Endogenous Gibberellins in Developing Fruits of Ziziph us mauritiana Lam. and Aegle marmelos Correa.

Endogenous Gibberellins in Developing Fruits of Ziziphus mauritiana Lam. and Aegle marme10s Correa. A. K. GHOSH, P. K. NAGAR and P. K. SIRCAR Plant Physiology Laboratory, Botany Department, University of Calcutta, 35, Ballygunge Circular Road, Calcutta-700019, India Received December 3, 1984 . Accepted April 11, 1985

Summary From immature young fruits of Ziziphus mauritiana and Aegle marmelos gibberellin-like factors were isolated. The factors a!, a2, a3, and <4 of Ziziphus were similar to gibberellins A!, A 3, A9, and ~. The factors XI, X2, X3 isolated from Aegle marmelos behaved similarly to gibberellins A!, A 3, and A9 on paper, thin layer, and silicic acid: celite column chromatographic techniques. In both fruits peak gibberellin activity occurred during the early period of fruit growth and decreased during rapid fruit growth. The possible significance of the finding is discussed in view of fruit development. Key words: Ziziphus mauritiana, Aegle marmelos, gibberellins, fruit development.

Introduction Growth substances in seeds and fruits may influence fruit growth. The active roles played by auxins, gibberellins, and cytokinins during fruit growth have been demonstrated in seeds and fruits of various species (Crane, 1969; Nitsch, 1970). The natural occurrence of hormones in fruits of Ziziphus mauritiana Lam. and Aegle mannelos Correa. have received considerably less attention than many other tropical fruits. Pramanik and Bose (1974) reported that exogenous application of GA3 can induce fruit set in the two species. These studies suggest that in these fruits naturally occurring gibberellins playa role in fruit development. In our previous papers cytokinins in fruits of Z. mauritiana (Ghosh et aI., 1981) andA. mannelos (Ghosh et aI., 1983) were studied. Reported here are the results of determination of gibberellin-like activity from the two fruits in relation to their growth and development.

Materials and Methods Ziziphus mauritiana Young immature fruits (1 kg) having the average diameter of 0.5 cm were used for qualitative study. To record the changes in gibberellin activity fruit samples from open-pollinated flowers were collected when the fruits were 10, 30, and 75 days old. At these periods the average diameters of fruits were 0.5 cm, 1.0 cm, and 1.5 cm, respectively.

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Fig. 1: Growth promoting activity as measured by lettuce hypocotyl test in the ethyl acetatesoluble acidic fraction of (A) Ziziphus mauritiana and (B) Aegle marmelos fruit extracts after paper chromatography in benzene: acetic acid: water (4:2: 1) solvent system. The acidic fraction after charcoal celite column chromatography, (C) and (D) 80 % acetone eluates of Z. mauri· tiana and A. marmelos after paper chromatography in the same solvent system. (E) and (F) 100 % acetone eluates of Z. mauritiana and A. marmelos after paper chromatography in the same solvent system. Broken and solid horizontal lines indicate control and LSD at 5 % level. Aegle marmelos

A large number of flowers were pollinated and tagged in the 2nd week of May. The resulting fruits were used for growth measurements (Gosh et aI., 1983) and for recording changes in endogenous gibberellin activity at 20 days intervals till maturity. The fruit samples for analysis of gibberellin activity were collected on the same dates when fruit diameters were measured. For qualitative determinations, a bulk sample of immature fruits (2 kg) was collected at the 40th day after pollination (av. diameter 2.4cm). Hormone extraction, purification, chromatography and bioassay

Fruits were blended with chilled 80% methanol in a Waring blender and the macerated tissues were re-extracted 5 times, each for 24 h at 3 °C with the same solvent. The methanolic extract was evaporated to an aqueous phase in vacuo, adjusted to pH 3.0 and the ethyl acetatesoluble acidic fraction was separated as described by Raja Rao and Nagar (1973 b). It was purified by a charcoal:celite (1:2) column (40x2.5cm). The extract, equivalent to 50g fresh weight of tissue, was chromatographed on the column, eluted with 100 ml of 80 % and 500 ml of 100 % acetone. Descending paper chromatography was mostly done on Whatman No. 1 paper strip (45 X 5 em) using benzene: acetic acid : water (4: 2: 1, v/v/v) as a solvent system. The charcoal: celite column eluates were further purified by silicic acid: celite column chromatography (Raja Rao and Nagar, 1973 a). For elution of the column chloroform (300 ml), ethyl acetate (200 ml), methanol (200 ml) and benzene (400 ml), acetonitrile (400 ml) and methanol (100 ml) systems described by Khalifa et aL (1963) were employed. The lettuce hypo-

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Plant Physiol. Vol. 120. pp. 381-388 (1985)

Gibberellins in developing fruits

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Fig. 2: Distribution of growth promoting activity as measured by lettuce hypocotyl test following silicic acid/celite column chromatography of 80 % acetone eluate of Zizipbus mauritiana in (A) chloroform gradient system and (B) benzene gradient systems. Same of Aegle marmelos in (C) chloroform gradient system and (D) benzene gradient system. Extract equivalent to 50g fresh weight of fruit tissue was chromatographed, the eluates were collected in 20 ml fractions and 10 ml of each fraction was bioassayed. Broken and solid horizontal lines represent same as in Fig.!. cotyl test (Frankland and Wareing, 1960) was u&ed to determine GA-like activity in column fractions or on paper chromatograms. The thin layer chromatography technique and sulfuric acid sprays described by MacMillan and Suter (1963) were used for detection of gibberellins. For the estimation of the gibberellin content (for Aegle marmelos), the ethyl acetate-soluble acidic fraction of each sample equivalent to 50 g fresh weight of tissue was chromatographed on a charcoal: celite column as described above. To estimate the total gibberellin content the fractions showing gibberellin activity were combined, dried in vacuo and tested for activity. The growth promoting responses of the extracts were evaluated by analysis of variance procedure and the means were compared using Hill and Wimble's method (1969).

Results Examination of the ethyl acetate-soluble acidic fraction of Z. mauritiana and A. marmelos fruits showed gibberellin-like activity on the paper chromatogram developed with the benzene: acetic acid: water solvent system. From each of the two types of fruits, two distinct zones of activity were evident. The zones of activity in the former showed Rr ranges at 0.0 - 0.1 and 0.7 - 0.9 (Fig. 1 A), in the latter at 0.0-0.2 and 0.8-0.9 (Fig. 1 B).

J. Plant Physiol. Vol. 120. pp. 381-388 (1985)

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Fig. 3: Distribution of growth promoting activity as measured by lettuce hypocotyl test foil"" ing silicic acid/ celite column chromatography of 100 % acetone eluate of Ziziphus mauritiana in (A) chloroform gradient system and (B) benzene gradient system. Same of Aegle marmelos in (C) chloroform gradient system and (D) benzene gradient system. Extract equivalent to 50 g fresh weight of fruit tissue was chromatographed and the eluates were collected in 20 ml fractions and 10 ml of each fraction was bioassayed. Broken and solid horizontal lines represent same as in Fig. 1.

Charcoal: celite column chromatography of the extracts and their elution with water, 80 % and 100 % acetone solutions showed growth-promoting activity confined only to two acetone eluates. Paper chromatography of the 80 % acetone eluate of Z. mauritiana extract showed one growth promoting zone at Rr 0.0 - 0.2 (Fig. 1 C) while in A. marme/os it was evident at Rr 0.0-0.1 (Fig. 1 D). The 100% acetone eluates of the two fruits also produced one growth promoting zone at Rr 0.8-1.0 (Fig. 1 E) and Rr 0.8-0.9 (Fig. 1 F). The growth-promoting zones of the 80 % and 100 % acetone eluates of the two fruits were chromatographed separately on silicic acid: celite columns using the chloroform and benzene gradient systems. The bioassay results show that the 80 % acetone eluate of Z. mauritiana (Figs. 2 A, 2 B) produced two gibberellin-like factors irrespective of the system used. The fractionation pattern of one (termed factor a2) was similar to that of GA3. In the case of A. marme/os (Figs. 2 C, 2 D) also two factors were evident and one of them (X2) had an elution pattern identical to that of GA3. The results obtained after silicic acid: celite column chromatography of the 100 % acetone eluate revealed the existence of three factors (a3' <4, and as) in Z. mauritiana (Figs. 3 A, 3 B) and only one factor (X3) in A. manne/os (Figs. 3 C, 3 D), irrespective of the gradient system used. Factors aJ, a2, a3, <4, and as of Z. mauritiana and Xi> X2, and X3 of A. marme/os were

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Gibberellins in developing fruits

Table 1: Rr values of the factors isolated from Ziziphus mauritiana and Aegle marmelos fruits and of some known gibberelins on thin layer chromatography developed in various solvent system. Factor

Isolated from Z. mauritiana al a2 a3 <4

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0.05 0.21 0.65 0.40

ND 0.19 0.19 0.15

0.53 ND ND ND

0.00 0.00 0.95 0.64

ND ND 0.95 0.67

ND ND 1.00 0.90

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0.45 0.38 1.00

0.00 0.08 0.66 0.90

0.47 0.52 0.94 0.95

0.11 0.14 0.35 0.82

0.07 0.21 0.38 0.70

ND ND 0.17 0.21

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0.23 0.16 0.87 0.98

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*) Systems (1) Benzene: acetic: water (8: 3: 5, upper phase); (2) Benzene: n-butanol: acetic acid (70: 25: 5); (3) Di-isopropyl ether: acetic acid (95: 5); (4) Chloroform: ethyl acetate: acetic acid (60: 40: 5); (5) Benzene: formic acid: water (8: 3: 5, upper phase); (6) Benzene: ethyl acetate (60: 40); (7) Carbon tetrachloride: acetic acid : water (8: 3: 5, lower phase); (8) system 7 + 10 % ethyl acetate; (9) system 7 + 20 % ethyl acetate. ND = Not done.

chromatographed separately on thin layer plates of silica gel G and Kieselguhr G in different solvent systems along with authentic samples of GAl, GA3, G~, and GA9 (Table 1). Factors aJ, a2, a3 and <4 produced fluorescence characteristics similar to the gibberellins AI, A 3, A 9, and A4 when treated with ethanol: sulfuric acid or water: sulfuric acid, and have similar Rr values. The factor as was in too small quantity to produce any fluorescence characteristics. Factors Xl, X2, and X3 have Rr values identical to those of GAl, GA 3, and GA9 (Table 1) and showed fluorescence characteristics similar to those of the above gibberellins.

Gibberellin changes during/ruit growth The data in Fig. 4 show that in Z. mauritiana gibberellin activities (in both 80 % and 100 % acetone eluates) were low at both the initial (Stage I) and mature (Stage ill) stages of fruit growth. The growth rate of A. marmelos was low during the first 20 days and high until 140 days. Beginning from 3 mm diameter the fruit reached its maximum size of 10 cm at the last stage of maturity (Fig. 5). On a concentration basis there was a peak in GA

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DAYS AFTER POLLINATION Fig. 5: Changes in the concentration 0.£ total gibberellins in Aegle marmelos fruit during its growth, as determined by growth promoting activity in the lettuce hypocotyl test. (-----.-----) activity ng/g of fresh wt. of tissue (--0--) activity ng/fruit, (--e--) average fresh weight in g.

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Gibberellins in developing fruits

387

content at 60 days which decreased rapidly and at the time of rapid fruit growth the gibberellin content was low. On a per fruit basis the gibberellin content reached maximum value at 100 days and then gradually decreased (Fig. 5). Discussion Four gibberellin-like factors were detected in the ethyl acetate-soluble fraction of Zizipbus mauritiana and three in Aegle marmelos fruit extracts. The factors at, a2, a3, and :l4 of the former may be the gibberellins AI, A3, A 9 , and ~ while Xt, X2 and X3 of the latter may be the gibberellins At, A3 and A 9 • Studies carried out on these factors showed that they possess properties similar to those of the above mentioned gibberellins in paper, thin layer, and silicic acid column chromatography systems. However, since the structural similarities between these factors and the said gibberellins have not been established, no definite conclusions can be drawn. At present more than 60 gibberellins are known to occur naturally and the chromatographic properties of some of them have not been studied in the systems employed in the present study. In the present study the endogenous gibberellins increased during the early stages of fruit growth and decreased during the period of rapid growth, especially in A. marmelos. Hence, no definite conclusion can be drawn regarding the involvement of these substances during the rapid fruit growth phase which involves mainly cell elongation. Gibberellins in conjunction with cytokinins have been implicated to play an active role in fruit development. It is of physiological interest to note that in Z. mauritiana andA. marmelos cytokinins (Ghosh et al., 1981; Ghosh et al., 1983) and gibberellins are present in relatively fair amounts during the early periods of fruit development. In many fruit species high levels of cytokinins and gibberellins do occur during the period of their active cell division (Crane, 1969) and in certain plant tissues they promote cell division (Wright, 1966). It has been conGluded (Nagar and Raja Rao, 1983) that in guava more cells might have been formed initially under the influence of high cytokinin and gibberellin content during the early stages of fruit growth whereas higher cell elongation might have taken place under the control of auxin. A similar explanation may perhaps be applicable for gibberellin changes observed in the present study. In both fruits gibberellin activity was evident even at the time of maturity (Figs. 4, 5). According to Goren and Goldschmidt (1970), a high gibberellin content at maturity in the peels of Citrus fruits could be due to the resumed growth activity. The low gibberellin activity is generally reflected by cessation of seed growth which is considered to be the chief centre of gibberellin production in fruit. However, in certain cases (Hayashi et aI., 1968; Nagar and Raja Rao, 1983) tissues of the fruit other than those in the seeds may also participate in gibberellin synthesis. Thus fruit growth may initially be regulated by gibberellins diffusing from seeds and at later stages the fruit may be self-sufficient by synthesizing its own gibberellins or importing them from other parts of the plant.

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A. K. GHOSH, P. K. NAGAR and P. K. SIRCAR

Acknowledgement We thank the Department of Science and Technology, Government of India and ICAR for financial assistance (HCS/DST/9/76), P.K.N. is grateful to CSJR, New Delhi for award of research associateship. Financial assistance from UGC, New Delhi to A.K.G. is gratefully acknowledged.

References CRANE, J. c.: The role of hormones in fruit set and development. Hort. Science 4, 108 -111 (1969). FRANKLAND, B. and P. F. WAREING: The effect of gibberellic acid on hypocotyl growth of lettuce seedlings. Nature 185, 255-256 (1960). GHOSH, A. K., P. K. NAGAR, and P. K. SIRCAR: Cytokinins in developing fruits of Ziziphus rnauritiana Lam. Science Horticulturae 14, 329-333 (1981). - - - A cytokinin complex from the developing fruits of Aegle marmelos. Physiol. Plant. 59, 165-170 (1983). GOREN, R. P. and E. E. GOLDSCHMIDT: Regulator systems in the developing citrus fruit: Hormonal balance in fruit tissues. Physiol. Plant. 23, 937 -947 (1970). HAYASHI, F., R. NAITO, M. J. BUKOVAC, and H. M. SELL: Occurrence of Gibberellin A3 in parthenocarpic apple fruit. Plant Physiol. 43, 448-450 (1968). HILL, T. A. and R. H. WIMBLE: A note on the precision of estimates of gibberellin concentration from regression lines calculated from bioassay data. Planta 87, 20-25 (1969). KHALIFAH, R. A., L. N. LEWIS, and C. W. COGGINS, Jr.: New natural growth promoting substances in young Citrus fruits. Science N.Y. 142, 399-400 (1963). MACMILLAN, J. and P. J. SUTER: Thin-layer chromatography of the gibberellins. Nature 199, 790-796 (1963). NAGAR, P. K. and T. RAJA RAo: Gibberellin-like substances in seedless guava (Psidium guajava L.) fruit. J. Hort. Science 56(4}, 339-343 (1981). - - Endogenous auxins in seeded and seedless guava. Scientia Horticulturae 18, 323 (1983). NITSCH, J. P.: Hormonal factors in growth and development. In: HULME, A. C. (ed.), The Biochemistry of fruits and their products, 427 - 472. Academic Press, London, 1970. PRAMANIK, D. K. and T. K. BOSE: Studies on effects of growth substances on fruit set and fruit drop in some minor fruits. South Indian Horticulture, 22, 117 -123 (1974). RAJA RAo, T. and P. K. NAGAR: Separation of gibberellins on silicic acid and celite columns. J. Exp. Bot. 24, 412-417 (1973 a}. - - Gibberellin-like substances in mature seeds of guava (Psidium guajava L.). J. Exp. Bot. 24, 418-424 (1973 b). WRIGHT, S. T. c.: Growth and cellular differentiation in the wheat coleoptile (Triticum vulgare) II. Factors influencing the growth response to gibberellic acid, kinetin and indolyl-3-acetic acid. J. Exp. Bot. 17, 165-176 (1966).

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