C 5.328b 0.45a 0.778 27 1.608 2.60b 1.90a 3.21a 1.28C 1.60b zWithin columns and years, means followed by the same letter are not significantly different from one another at the 5% level. 35 Fig. 3. Concentrations of free and bound ABA in extracts of embryonic axes during maturation vs, germination of excised embryos. (%) 00140111111169 22. 053.com 5m cud nu ma .3 km mud «Me as em .3. o. 0.6. . . . oo. o... . «2 out... 0v .