Lipid photosynthesis in olive fruit

Prog. Lipid Res. Vol. 33, No. I/2, pp. 97-104, 1994 Copyright ~ 1994 ElsevierScienceLtd Printed in Great Britain. All rights reserved 0163-7827/94/$24.00

Pergllmoll

LIPID PHOTOSYNTHESIS IN OLIVE FRUIT J U A N SANCHEZ

Instituto de la Grasa, C.S.I.C. Av. P. Garcia Tejero, 4, 41012 Sevilla, Spain

CONTENTS ABBREVIATIONS

I. INTRODUCTION II. THEOLIVEFRUIT A, Botanicalcharacteristics B. Fruit development IlL FRUITPHOTOSYNTHESIS IV. EFFECrOFLIGHT ON OLIVEGROWTH AND OIL FORMATION A. Influence of the canopy structure

B. Labellingstudies with tissue slices C. Labellingstudies with 14CO2 D. Effectof defoliation V. CONCLUSIONS AND PROSPECTS ACKNOWLEDGEMENTS REFFJtENCeS

97 97 98 98 98 99 100 100 101 101 102 103 104 104

ABBREVIATIONS TAG Triacylglycerols WAF Weeks after flowering

I. INTRODUCTION Olive (Olea europaea) was one of the first plants to be cultivated for the production of oil, there being records of its existence in the most ancient civilizations.I Its Latin name, olea, which derives from the Greek elaia, is the etymological root of oil, and the same root is recognized in most of the European languages to designate the same product. (The exceptions are the Spanish aceite and the Portuguese azeite, which derive from the Arabic al-zait, and reflect the strong influence of the long lasting Muslim domination of the Iberian peninsula.) Among vegetable oils, virgin olive oil is unique because it is extracted by using gentle physical procedures only, which results in a genuine fruit juice having excellent organoleptic and nutritional properties. The extraordinary dietary properties of olive oil are conferred by its well balanced fatty acid composition, of which oleate (the etymological root is also apparent here) is the main component, as well as the presence of minor components such as natural antioxidants and vitamins. These characteristics, as a whole, make olive oil a premium oil, and hence a product of the major economical importance for most of the Mediterranean countries, which are the main olive oil producers. In olive fruit, as in oilpalm fruit, triacylglycerols (TAG) are formed and stored in both the mesocarp and the seed. These two compartments display significant differences. First of all, storage T A G are synthesized in the seed with the purpose of nourishing the embryo during the initial stages of germination. On the contrary, in the mesocarp, storage TAG do not serve any physiological purpose. Instead they are formed and stored to attract animals, hence aiding seed dissemination. Such a selective advantage was first observed by 97

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Darwin: "But this (a fruit's) beauty serves merely as a guide to birds and beasts in order that the fruit may be devoured and the manured seeds disseminated".~ Moreover, the fact that TAG stored in the mesocarp are not utilized to nourish the embryo make them more amenable to manipulation of their fatty acid composition, since such fatty acids will not interfere with embryo development. Another important difference between olive seed and fruit lies in the fact that the latter, possessing active chloroplasts, has the capability of fixing atmospheric CO2 and incorporating it into storage material, whereas oilseeds depend completely on the import of photosynthates for the synthesis of storage TAG. This means that fruits occupy an intermediate position between heterotrophic tissues, like seeds, and autotrophic organs, like leaves. It is this aspect which is dealt with in this paper. II. THE OLIVE FRUIT A. Botanical Characteristics The olive is an evergreen tree reputed for both its rusticity and longevity, characteristics which are likely related to the extensive root system that it develops. Due to climatic constraints olive trees grow in temperate subtropical areas, 30° to 45 ° latitude, and are concentrated in the Mediterranean basin, where they constitute important elements of the landscape. Many cultivars, differing in height, resistance to drought and diseases, fruit quality, oil content, size, etc.) have been developed during the millennarian history of olive cultivation. The fruit is a drupe consisting of a fleshy pericarp (pulp) and a woody endocarp (stone) which encloses a single seed. The pericarp in turn consists of an outer epicarp, which is composed of a layer of small cells rich in chloroplasts, and an inner mesocarp composed of parenchymatous cells rich in oil, whose sizes increase radially from outside to inside. 4 The oil content of olives can reach over 30% (fresh weight) at the end of the maturation period. B. Fruit Development Olive trees bloom in the Spring, the exact date being related to the average daily temperatures during the previous vegetative season. Small whitish flowers are borne in inflorescences in the axils of the leaves. A period of 2-3 weeks is required to reach full bloom from the time the first flowers appear from the buds. This makes it difficult to define a sharp date of flowering in a particular grove or even within one tree. Fruit setting in olives is rather erratic; an alternate year fruit-bearing pattern, where a tree may set a heavy crop one year and not even bloom the next, is the rule in many areas, especially those where fertilization and irrigation are not practised. The development of olive fruit from anthesis to full maturity is a long process which lasts 35--40 weeks (Fig. 1). This is one of the longest growth periods among common commercial fruits. 5 During the first half of the developmental period, olive fruit increase their weight at more or less linear rates, so that some 25 weeks after flowering (WAF) they reach their final size. No oil deposition is observed during the first 10 WAF, either in the seed or in the pulp. The lignifieation of the endocarp is an important event which takes place between 10 and 12 WAF. This event marks the start of TAG synthesis and accumulation in both the seed and the pulp. Oil accumulation in the seed is relatively fast and is completed in about 10 weeks. In the pulp, on the other hand, the oil content increases more slowly and takes more than 20 weeks to reach a plateau. It is, however, a period of intense TAG synthesis: a rate of about 40 mg oil per fruit per week can be derived from the data in Fig. 1 during the period of maximal oil accumulation. Similar results have been reported previously.6,7 The ripening process, which starts some 30 WAF when the rate of oil accumulation is reaching a plateau, is characterized by a change in the colour of the fruit, which turn from

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Weeks After Flowering FIG. 1. Profile of 5pid accumulation in the pulp and seed of olive fruits along the developing period. Abbreviations: Lig, lignification of the endocarp; Rip, start of the ripening process.

green to purple and then black. It is of interest to emphasize that most of the T A G formation in olives takes place before the start of the ripening process, when the fruit is green and photosynthetically competent. III. F R U I T P H O T O S Y N T H E S I S

The most immediate observation is that many fruits are green during the longest part of their maturation period. It would be expected, therefore, that developing fruits are capable of CO2 fixation. Pioneering experiments by Bean and Todd s showed that detached developing oranges were able to fix ~4CO2into a variety of carbohydrates, amino acids and organic acids, and that the overall rate of incorporation of label was higher in the light than in the dark. The outer green peel (flavedo) was shown to be more active than either the inner white peel (albedo) or the juice vesicles when incubated in the light, but showed only minor differences when incubations were carded out in the dark. Interestingly, fruit incubated in the light incorporated most of the label into carbohydrates, mainly sucrose, whereas in the dark most of the label appeared associated with malate. Subsequent studies by the same group 9'~° showed that the photosynthetic activity of fruit declined during the developmental period, especially during the period of colour change, and that the rate of photosynthesis rarely exceeded the compensation point. It was concluded that the contribution of photosynthesis of fruit towards its own development was small. The high levels of C4 organic acids, especially malate, TM and enzyme activities related to C4 metabolism, particularly the high phosphoenolpyruvate carboxylase, malate dehydrogenase and malic enzyme, found in fruit in comparison to leaves, ~2-j4were the basis for the suggestion that photosynthesis of fruit was a special case of C4/CAM type of photosynthesis. However, discrepancies such as the lack of 'Kranz' anatomy and the absence of significant fluctuations in acidity, indicated the lack of C4 photosynthesis in fruit.~2 These discrepancies have been the basis for the suggestion that 'fruit photosynthesis' should be considered as a new type of photosynthesis, m5According to this concept fruits are considered to be highly heterotrophic, acting as strong sink organs; during the fruit growth, and due to both mitochondrial respiration of imported photosynthates and the

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impermeability of the cuticle, the fruit accumulates high levels of C02 in its free space. Thus, the role of the h i g h phosphoenolpyruvate carboxylase activity in the fruit would be to refix the respired CO2, which is stored in the vacuole in the form of organic acids, commonly malate, minimizing losses of CO2. During the light period malate can be mobilized through malic enzyme, and the resulting CO2 recycled through the Calvin cycle, in parallel to direct fixation of atmospheric CO2. Therefore, the role of photosynthetic carbon metabolism in fruit is the reassimilation of respired CO2.

IV. EFFECT OF LIGHT ON OLIVE GROWTH AND OIL FORMATION

A. Influence of the Canopy Structure Canopy structure refers to the organization of the above-ground plant material, including the size, shape and orientation of organs such as leaves, stems and fruit, and it determines the magnitude of light irradiance on the different plant organs, influencing their photosynthetic activities. Experiments carried out by Ortega-Nieto 16in Spain some 50 years ago showed that the average size and oil content of olives were related to their position in the canopy of the tree: the biggest olives were harvested from the upper part of the canopy, which is exposed to the highest irradiance, and these olives showed the highest oil content (Fig. 2). These studies, which were conducted in association with the effect of pruning and training on olive yield per tree, illustrate the important role of canopy structure on olive growth and oil formation, although they provide no information about the contribution of fruit photosynthesis to oil formation.

FIG. 2. Influence of the position in the canopy on fruit weight and oil content. (From OrtegaNietol~.)

Lipid photosynthesisin olive fruit

101

B. Labelling Studies with Tissue Slices Previous studies have shown that developing olives are able to incorporate the label from [~4C]acetate into TAG and other glycerolipids,~7'~s although the role of light was not directly investigated. First attempts to assess lipid photosynthesis in olives used [14C]acetate labelling in experiments carried out with tissue slices from pulp of maturing olives, t9 It was observed that the rate of incorporation of [~4C]acetate into TAG was higher in the light than in the dark. In addition, only the outer green epicarp of the fruit was found to be stimulated by light, whereas illumination did not cause any significant effect on the rate of TAG synthesis in the mesocarp. Similar results have been obtained by using [~4C]pyruvate as an alternative precursor of fatty acids, indicating that fatty acid synthesis in olives is light dependent. Both acetate and pyruvate are convenient precursors to study TAG formation, because they are efficiently channelled to fatty acid synthesis. However, more clear-cut results on the photosynthesis of lipids were to be obtained by using [t4C]bicarbonate as the labelled substrate. Tissue slices from developing olives were found to be capable of fixing [~4C]bicarbonate into both water-soluble products (carbohydrates, organic acids and amino acids) and glycerolipids, including TAG. 2° Glycerolipid synthesis from [~4C]bicarbonate was found to be highly dependent on the light and concentrated in the epicarp. :~ Moreover, nbulose-l,5-bisphosphate carboxylase was found to be concentrated in the epicarp, whereas phosphoenolpyruvate carboxylase was equally distributed between epicarp and mesocarp, indicating that the incorporation of [~4C]bicarbonate into lipids takes place through the Calvin cycle and was, therefore, directly linked to photosynthesis.

C. Labelling Studies with

14CO2

The use of tissue slices, although methodologically convenient, has the disadvantage of introducing disturbing factors into the results as a consequence of wounding metabolism elicited upon excising the tissue. The next approach was, therefore, to use whole fruits and 14CO2 as the radiolabelled precursor. When detached whole olives were exposed to 1aco2 in a closed chamber under illumination, the label was actively incorporated into lipids, with TAG being the main product (Table 1). The distribution of label among different acyl lipids was similar to that observed when tissue slices were incubated with labelled acetate 19 or bicarbonate. 2° When the epicarp was separated from the mesocarp at the end of the exposure period, it was found that the former accumulated most of the label, with the lipid pattern being similar in both tissues. No incorporation of label into seed lipids was observed in this kind of experiment with detached fruits. It should be noted that the rates of incorporation of ~4CO2 determined in these experiments are likely to be underestimated, due to the fact that radioactivity in the atmosphere of the sealed chamber decreased to half in the course of the incubation. On the other hand, levels of CO: above 1% have been measured in the atmosphere of the cell free space of olives used in these experiments (results not shown), and this endogenous CO2 would dilute the uptaken radiolabelled precursor. Nevertheless, results in Table 1 clearly show the ability of detached olives to fix atmospheric CO2 and TABLE1. In I/ivo Labelling of Detached Olives with 14CO2 Distribution of label (nmol/g) Incorporation Tissue (nmol CO2/g FW) TAG DAG PC OtherPLs Whole 25.44- 1.4 15.8 5.1 1.4 3.0 Mesocarp 8.5 4- 1.2 4.3 2.3 0.6 1.3 Epiearp 65.3 4- 6.9 39.6 13.1 4.3 8.3 Detached fruit harvested 21 WAF were exposed to t4CO2for 18 hr under illumination (4000Ix). Abbreviations: DAG, diacylglycerol;FW, fresh weight; PC, phosphatidylcholine;PLs, polar lipids. JPLR33/I-2--H

102

J. SANCI~Z TASTE 2. :Distribution

Conditions Autotrophic

Intermediate

of Label among Different Parts of the Plant after In Vivo Labellingwith I~CO2 Pulp Seed Leaf Fraction (nmol/mg Chl) (nmol/g FW) (nmol/mg Chl) Total 231 17 Aqueous 70 nd Lipid 161 nd

Total Aqueous Lipid

593 353 240

480 302 178

1006 813 193

Heterotrophic

Total 293 563 1192 Aqueous 262 335 992 Lipid 31 228 200 Material used in this experimentwas collected 12 WAF and exposedto t4CO2 for 18 hr. At the end of the exposurethe differenttissues were extractedwith hexane--isopropanol(3 : 2) accordingto Hara and Rading [Anal. Biochem. 90, 420-426 (1978)];radioactivitywas measuredin the total extract and then, after separation of phases, in the aqueous and lipid layers. See text for description of the exposure conditions. incorporate it into storage TAG, although they tell nothing about the relative contribution of fruit photosynthesis, as compared to leaf photosynthesis, to T A G formation in olives. As discussed above, fruits act as sink organs attracting photosynthates from adjacent leaves, but at the same time they are capable of fixing atmospheric CO2. Experiments have been carried out to try to assess the relative contribution of fruit and leaf photosynthesis to the formation of T A G in both the pulp and the seed of olives. Pieces of stem beating one fruit with or without its subtending leaf (the other being excised) were cut from apical branches and exposed to ~4CO2 as described previously. The experiments included three different treatments. In the first treatment olives, deprived of their subtending leaf, were kept under autotrophic conditions; in the second treatment, olives and their subtending leaves were exposed to 14CO2 under illumination, so that the fruit could both fix atmospheric CO2 and import photosynthates from the leaf, a situation which is close to physiological conditions in which the fruit share characteristics of both source and sink organs; in the third treatment, olives were maintained under heterotrophic conditions, because they were kept in the dark and depended on their subtending leaves for the import of photosynthates. Results in Table 2 show how lipid synthesis from ~4CO2in the pulp was mainly dependent on the light; the presence of the subtending leaf caused a 2.5-fold increase in the total radioactivity incorporated by the pulp in the light, but such an increase affected mainly the labelling of water-soluble products (5-fold increase), whereas the effect on lipid synthesis was smaller (1.5-fold increase). On the other hand, fruit maintained in the dark showed a remarkable decrease in the formation of lipids by the pulp, whereas radioactivity associated with water-soluble products was not affected to the same extent. These results indicated that fruit photosynthesis played a more than significant role in the formation of storage T A G by olive pulp. On the other hand, synthesis of lipids in the seed, as would be expected, was absolutely dependent on the presence of the subtending leaf, from which photosynthates were imported, and independent of light.

D. Effect of Defoliation Although defoliation experiments have shown that the growth of fruit can be sustained by leaves situated a considerable distance away, 5 there was a marked tendency for leaves close to a fruit to supply that fruit. 22 Once it was established that fruit photosynthesis plays a significant role in T A G synthesis by olive pulp, it was of interest to study the growth of olive fruit under conditions

Lipid photosynthesis in olive fruit

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TAItLB 3. Effect of Defoliation on Olive Growth and Oil Formation

Parameter Fresh weight (g) Oil content (% FW) Oil content (ms/fruit) Incorporation of [14C]acetate* Incorporation of [14C]bicarbonate* Incorporation of I~:~O2.

Control

Defoliated

3.05 19.8 495 27.1 + 0.4 10.3 + 0.7 0.51 4- 0.06

1.85 16.4 236 21.1 + 1.7 37.2 + 5.5 0.89 + 0.06

Defoliation was carried out on selected branches 4 WAF; fruit were harvested 26 WAF. *Results are in nmol/hr/g FW.

of low (or no) carbohydrate supply from the leaves, that is, under (quasi-)autotrophic conditions. To this aim, whole branches of olive trees were defofiated at an early stage of fruit development, and olives were allowed to develop until the onset of the ripening process. As shown in Table 3, fruit which developed on defoliated branches were clearly smaller than those which had grown on control branches having an average of 7-8 leaves per fruit. Moreover, determination of oil content showed that fruit from defoliated branches had accumulated about half the amount of oil measured in control fruit. These results indicated that leaves supply photosynthates to the fruit to make at least half of their storage TAG. Interestingly, when comparing biosynthetic activities of fruit by labelling experiments, it was found that fruit from defoliated branches were more active in incorporating the label from both [14C]bicarbonate and ~4CO2 into lipids in the light, whereas minor differences were observed by using ["C]acetate. These results suggested that fruit deprived of the suitable supply of photosynthates enhanced their photosynthetic capacities, thus allowing them to partially cope with the demands of the growth process. V. C O N C L U S I O N S

AND PROSPECTS

During the long history of olive cultivation, new cultivar varieties with high oil content have been developed through empirical breeding. Nowadays, the use of high yield varieties, together with good farming practices, allow olive production above, 10,000kg/ha, equivalent to about 2000 kg oil per hectare in good years, although such a figure is halved in the long term average due to the already mentioned alternate year fruiting of olive trees. However, there is still room for increasing the oil content of some cultivars that have adapted particularly well to their environment, and this could be achieved in a straightforward manner by genetic engineering. The results of the experiments described here demonstrate that developing olives are able to fix atmospheric CO2 into storage TAG, and that this process might contribute significantly to the overall TAG formation in the fruit. Therefore, any programme aimed at increasing or decreasing the oil yield of olives should take into consideration the photosynthesis of fruit as well as that of leaves. In this regard, it is of interest to mention the efforts currently being invested in the development of olive cultivars with low oil content, which have the potential to be marketed as low-calorie table olives ('light olives'), such as the Kadesh variety developed in Israel. 23 Future research should focus on the characterization of enzymes directly involved in fruit photosynthesis, in particular ribulose-l,5-bisphosphate carboxylase and phosphoenolpyruvate carboxylase. Results from this laboratory have shown that phosphoenolpyruvate carboxylase is an abundant enzyme in olive pulp, having a high affinity for bicarbonate. A more refined experimental protocol will be developed in order to determine the contribution of fruit photosynthesis to the overall TAG synthesis in olives. There also exists a notable lack of knowledge regarding the import of photosynthates from the leaves into the fruits. Published results indicate that mannitol serves as a translocable compound in olives, 24 whereas a more recent report has implicated the tetrasaccharide stachyose in the process. 25 More research should be carried out, therefore, to determine the nature of the incoming photosynthates into developing olives, and then to characterize the enzymes

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involved in the metabolic route leading to lipid formation. Such knowledge would provide useful hints on the target enzyme(s) to address when trying to manipulate oil content of olives, either to increase or to decrease it. Acknowledgements--The financial support of the Comisi6n Interministeriai de Ciencia y Tecnologia (CICYT) of Spain and the European Community (ECLAIR 209) is gratefully acknowledged. Thanks are also due to Dr Nora W. Lem for revising the manuscript.

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