Contents and Recovery of Gibberellins in Monoecious and Gynoecious Cucumber Plants 1

Diffusetes from seedlings and root exudates from 6-week-old plants of a monoecious line of cucumber, Cucumis sativus L., contained considerably higher levels of gibberellin.(GA-)like substances than did those from plants of an isogenic gynoecious line. Most of the GA4ike activity was found in a chromatogram region ty.pical of GA1 and GA3; some activity, particularly in root exudates, appeared also at an RF similar to that of GA4 and GA7. When seedlings were treated with 3H-labeled GA1, more radioactivity was found in the diffusates from monoecious seedlings than from gynoecious ones. The same was true of biological activity in root diffusates from older plants which had been treated with gib!erellin A4+,. In conjunction with evidence present in literature, these resulits support the idea that endogenous GAs play a part in the regulation of sex expression in cucumber, relatively high levels favoring the formation of staminate flowers. Auxins have long been known to enhance the female sex "tendency" in cucumber, that is, to promote the formation of pistillate flowers (14). Gibberellins (GAs) have the opposite effect, favoring the formation of staminate over that of pistillate flowers (4) and moreover inhibiting the further development of the latter (Atsmon, unpublished data). These findings were obtained with exogenous hormones. However, in the case of auxin, both, indirect studies concerning relations between growth rates and sex expression (2,7) and direct analvses of the auxin content of plants with different sex expression (8), have provided evidence that this hormone participates in the endogenous regulation of sex expression in cucumber. In the case of GAs, there is so far only indirect evidence for such an endogenous function. Part of it comes from studies on hypocotyl and internode length in plants of different sex types. These parameters can be used as indicators of the effective endogenous GA level in the plants, and the results show that this level increases with increasing male tendency (Atsmon, unpublished data). Another part of the evidence comes from work with the growth retardants AMO-1618 and OCC (Cycocel) which are known to inhibit GA biosynthesis in plants and which were found to reduce the male tendencv in cucumber (Galun and Lang, unpublished data). 1 This research was ca(rried out under Unit-d States Atomic Energy Commission Contract No. AT(11-1) 1338. 2 Present address: Plant Genetics Section, The Weizmann Institute of Science, Rehovot, Israel. The experiments described below were designed to obtain more direct evidence for differences in GA content in cucumber plants of different sex types; and also to determine whether such plants differ with respect to recovery of applied GA, that is, presumably, in their metabolism of GAs. Both types of experiments were done with 4-dayold seedlings and with 6-week-old plants. In the seedlings, we determined the quantities of GAs, either endogenous or applied, diffusing from the shoot into agar blocks. In the older plants, the GA content of root exudates was determined, this latter approach being based on the discovery that root exudates may contain significant levels of GA-like substances (16, 18) synthesized in the root (10, 11). Materials and Methods Plant Alaterial. A homozygous gynoecious and a monoecious line of cucumber (Cucumis sativuts L., cv. "Beit Alpha") were used throughout this study. The 2 lines are practically isogenic, differing only in the alleles of 1 of the 2 major genes (st-/st and M/m; see 6) which determine sex expression in cucumber. The monoecious plants have the genic constitution st-/st+ M/M. They produce only staminate flowers on the first flower-bearing nodes of the stem; on the latter nodes, an increasing proportion of pistillate flowers are formed. The homozygous gynoecious plants, with the constitution st/st M/M, form exclusively pistillate flowers; formation of staminate flowers on the first flower-bearing nodes can be induced only by treating the plants with GA (15). Collection of Diffutsates and Exudates. The procedures for obtaining diffusates and exudates used 806 www.plantphysiol.org on October 15, 2017 Published by Download d from Copyright © 1968 American Society of Plant Biologists. All rights reserved. ATSMON iEU AL.-GIBBERELLINS IN CUCUMBER PLANTS 807 in our work were essentially those (lescribed by Jones and Phillips (9), and Phillips and Jones (16), respectively. Diffusates were obtained from seedlings which were grown from seeds soaked for 2 hours in water, then planted on moist facial tissue and kept, the first 48 hours, in the dark and at 320, the following 48 hours in the greenhouse (natural light, extended to a total of 16 hours per day by light from Gro-lux fluorescent lamps). For each experiment, 50 seedlings of either sex type were selected for equal size, cut at the base, and placed with the cut ends into a layer of 1.5 % agar. After a diffusion period of 24 hours, the seedlings were discarded and the agar frozen and stored until further use. For exudate collections, plants were grown in the greenhouse, under the conditions described above, in plastic containers with vermiculite, and were watered with half-strength Hoagland nutrient solution. The culture period was 6 weeks. When ready for exudate collection, a plant was cut at the base of the main shoot, the stump provided with a tightly fitting rubber tube, and the exudate collected in glass containers over a period of 24 hours. After collection, the exudate was frozen and then lyophilized. Each determination is based on exudate collected from 15 plants and totalling ca. 900 ml. GA Extraction from Diffusates and Exudates. The frozen agar from the diffusion treatments was allowed to thaw and was then extracted with methanol. The extract was filtered through a Buchner funnel, the methanol evaporated under vacuum, and the aqueous residue acidified to pH 2.7 and extracted 3 times with ethyl acetate. The ethyl acetate was dried with Na,SO4, reduced to dryness in a flash evaporator and taken up in a small volume of methanol for thin-layer chromatography. The dried exudate was dissolved in phosphate buffer, pH 8.2, partitioned 3 times against ethyl acetate, then acidified and further treated the same way as the diffusate extract. Chromatography and Bioassays. The final methanol solutions obtained from diffusates and exudates were streaked on Silica gel G plates and developed in chloroform :ethyl acetate :acetic acid (60:40:5), according to Sembdner et al. (17). The chromatogram fractions were eluted with water-saturated ethyl acetate, and the eluates reduced to dryness under vacuum and redissolved in 0.7 ml water. Subsequently, 0.2 ml of each fraction were tested by nieans of the barley half-seed a!-amylase test (12) and 0.4 ml by the lettuce seedling test (5). The barley used was the cultivar "Himalaya", the lettuce, '"Cabbage lettuce Arctic" ('Carter's Tested Seeds, Ltd., London). No attempt was made to identify the endogenous cucumber gibberellins chemically or bv fluorescence. Application of Exogenous GA. Short-term experiments were carried out with 3H-labeled GA1, prepared by Dr. H. Kende (13). The hormone was applied to the apices of 4-day-old, light-grown seedlings in 5-,ul droplets, the concentration being 20 ,pM. The number of seedlings used, diffusion time, and further handling including chromatography were as described above for ordinary agar diffusion. The dried chromatogram fractions were redissolved in water and an aliquot was counted in a scintillation counter (Packard Tri-Carb Model 2211). In long-term experiments, a mixture of GA4 and GA7 (approximately equal parts) at 10 ,zM in 0.05 % Tween was applied to plants grown in the greenhiouse, as 10-,ul droplets placed onto the youngest leaf, 3 times a week, starting with the first true leaf and continuing for a period of 6 weeks. Exudate was obtained and handled as described above and was tested for biological activity. The aqueous phases remaining after the ethylacetate extractions wvere not tested for activitv.