Variations in carbohydrate and lipid content and in osmotic potential of watermelon cotyledons treated with benzyladenine

Plant Science Letters, 12 (1978) 199--207

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© Elsevier/North,HollandScientific Publishers Ltd.

VARIATIONS IN CARBOHYDRATE AND LIPID CONTENT AND IN OSMOTIC POTENTIAL OF WATERMELON COTYLEDONS TREATED WITH BENZYLADENINE

G I O V A N N A P. L O N G O , C L A U D I O P. L O N G O , G I A N F R A N C A V I T A L E and M A R Z I O PEDRETTI

ROSSI, A L E S S A N D R O

Centro di Studio del C.N.R. per la Biologia Cellulare e Moleeolare delle Piante, Universitd

degli Studi di Milano, Istituto di Scienze Botaniche, Via G. Colombo, 60, 20133 Milano (Italy).

(Received December 21st, 1977) (Revision received February 14tb, 1978) (Accepted February 14th, 1978)

SUMMARY Benzyladenine (BA)-stimulated growth of excised watermelon cotyledons is accompanied by a stimulation of the conversion of reserve lipids to soluble sugars. After the 3rd day of development most of the carbohydrate is found as starch in the controls and as soluble sugars in treated cotyledons. As a result of these two effects the amount of soluble sugars per cotyledon is always higher in BA-treated cotyledons.. Osmotic potential (measured as variation in fresh weight in presence of mannitol solutions or as osmolarity of cell sap) is, however, always lower in BA-treated cotyledons. These data suggest that BA increases the capacity of water uptake not only by build-up of solutes, but also by a direct influence on cell walls.

INTRODUCTION Exogenously-supplied cytokinins promote growth of cotyledons from several plant species [1--4]. Cytokinin-induced growth in cotyledons occurs mostly by cell enlargement [1] and necessarily involves a stimulation of water uptake. The greater capacity to take up water could be explained by the presence of a more negative osmotic potential in cytokinin-treated cotyledons. More negative osmotic potential could in turn depend on the conversion of insoluble

Abbreviation: BA, Benzyladenine.

200 storage material to soluble molecules. Some observations suggest indeed that mobilization of reserve material is accelerated by cytokinins. We have found that 0.1 mM benzyladenine (BA) enhances the activities of glyoxylate cycle enzymes in sunflower cotyledons [5]. A higher activity of these enzymes could possibly accelerate the conversion of lipids to soluble sugars. H u f f and Ross [6] have found indeed that treatment with zeatin raises the level o f reducing sugars in radish cotyledons. Direct evidence for a cytokinin-induced acceleration of fat-breakdown has however not been presented. This paper presents some data a b o u t the effect of BA on lipid and carbohydrate levels in excised watermelon cotyledons. We have also investigated whether changes in the breakdown of storage material induced by cytokinins can be correlated with changes in the osmotic potential of the cotyledons during development. MATERIALS AND METHODS

Plant material Watermelon (Citrullus vulgaris Schrad., var. Fairfax) seeds were bought from SRS seeds, Modesto, California. The cotyledons were excised from seeds soaked for 24 h and cultivated at 27°C in the dark on filter paper saturated with distilled water or with a 0.1 mM BA solution.

Determination of lipids and carboyhdrates Lipid content was measured following the m e t h o d of Bligh and Dyer as reported by Breidenbach [ 7]. Reducing sugars and starch were assayed colorimetrically as described by Marr~ [8].

Determination of water potential of cotyledons Cotyledons were incubated in Petri dishes on filter paper saturated with mannitol solutions of stepwise increasing concentrations ( 0 . 1 - 0 . 6 M). They were blotted and weighed after 90 min a time sufficient to attain equilibrium with the external solution. The concentration of the mannitol solution that did n o t induce a measurable variation in wt. was taken as an index o f the water potential of the cotyledons.

Determination of osmotic potential of cleared homogenates Determinations of osmolarity of cell sap were performed by determining the freezing point of the sap with an Halbmikro-Osmometer (Knauer). Batches of 3--10 cotyledons were thoroughly homogenized in small mortars w i t h o u t adding any liquid. An aliquot of the homogenate was centrifuged at t o p speed in a Beckman microcentrifuge for 5 mifi. A measured volume of the clear supernatant was carefully withdrawn with a syringe w i t h o u t disturbing the floating lipid layer. The supernatant was diluted with a known volume of bidistilled water before measuring osmotic pressure. Each experiment was repeated at least 3 times with 3 replicates.

201 RESULTS

Fig. 1 shows the effect of BA on increase in fresh wt. of excised watermelon cotyledons.Maximal stimulation of fresh wt. increase was obtained with a 0.1 mM concentration. This concentration was used in all of the following experiments. No significant changes in dry wt. were observed over the growth period considered, both in treated cotyledons and in water controls.

Lipid and sugar content. The lipid content of cotyledons gradually decreases in the 5 days following excision. This decrease is rather slow in the water controls: after 5 days 50% of the original lipid content is still present. Lipid depletion is greatly accelerated by BA: in treated cotyledons nearly all of the lipid has disappeared at the 5th day (Fig. 2). This stimulation by BA is evident after a lag phase of about 24 h. Electron microscopical observations clearly confirm the hormonal effect on lipid disappearance (Fig. 3 and 4). Reducing sugar content (per cotyledon) increases steadily during the first 5 days. This increase is enhanced by BA. At the 4th day of incubation in BA the amount of reducing sugars is nearly 3 times as high as in the water controls (Fig. 5A). Sucrose is present in considerable amounts already in the dry cotyledon. In the water controls sucrose content slowly increases until day 5. In BA-treated cotyledons the level of sucrose follows a more complex pattern: the initial drop during the first day is followed by a maximum at day 3 and by a !Fig. 1

Fig. 2

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Fig. 1. E f f e c t o f 0.1 m M B A o n t h e g r o w t h o f excised w a t e r m e l o n c o t y l e d o n s in t h e dark. 24 h o f i m b i b i t i o n ( t i m e o f e x c i s i o n o f c o t y l e d o n s ) were t a k e n as z e r o time. o o, w a t e r control; • 6, BA. Fig. 2. E f f e c t o f 0.1 m M B A o n t h e d i s a p p e a r a n c e o f reserve lipids in excised w a t e r m e l o n cotyledons, o o, w a t e r c o n t r o l ; • 6, BA.

Fig. 3. Low power view of cells from a water control cotyledon at day 4 after excision. Most of the field is filled with lipid bodies (L) and partially digested protein bodies (PB). Two plastids are indicated by arrows. (× 4000).

Fig. 4. Low power view of cells from a BA-treated cotyledon at day 4 after excision. Most of the lipid bodies (L) have disappeared. Protein bodies have been digested and large vacuoles (V) have taken their place. Some remainders of protein bodies (PB) can still be seen. Plastids (PL) arc the most conspicuous cytoplasmic organelles. (x 4200).

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Reducing sugars

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Fig. 5. E f f e c t o f 0.1 m M B A o n t h e c a r b o h y d r a t e c o n t e n t in excised w a t e r m e l o n cotyledons. ~ o, w a t e r c o n t r o l ; • e, BA. T h e level o f t h e d i f f e r e n t sugars in t h e dry seed ( n o t s h o w n ) is t h e s a m e as t h a t in t h e c o t y l e d o n a t z e r o time.

second decline. At day 5 sucrose has practically disappeared in the BA-treated cotyledons, at the same time when the maximal levels are reached in the water controls. In contrast to sucrose, starch is very scarce in dry watermelon seeds (the major reserves are lipids and proteins). During the first 3 days there is a parallel increase in starch content in the water controls and in the treated cotyledons; during the 2 following days the level of starch remains nearly constant in the water control while dropping to a very low level in the treated cotyledons (Fig. 5C). The difference in starch content after day 3 is confirmed by electron microscopical findings. At day 4 the plastids of the water control contain a high amount of starch (5--7 grains per plastid); in the treated cotyledons the plastids are larger and more numerous, but there is on the average 1 starch grain per plastid section (Fig. 6 and 7). All data about carbohydrates reported so far were expressed on a cotyledon basis. A quite different developmental pattern emerges if sugar content is related to fresh wt. so that water uptake during growth is taken into account. Fig. 8 shows developmental patterns of soluble sugars (reducing sugars + sucrose) related to fresh wt. (A) and to cotyeldon (B). If related to fresh wt. all values for soluble sugars are much lower in the treated cotyledons. Since the related to fresh wt. so that water uptake during growth is taken into account. uptake these values should be proportional to the effective concentration of soluble sugars in the cells. (At day 1, however, the difference in soluble sugar concentration between treated cotyledons and water controls cannot be accounted for by water intake since the fresh wt. is approximately the same for both groups of cotyledons).

Water potential of intact cotyledons and osmotic potential of homogenate. Measurements of soluble sugars alone should not be a reliable index for the

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Fig. 6. Plastids f r o m a w a t e r c o n t r o l c o t y l e d o n at day 4 a f t e r excision. M o s t o f t h e p l a s t i d ' s i n n e r space is filled w i t h s t a r c h granules. (x 29 500~.

Fig. 7. Plastids f r o m a B A - t r e a t e d c o t y l e d o n a t d a y 4 a f t e r excision. O n l y small s t a r c h grains are visible. (x 16 000).

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Fig. 8. Effect of 0.1 mM BA on the amount of soluble sugars per unit fresh wt. (A) and per cotyledon (B). o o, water control; a a, BA.

concentration of osmotically active solutes since other solutes (e.g. aminoacids derived from hydrolysis of storage proteins) are likely to be present. In spite of these facts measurements o f water potential agree fairly well with the observed concentrations of soluble sugars. This agreement can be appreciated by comparing the values for water controls and treated cotyledons in both sets of data (compare Fig. 8A and Table I). As shown in Table I water potential is always smaller (less negative) in the treated cotyledons with the exception of day 1 when both values almost coincide. Direct measurements of the osmotic potential of cotyledon homogenates c l o s e l y agree with the measurements of the water potential performed on the intact organs ( F i g . 9). The value of osmotic potential at day 1 is approximately the same in controls and treated cotyledons: in the following days its value gradually increases in the water controls and severely d e c l i n e s i n the treated cotyledons. TABLE I CONCENTRATION OF MANNITOL SOLUTION THAT DOES NOT INDUCE A MEASURABLE VARIATION IN FRESH WT. OF WATERMELON COTYLEDONS GROWN II~ BA OR IN WATER. Mannitol concentration (M) Day

1 2 3 4 5

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Fig. 9. Changes of intracellular osmotic potential in excised watermelon cotyledons grown in BA or in water for different days. o, water control; o, BA.

DISCUSSION

This is to our knowledge the first report showing that the effect of cytokinins on c o t y l e d o n growth is associated with accelerated depletion of lipid reserves (see also [9] ). Since BA also increases the level o f soluble sugars it is reasonable to assume that the increase in sugars is a consequence of accelerated fat breakdown. The glyoxylate cycle provides a well-known route for conversion of fat to sugars. Moreover, the a m o u n t of starch in the dry cotyledons is t o o low to account for the increase in sugar content. Since BA-treated cotyledons contain higher amounts of soluble sugars one could expect also more negative values for water potential in comparison to the water controls. However our data show that at any time of development both water potential of intact cotyledons and osmotic potential of sap are less negative in the treated cotyledons. This means that in presence of BA the increase in osmotically active solutes does not keep pace with the enhancement in the rate of water uptake. A dilution of solutes necessarily takes place. If stimulation of water uptake by BA had to depend entirely on the production of osmotically-active solutes one should expect that the two processes

207

should run parallel and water potential should remain constant. This has been indeed observed in the sunflower h y p o c o t y l [10]. The different behaviour of watermelon cotyledons suggest that in this case BA increases the capacity of water uptake n o t only by a build-up of solutes, but also by a direct influence on cell wall extensibility. Marr~ et al. [11] have shown that the stimulation of growth induced by BA in radish and squash cotyledons is correlated with an increase in the rate of proton extrusion. Proton extrusion is assumed to cause loosening of cell wall [12]. The possibility of wall loosening is supported by the finding of Halmer and Thorpe who observed that kinetin affects cell wall composition in tobacco callus cultures [ 13]. A plasticizing effect of BA on cell wall seems likely in our material since the water controls break easily when bent while the treated cotyledons bend without breaking. Unfortunately it will not be easy to analyze how BA affects the mechanical properties of cotyledons since this material is not easily amenable to measurements of stresses and deformations because of its unfavourable shape. REFERENCES

1 2 3 4 5 6 7 8 9 10 11 12 13

D.S. Letham, Physiol. Plant., 25 (1971) 391. M.E. Gordon and D.S. Letham, Aust. J. Plant Physiol., 2 (1975) 129. O. Servettaz, D. Castelli and C.P. Longo, Plant Sci. Lett., 4 (1975) 361. A. Narain and M.M. Laloraya, Plant and Cell Physiol., 11 (1970) 173. O. Servettaz, F. Cortesi and C.P. Longo, Plant Physiol., 58 (1976) 569. A.K. Huff and C.W. Ross, Plant Physiol., 56 (1975) 429. R.W. Breidenbach, in A. San Pietro (Ed.), Experimental Plant Physiology. The Mosby Company, Saint Louis, 1974, p. 108. E. Mart6, N. Giorn. Bot. Ital., n.s., 60 (1953) 914. G.P. Longo, M. Olginati, G.F. Rossi, M. Valente and C.P. Longo, Plant, Cell and Environment, 1 (1978) 39. D.L. Mc Nell, Aust. J. Plant Physiol., 3 (1976) 311. E. Marr6, R. Colombo, P. Lado and F. Rasi-Caldogono, Plant Sci. Lett., 2 (1974) 139. D.L. Rayle and R. Cleland, in A.A. Moscona and A. Monroy (Eds.), Current Topics in Developmental Biology, Academic Press, New York, 1977, p. 187. P. Halmer and T.A. Thorpe, Phytochemistry, 15 (1976) 1585.