Scientia Horticulturae, 36 (1988) 47-54 47 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

Ident i f icat ion and Seasonal Changes of Glucose, Fructose and Mannito l in Relat ion to Oil Accumulat ion during Fruit Deve lopment in Olea e u r o p a e a (L.)

MARIA WODNER, SHIMON LAVEE 1 and EPHRAIM EPSTEIN

Department of Olei- and Viticulture, Volcani Center, Bet Dagan (Israel) Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 1928-E. 1987 Series

(Accepted for publication 15 February 1988)

ABSTRACT

Wodner, M., Lavee, S. and Epstein, E., 1988. Identification and seasonal changes of glucose, fruc- tose and mannitol in relation to oil accumulation during fruit development in Olea europaea (L.). Scientia Hortic., 36: 47-54.

The amounts of glucose, fructose and mannitol, the main sugars in olive fruits, were determined in 'Kadesh', 'Uovo di Piccione' and 'Manzanillo' which have 8, 15 and 20% oil, respectively, at full black maturation. The sugars were separated by high-pressure liquid chromatography and char- acterized by ultraviolet detection after derivatization with nitrobenzoate. The total amount of sugar in 'Kadesh' was more than double that in the two other cultivars. A parallel rise in oil and sugar levels was noticed at the beginning of the fruit ripening period of 'Kadesh', while the sugar level of the other cultivars decreased during most of that period. Glucose was the predominant sugar in all cultivars, followed by fructose and mannitol. The relative amounts of mannitol were only 2% in 'Kadesh' and 10% in 'Manzanillo' and 'Uovo de Piccione'. It is suggested that mannitol levels and oil accumulation are related.

Keywords: mannitol; maturation; oil accumulation; olive fruit; sugar.

Abbreviations: DW = dry weight; FW = fresh weight.

INTRODUCTION

The relationship between oil and sugar content in the ripening olive fruit has not been studied extensively. Donaire et al. (1975, 1977) measured the

1Author to whom correspondence should be addressed at: Department of Horticulture, Volcani Center, Bet Dagan, P.O.B. 6, Israel.

0304-4238/88/$03.50 © 1988 Elsevier Science Publishers B.V.

48

total sugar content during the period of fruit maturation. Borbolla et al. ( 1955 ) and Fernandez-Bolanos et al. ( 1982, 1983 ) measured both total and individual sugars (glucose, fructose, sucrose and mannitol ), but only at the beginning and end of the fruit development period. Fernandez-Bolanos et al. (1982) also identified xylose and rhamnose in the fruit, but did not measure their quantities.

The olive 'Kadesh' is characterized by a very low oil content (Lavee, 1978). Even at full black maturation, the oil content of 'Kadesh' fruits did not exceed 8% on a FW basis vs. 20-22% in 'Manzanillo' and 14-16% in 'Uovo di Pic- cione'. In order to obtain a better understanding of the metabolic basis for this unusually low amount of oil, we decided to study the sugar content of the fruit throughout the growth and maturation process, and to determine its relation- ship to the oil content during this period. Up to 98% of the oil in olive fruits is located in the mesocarp and only 2-4% in the kernel. Depending on cultivar, 70-85% of all fatty acids in the olive is oleic acid its distribution on the tri- glyceride is at random. (Fedeli, 1977). The other major fatty acids reported in olive oil are palmitic 10-15%, stearic 2-3%, linoleic 5-10% and small amounts or traces of various acids from C14 to C22. In addition, some non-glyceric esters of the fatty acids, aromatic hydrocarbons, hydroxytriterpenes and many dif- ferent flavor compounds were identified. It has been established that the fatty acids, particularly oleic acid, are formed in the chloroplasts of leaves or pro- plastids of seeds (Stumpf, 1983). Acetyl co-enzyme A is the basic unit for fatty acid biosynthesis. The source of the acetyl units of the fatty acids in the plas- tids is not entirely known (Givan, 1983). While the olive oil accumulation occurs in the fruit, the precursors for its synthesis have to be imported from the leaves. The carbohydrates in the olive fruit pericarp evidently reflect the influx of metabolites from the leaves to the fruits and serve as a source for the acetyl needed for fatty acid biosynthesis, as demonstrated in castor seeds (Simcox et al., 1979 ). Thus, the changes in olive fruit sugar content were stud- led during the oil accumulation period to determine the quantitative correla- tion between the two processes. Special emphasis was put on the content of the sugar alcohols, which are a translocation form of carbohydrates (Lewis and Smith, 1967 ). The relationship between the three major sugars of the fruit (glucose, fructose and mannitol) and its oil content was studied in the low oil- producing 'Kadesh' in comparison with 'Uovo di Piccione' and 'Manzanillo'.

MATERIALS AND METHODS

Fruits were sampled from irrigated trees growing at the Lachish Experiment Station in the southern coastal plain of Israel. Samples were taken at 2-week intervals from early August to the end of November (full maturity) during 1984. Five-gram fresh-fruit mesocarp were used (taken from 10-15 fruits) to determine dry weight, oil and sugar content. For determination of oil content, dried samples were ground with an Ultra Turrax homogenizer (IKA Labor-

49

technik, W. Germany) in 1-chloronaphthalene and the refraction index of the filtered solution was read. Oil percentage was calculated from predetermined calibration curves (Lavee et al., 1988). Standard error was calculated from measurements of 4 independent samples for each cultivar on each date.

Sugars were extracted by grinding the samples with sufficient 95% ethanol to make a final concentration of 80% ethanol. After shaking overnight on a reciprocal shaker at 4°C, the supernatant was decanted and the residue was re-extracted twice with 80% ethanol. The supernatants were combined and evaporated to a small volume with a Buchi rotary evaporator at 40 ° C. Fatty materials were removed by shaking the extract with chloro- form:methanol:distilled water (13:3:4, v/v) (Hageman and Rinne, 1976) and the aqueous solution was evaporated to 10 ml. Each sample was shaken with 1 g of charcoal powder (Ebell, 1969 ), filtered with vaccum suction, and the char- coal residue washed 4 times with 25 ml distilled water. The filtrates were com- bined and evaporated in vacuo to 10 ml. Sugars were converted to their 4- nitrobenzoate derivatives (Nachtmann and Spitzy, 1976; Nachtmann and Budna, 1977). A 100-~l aliquot of the extract was freeze-dried, dissolved in pyridine, and the reagent 4-nitrobenzylchloride was added. The reaction was monitored by thin-layer chromatography, viewed under UV light and found to reach completion after 30 min. The derivatized sugars were separated using a Varian 5000 high-pressure liquid chromatograph equipped with a Rheodyne 7125 valve, and a 4.6 X 250 mm Merck Si60 column. Elution was made with a mixture of 55% n-hexane and 45% chloroform:dichloromethane:acetonitrile, 45:45:10, at a flow rate of 0.8 ml min-1 at 30 ° C. The eluant was monitored by a Varian UV-100 detector at a wavelength of 254 nm. Known concentrations of derivatized sugars were used as external standards for quantification of the individual sugars in the fruit extract.

To relate the oil and sugar content to the development and maturation of the fruit, which is influenced by environmental and cultivar differences, changes in fruit color have been included in all figures. This allows comparison of re- sults on the basis of physiological stages of development as well as on the basis of a calendaric date. Sugar determinations were made until full black ripening in each cultivar. The standard errors were calculated from measurements of 2 separate fruit samples and 2-3 independent derivatizations of each.

RESULTS

The initial sugar content in August, based on FW, was lowest in 'Kadesh' (Fig. 1 ). In this cultivar it increased during maturation before fruit color change, and dropped back thereafter to approximately its initial value. In 'Manzanillo' and 'Uovo di Piccione', the sugar content in August was high and decreased continuously until full maturation in November. A marked temporary increase

50

5 .~. Uovo

~I~ ~- ~ Manz

"~ 2

0 , i , ,

Auq. Sep. Oct. Nor.Dec.

Fig. 1. Sugar content during the maturation period of olive fruits as a percentage of FW from 3 cultivars. Open arrow = time of 50% black fruit; solid arrow = time of 100% black fruit.

20 ).,_ Uovo l~Manz "~ Kadesh ~

0 ' ,

Aug. 'Sep. ' Oct.' Nov.Dec.

Fig. 2. Oil accumulation during maturation of olive fruits from 3 cultivars on a FW basis. Open arrows = time of 50% black fruit; solid arrows = time of 100% black fruit.

70

60 ~

~ 50 e..,

~ 40

~ 30 o 2 0

I 0 J ~ Manz KacIesh

o Aug. Sep. Oct. Nor.Dec_

Fig. 3. Oil accumulation as a percentage of DW during the maturation of olive fruits from three cultivars. Open arrows = time of 50% black fruit; solid arrows--time of 100% black fruit.

w a s n o t i c e d in ' M a n z a n i l l o ' d u r i n g S e p t e m b e r a t t h e b e g i n n i n g o f f r u i t c o l o r

c h a n g e . N e a r l y 80% o f t h e oi l c o n t e n t w a s a c c u m u l a t e d b y t h e t i m e f r u i t s r e a c h e d

t h e 50% b l a c k s t age , a l t h o u g h q u a n t i t a t i v e l y t h e r e w e r e m a j o r d i f f e r e n c e s be -

t w e e n c u l t i v a r s (F ig . 2 ). A f t e r t h e h a l f - b l a c k s t age , oi l a c c u m u l a t e d a t a con - s i d e r a b l y s l o w e r r a t e in a l l 3 c u l t i v a r s .

51

The percentage oil content on a DW basis increased during the entire sam- pling period in all 3 cultivars (Fig. 3 ). The total dry material content, however, did not change during the sampling period in 'Kadesh' and increased only slightly in 'Manzanillo' and 'Uovo di Piccione'.

Of the three sugars determined, the level of glucose was highest, followed by fructose and mannitol (Fig. 4). In 'Uovo di Piccione', however, at full black maturation the level of fructose was the same or somewhat higher than that of glucose. In 'Kadesh', the glucose content increased to a peak before fruit color change, and then decreased to a final value of 1.9%. In 'Uovo di Piccione', the

0

3-4, ~ / /~ GLUCOSE

!

4 MANNITOL

3"

2-

I

Aug. Sep. Oct. Nov.Dec.

FRUCTOSE

Aug. 'Sep.' Oct.' Nov.'Dec

.~ Uovo di piccione

Manzonillo

Kadesh

Fig. 4. Amount of glucose, fructose and mannitol in olive fruit of 3 cultivars during the maturation period as a percentage of FW.

co : Auq. Sep. Oct "~ 80

60 .E 4o • Manmtol

[] Fructose 20 0 [] Glucose

Aug. Sep. Oct. Nov. Fig. 5. The relative amounts of mannitol, fructose and glucose during maturation of fruits from 3 cultivars as a percentage of total sugars.

52

initial glucose level was higher than in the other two cultivars, but decreased continuously to a final value of 0.6%. In 'Manzanillo', the glucose level de- creased during the sampling period to about the same final value as 'Uovo di Piccione'. However, it increased again in September at the beginning of fruit coloration, parallel to the temporary increase in total sugar content (see Fig. 1 ). The level of fructose remained relatively constant in 'Kadesh', whereas it decreased during fruit development in 'Manzanillo'. Fructose content in 'Uovo di Piccione' was inconsistent and variable. The mannitol content in 'Kadesh' was low during the whole fruit ripening period, while in 'Manzanillo' and 'Uovo di Piccione' the initial mannitol level was about 10 times higher and then it decreased to the level of that in 'Kadesh'.

The relative amounts of the three sugars in each cultivar are demonstrated by plotting the different sugars as a percentage of the total sugar content (Fig. 5). In 'Kadesh', glucose accounted for almost 70% of the total sugars through- out the fruit development period studied, while mannitol accounted for only a small fraction (2%) of the total. In the other cultivars, mannitol constituted almost 10% of the total amount of sugar, while glucose fluctuated between 40 and 60%. In 'Uovo di Piccione', the relative amount of fructose increased con- siderably during the advanced maturation period. This was particularly noted after the onset of color change in the fruit.

DISCUSSION

Oil accumulation increased in 'Manzanillo' and 'Uovo di Piccione' concom- itantly with a decline in total sugar content. A similar inverse relationship between oil and sugar in two Spanish cultivars ('Zorzaleno' and 'Gordal' ) was described by Borbolla y Alcala et al. (1955). In 'Kadesh', however, which is characterised by an extremely low oil content, oil and sugars accumulated si- multaneously through September; later the sugar content decreased as oil con- tinued to accumulate. Furthermore, while the amount of oil in 'Kadesh' was only one-third of that in the other cultivars, the amount of sugar was more than double and a sweet taste was noted in addition to the bitterness of the uncured fruits.

Analysis of the major sugars of the three cultivars up to full fruit color change showed that glucose was the predominant sugar, followed by fructose and man- nitol. Fernandez-Bolanos et al. (1983) also found glucose to be the major sugar in olive fruit of 'Manzanillo', 'Gordal', 'Hojiblanca' and 'Verdial'. They noted that fructose and mannitol were present in similar amounts.

In our study, however, only very small amounts of mannitol were found in all cultivars. Even during August, when the amounts were relatively high in 'Manzanillo' and 'Uovo di Piccione', they were still less than half the amounts of glucose and fructose in the same cultivars. In the low-oil-containing 'Ka- desh', the mannitol content was also very low in August. Furthermore, in this

53

cultivar the relative amount of mannitol, calculated as a percentage of the total sugar content, was considerably lower than in the other two cultivars. This was noted throughout the whole period of fruit development and oil accumulation. The fruit is fed by translocation of metabolites produced in the leaves. The accumulated reducing sugars which were found in the fruits (glucose and fruc- tose) are not translocated in the phloem of higher plants. It has been shown, however (Lewis and Smith, 1967), that in some plants the polyols serve as translocatable carbohydrates in addition to sucrose and other non-reducing oligosaccharides. Thus the mannitol in the olive fruit, as well as mannitol and other polyols in the fruits of many other plant groups (Bourne, 1958), might be of specific importance in the metabolic transformation and synthesis of the specific fruit storage material.

Furthermore, it has been shown in the Oleaceae (Trip et al., 1964) that mannitol is readily metabolized to C02 and translocated or converted to car- bohydrates (Trip et al., 1965 ). The total amount of sugars in the olive is rather low. Even 'Kadesh', which has a relatively high sugar content in comparison to the other olive cultivars, contains only 3% of sugars at maturation. Oil ac- cumulates in 'Kadesh' during fruit development as in the other cultivars, though at a very low rate, and reaches only a low level at full maturation. Thus the energy stored as oil and sugar in 'Manzanillo' and 'Uovo di Piccione' fruits at maturation is at least three times higher than that in 'Kadesh'. The major sugar in the olive cultivars was found to be glucose, which is the main sugar that is metabolised via acetyl CoA to oil (Stumpf, 1983 ). The high level of this sugar in 'Uovo di Piccione' and of fructose in 'Manzanillo' at the beginning of August, followed by their sharp decrease, might be responsible for the rapid oil accumulation in those cultivars. If that is so, the question remains why oil accumulation is small in 'Kadesh' although the glucose reaches its high level in the pericarp in September. This might be explained either by a genetically determined slow metabolism of glucose to oil in 'Kadesh', or by slower rates of translocation of the carbohydrates from the leaves to the fruits in this cultivar. This second hypothesis is primarily supported by the slow glucose accumula- tion, which reaches a peak 6 weeks later than in the other two cultivars. The first hypothesis, however, is supported by the relatively low mannitol concen- tration calculated on the basis of total sugars in 'Kadesh' fruits throughout their period of development. Since any analysis reflects a momentary situa- tion, and mannitol is not a final reaction product, we would expect to find a similar amount of translocated mannitol in the fruit at any given time. How- ever, if in 'Kadesh' the glucose is metabolized at a slower rate than in 'Man- zanillo', we would expect the relative amount of mannitol to be lower due to the slow reduction in the glucose level. The involvement ofpolyols as acceptors in the reduction processes of glucose (Lewis and Smith, 1967) would not affect the turnover rate, which can be detected only by the amount of the final reac- tion product.

54

I t is possible t h a t bo th the rates of ca rbohydra te t r ans loca t ion and glucose tu rnover are i m p o r t a n t in the process of oil accumula t ion in the olive fruits. T h u s the relative a m o u n t of mann i to l calculated f rom the tota l a m o u n t of sugars in the fruit migh t indicate the po ten t ia l of a cul t ivar for oil b iosynthesis . I t would be in teres t ing to tes t whe the r exogenous feeding of mann i to l would increase the oil con ten t of the olive fruit. I t is clear t h a t fu r ther research is needed, as conclus ions about oil b iosynthes is and different oil accumula t ion levels c anno t be based only on changes in the sugars and polyol composi t ion.

REFERENCES

Borbolla y Alcala, J.M.R., Fernandez Diez, M.J. and Gonzalez Pelliso, F., 1955. Cambios en la composicion de la aceituna durante su desarrollo. Grasas Aceites (Seville), 6: 5-22.

Bourne, E.J., 1958. The polyhydric alcohols. Acylic polyhydric alcohols. In: W. Ruhland (Editor), Encyclopedia of Plant Physiology. Vol. VI. Springer, Berlin, pp. 345-362.

Donaire, J.P., Sanchez-Raya, A.J., Lopez-Gorge, J. and Recalde, L., 1975. Metabolic changes in fruit and leaf during ripening in the olive. Phytochemistry, 14:1167-1169.

Donaire, J.P., Sanchez-Raya, A.J., Lopez-Gorge, J. and Recalde, L., 1977. Etudes physiologiques et biochimiques de rolive. I. Variation de la concentration de divers metabolites pendant son cycle evoluti£ Agrochimica, 21: 311-321.

Ebell, L.F., 1969. Variation in total soluble sugars of conifer tissues with methods of analysis. Phytochemistry, 8: 227-233.

Fedeli, E., 1977. Lipids of olives. Prog. Chem. Fats Other Lipids, 15: 57-74. Fernandez-Bolanos, J., Fernandez Diez, M.J., Rivas Moreno, M. and Gil Serrano, A., 1982. Azu-

cares y polioles en aceitunas verdes. II. Identification y determinacion cuantitativa por cro- matografia sobre papel. Grasas Aceites (Seville), 4: 208-211.

Fernandez-Bolanos, J., Fernandez Diez, M.J., Rivas Moreno, M., Gil Serrano, A. and Perez Rom- ero, T., 1983. Azucares y polioles en aceitunas verdes. III. Determinacion cuantitativa por cromatografia gasliquido. Grasas Aceites (Seville), 3: 168-171.

Givan, C.V., 1983. The source of acetyl coenzyme A in chloroplasts of higher plants. Physiol. Plant., 57: 311-316.

Hageman, M.E. and Rinne, R.W., 1976. Glucose, pyruvate and acetate metabolism by developing soybean seeds. Plant Cell Physiol., 17: 501-507.

Lavee, S., 1978. 'Kadesh' table olive. HortScience, 13: 62-63. Lavee, S., Wodner, M. and Avidan, B., 1988. A rapid refractometric method for determination of

the oil content in olive (Olea europea L.) Fruit. Adv. Hortic. Sci.: in press (in Hebrew, Has- sadeh, 68: 87-90).

Lewis, D.H. and Smith, D.C., 1967. Sugar alcohols (polyols) in fungi and green plants, New Phy- tol., 66: 143-184.

Nachtmann, F. and Budna, C.W., 1977. Sensitive determination of derivatized carbohydrates by high performance liquid chromatography. J. Chromatogr., 136: 279-287.

Nachtmann, F. and Spitzy, H., 1976. Ultraviolet derivation of digitalis glycosides as 4-nitroben- zoates for liquid chromatographic trace analysis. Anal. Chem., 48: 1576-1579.

Simcox, P.D., Garland, W., DeLuca, V., Canvin, D.I. and Dennis, D.T., 1979. Respiratory path- ways and fat synthesis in the developing castor oil seeds. Can. J. Bot., 57: 1008-1014.

Stumpf, P.K., 1983. Plant fatty acid biosynthesis - present status, future prospects. What's New Plant Physiol., 14: 25-28.

Trip, P., Krotkov, G. and Nelson, C.D., 1964. Metabolism of mannitol in higher plants. Am. J. Bot., 51: 828.

Trip, P., Nelson, C.D. and Krotov, G., 1965. Selective and preferential translocation of C14-1abeled sugars in white ash and white lilac. Plant Physiol., 40: 470.