Occurrence of Gibberellin A 3 in Parthenocarpic Apple Fruit

Abstract

The gibberellins 'have ibeen established as effective fruit setting agents (5). In the apple, where auxins are generallly inactive, the gi'bberellins induce parthenocarpy and sustain fruit growth to maturity (2,6, 10). This pronounced fruit-setting activity and the estafblished observation that asymmetric growtth of apple fruit is related to incomplete seed development,-has encouraged investigations on the rela.tionship of endogenous seed gi!bberellins to fruit-set and growth. Gibbetrel'lin-like activity 'has been demonstrated in endosperm (11) and GA4 and GA7 identified in immature apple seed (8). The biosynthesis of gib-berellins by 'seeds is viewed as a specific role of seeds in fruit-set o.f the apple (7). However, some cultivars of Malus are prone to produce seedless fruit after exposure to 'low temperature during the fruit-setting period. The presence of gibberellin-like substances in such parthenocarpic fruiit is, herein, reported. Immature seedless apple fruits (Malus sylvestris iMill., selection New Jersey 12) developing subsequent to 'a severe 'frost, 4 days after full bloom, were harvested (6-wk post-tbloom), frozen immedi-.altely and stored at-300 until used. Frozen fruits (6.9 kg) were immersed in 100 % acetone (7.6 liters), then each fruit was halved to check for seedlessness (no seeds were found), homogenized in acetone and extracted twice 'by stirring tinder nitrogen for 24 hours at 2°. The filltrates were comibined and concentrated to an aqtueoous solultion (3.5 liters, pH of 2.8) in vacuo. The pH of the filtrate was increased to 6.0 with 10 % KOH (13) and centriffuged to remove proteins and other insoluble substances. The super-nate was washed with n-hexane and adjusted to pH 2.0, stiirred for 2 hours a'fter a'ddition of 70 g of activated charcoal and filtered. The charcoal was washed several times with distilled water and eluted with acetone. The eluate (acetone) was evaporated 'to dryness, the residue (2.62 g) stus-1 Michigan Agricultural Experiment Station Journal Article No. 4227. 448 pended in phosphate buffer (0.2 M, pH 6.2) and extracted with chloroform (9). The aqueous phase was adjusted 'to pH 2.5 with su!lfuric acid and extracted with ethyl acetate. T'he chloroform and ethyl a'cetate extracts were dried over anhydrous sodium sulfate and evaporated to dryness yielding residues of 0.108 and 0.690 g, respectively. These residues were taken tup in ethanol an'd lanolin paste preparations of either fraction were found to induce parthenocarpy in tomato. The chloroform fraction was more active than the ethyl acetate fraction. Considerable quanti,ties of inhiibitors were present in the ethyl acetate fraction as was evidenced by injury of treated ovaries. Further purification was aohieved by streaking the extracts on Whatman 3 MM paper (57 X 46 om) and developing with isopropanol :ammonium hydroxide :water (10:1:1 v/v). The chromato-grams were cut transversely, at interval's of 0.1 RF, and eluted with 80 % ethanoll. The gibiberellin activity of each eluate was assessed using dwarf pea (9) (cv. Morse's Progress No. 9) and cutcutmber seedling (4) (cv. Burpee Hybrid) assays. The chloroform fraction contained an active component(s) migrating to RF 0.4 to 0.7 and which was similar in activi'ty and RF with autihentic GA3 trea,ted tinder identical conditions (fig 1). A greater quantitative response was obtained from the chloroform than ethyl acetate fraction (approx 10-fold) in the dwarf pea assay. Little or no activity was observed for both fractions in the cucumber assay. Fuirther pturificationi was limited to the active component(s) of the chloroform fraction migrating to RF 0.4 to 0.7. The eluate was streaked on silica gel-G,plates (E. Merck F-254, 250 4). Plates were developed with the lower phase of carbon tetrachloride :acetic acid water (8:3 :5, v/v) pluts 20 % ethyl acetate. The active component(s) did not migrate, thus it was elutited from the origin with et-hano,l and rechromatographed utsing benzene: n-1butanoIl:acetic acid (70:25:5 v/v) as the developing so,lvent. The active component(s) was identical to GA1 and/or GA3 with respect to RF (0.54) and color of fluoirescence and differed in 1)oth respects www.plantphysiol.org on April 9, 2018-Published by Downloaded from