Lipid mobilization in Citrus cotyledons during germination

J. Plant Physiol. Vol. 140. pp. 1-7{1992}

Lipid Mobilization in Citrus Cotyledons during Germination P. GARcIA-AGUSTIN, M.]. BENACHES-GASTALDO,

and E.

PRIMO-MILLO*

Instituto Valenciano de Investigaciones Agrarias, Departamento de Citricultura, Apartado OficiaI46113-Moncada (Valencia), Spain Received July 29, 1991 . Accepted December 4, 1991

Summary

In the present work, lipid mobilization in C. limon (L.) Burm. f. cotyledons and the axial control of this process were studied. Two lipases were found in extracts from cotyledons: one with optimal activity at pH 5.0 (acid lipase) and another with a pH optimum between 7.5 - 8.0 (alkaline lipase). Both lipase activities showed a parallel pattern of evolution, increasing to a peak by the 16th day of germination. Most acid lipase activity was recovered in the fat layer obtained from crude extracts, and the alkaline lipase was located mainly in the glyoxysomes. The rate of lipid breakdown was lower in excised cotyledons when compared with intact ones. However, substantial hydrolysis of lipids occurs in the absence of the axis. In excised cotyledons, both acid and alkaline lipase activities were markedly reduced, indicating that the presence of the axis was necessary for maximum enzyme formation. Also, development of isocitrate lyase activity was partially inhibited by removal of the axis. The inhibition of isocitrate lyase activity observed when cotyledons were detached from the embryo cannot be explained by the source-sink model, as glucose and sucrose levels were lower in excised cotyledons than in intact ones. Incubation of excised cotyledons in gibberellic acid (10- 5 M) or kinetin (10- 4 M) solutions was effective in maintaining the development of lipase activities at similar levels than in intact cotyledons. Gibberellic acid (10- 5 M) produced a stimulation of isocitrate lyase activity in excised cotyledons, but not reaching the activity level developed by intact cotyledons. The data presented indicate that lipolysis in Citrus cotyledons seems to be only partially dependent on the axis. It is concluded that enzyme regulation in Citrus cotyledons cannot be entirely explained by hormonal control or source-sink relationships.

Key words: Citrus limon (L.) Burm j, axial·control, germination, lipid·mobilization. Abbreviations: GA3 = gibberellic acid; Kin = Kinetin; BA = Benzylaminopurine; AMO-1618 = 2-isopropyl-4-dimethylamino-5-methyl-phenyl-1-piperidinecarboxylate-methyIchloride. Introduction

Some seeds contain triacylglycerols as a major food reserve for germination. The storage triacylglycerols are localized in lipid bodies Oacks et aI., 1967; Ory et aI., 1968; Yatsu and Jacks, 1972). During early seedling growth, the triacylglycerols in the storage tissues are mobilized to support the growth of the embryonic axis (Muto and Beevers, 1974; Davies and Chapman, 1979 a, b; Lin et aI., 1982, 1983; Lin and Huang, 1983).

* To whom correspondence should be addressed. CS 1992 by Gustav Fischer Verlag, Stuttgart

The initial step in the mobilization of triacylglycerols is their hydrolysis to fatty acids and glycerol, which are then converted to sucrose by the gluconeogenic pathway (Hutton and Stumpf, 1969; Huang, 1975 a). The lipase activity that is responsible for the initial lipid hydrolysis has been described for some oil seeds. In castor bean seeds, an acid lipase is associated with the membrane of the lipid bodies, and the activity of the enzyme decreases sharply during germination (Ory et al., 1962, 1968; Ory, 1969). An alkaline lipase is associated with the membrane of the glyoxysomes and its activity is absent in the dry seed but increases markedly during germination (Muto and Beevers, 1974). It has been suggested that the two enzymes cooperate to hydrolyze the reserve triglyceride

2

P. GARciA-AGUSTIN, M. ]. BENACHES-GASTALDO, and E. PRIMo-MILLO

during germination. Huang and Moreau (1978) observed that the storage tissues of other oil seeds, namely peanut (Arachis hypogaea L.), sunflower (Helianthus annuus L.), cucumber (Cucumis sativus L.), cotton (Gossypium hirsutum L.), corn (Zea mays L.) and tomato (Lycopersicon esculentum Mill), contained only alkaline lipase activity, which increased during germination. In peanut cotyledons, most activity of the enzyme was found to be associated with the glyoxysomes. A glyoxysomal alkaline lipase has been also reported in cotyledons of soybean. This lipolytic activity was absent in the dry seeds and increased after germination, concomitant with the decrease in total lipids (Lin et aI., 1982). However, Lin and Huang (1983) reported the presence of lipase activity in the lipid bodies from the seed of rape (Brassica napus L.) and mustard (Brassica juncea). The lipase activity is absent in the ungerminated seed and increases in seedling growth. The lipase in the lipid-bodies from the scutella of corn was purified and characterized by Lin and Huang (1983). The purification and properties of alkaline lipase in the glyoxysomes from the endosperm of castor bean were reported by Maeshima and Beevers (1985). The regulation of lipid reserve mobilization has been subject of controversy, especially with concern to the role of the embryo or embryonic axis in the breakdown of lipid stores of oil seeds. Penner and Ashton (1967) suggested that the embryonic axis could control the level of glyoxylate cycle enzymes, notably isocitrate lyase in squash (Cucurbita maxirna) cotyledons. In the megagametophytic tissue of pine (Pinus ponderosa) the complete absence of the embryo reduced isocitrate lyase activity. In castor bean (Ricinus communis) cotyledons, isocitrate lyase and malate synthase activities were lower in excised material when compared with cotyledons obtained from intact seedlings (Tarpley and Choinski, 1986). In contrast, the embryo or embryonic axis appears to have no influence on the levels of isocitrate lyase and ~-oxidation enzymes in the endosperm of castor bean (Huang and Beevers, 1974; Marriot and Northcote, 1975 a, b; Tarpley and Choinski, 1986) or isocitrate lyase in soybean (Glycine max) and cucumber (Cucumis sativus) cotyledons (Slack et al., 1977). The major food reserves in Citrus seeds are lipids, which are stored in cotyledons (Braddock and Kesterson, 1973 a; Garda-Agustin and Primo-Millo, 1989). In the present work, aspects of lipid mobilization in Citrus cotyledons and in particular the axis control of this process are studied.

Material and Methods

Material Mono-embryonic seeds of Citrus limon were used in all experiments. Seeds were sterilized in 2 % commercial bleach for 5 min after removal of seed coats and rinsed three times with sterilized water. Germination was accomplished in 200 x 16 mm culture tubes containing 20 mL sterilized distilled water and seeds were maintained upon the upper surface of moistened filter paper. Culture tubes were maintained at 25°C and a light intensity of 40 J,1molm- 2 s- 1 during a 16h-day. In some experiments, cotyledons were detached from seedlings and were incubated under the same conditions as germinating seeds.

To test the effect of hormones on enzymatic activities in intact or excised cotyledons, distilled water was replaced by solutions of GA, or Kinetin (10-5, 10- 4 and 10-' M).

Lipids analysis Lipids were extracted from dry material with petroleum ether in a Soxhlet apparatus. Solvent was evaporated and the fat residue weighed.

Lipase assay One gram of fresh weight from cotyledons was homogeneized with 5 mL of grinding medium containing 0.4 M sucrose, 10 mM KCI, 1 mM EDTA, 10 mM dithiothreitol, 1 mM MgCl and 165 mM Tricine-NaOH buffer (pH 7.5). The homogenate was filtered through a layer of nylon cloth and centrifuged at 4000 g for 10 min. The pellet and residues on the nylon cloth were combined and rehomogenized with 3 mL of grinding medium. The two supernatants were combined and fractionated into fat layer, supernatant and particulate fractions by centrifugation at 10,000 x g for 30 min at 4°C. The fat layer and the particulate fraction were each suspended independently in grinding medium. Then, the fat layer, particulate fraction and the supernatant fraction were each recentrifuged. The resulting fat layer, particulate fraction and supernatant fraction were once again recombined and resuspended in grinding medium. These fractions were used as an enzymatic extract. Lipase activities were assayed by a colorimetric method (Duncombe, 1959; Muto and Beevers, 1974). For alkaline lipase determination, the reaction mixture in a final volume of 1 mL contained: 83 mM glycine-NaOH (pH 8.0), 5 mM dithiothreitol, 10 mM substrate and enzyme extract. To determine the activity of the acid lipase 83 mM citrate buffer (PH 5) was used in the reaction mixture. The fatty acids released were converted to copper soaps and measured using sodium dithiocarbamate as the color reagent. Emulsion of the substrate monopalmitin was prepared daily. For a typical emulsion preparation, 2.0 mL of 5 % gum arabic and an aliquot of the chloroform solution containing the substrate were emulsified for 2 min in an ultrasonic generator (Sowfer B-12). The emulsion was evaporated to remove all traces of chloroform. A standard curve of palmitic acid was used as a reference.

Sucrose density gradient centrifugation of the total homogenate This was carried out following the procedure of Lord et al. (1972), modified by Muto and Beevers (1974). Two hundred cotyledons (after 8 days from the start of germination) were homogenized by chopping for 15 min with a razor blade in 30 mL of the grinding medium described above. The crude extract was filtered through two layers of nylon cloth, and cell debris was removed by centrifugation at 2500 g for 10 min. Four mL of supernatant was layered onto a sucrose gradient consisting of 28 mL of sucrose solution increasing linearly in concentration from 30 to 60 % (w/w) over a 2 mL cushion of 60 % (w/w) sucrose, and topped with a 5 mL layer of 20 % (w/w) sucrose. All sucrose solutions were prepared in 1 mM EDTA (pH 7.5). Gradients were centrifuged for 4 h at 20,000 x g with a SW27 rotor in a Sorvall OTD-65B ultracentrifuge. After centrifugation 1.2 mL fractions were collected with an ISCO density gradient fractionator, Model 185. All steps were carried out at 0° to 4°C. Each fraction was assayed for lipase and isocitrate lyase activities.

Isocitrate lyase assay Homogenates were prepared by vigourously grinding batches of 2 g cotyledons with 2.5 mL of a solution containing 0.3 M sucrose

Lipid mobilization in Citrus cotyledons during germination 20

c:

15

0

"0 Q)

j1 '

~ 0 0

"''"E

10

1'-.

e~

9~1

~ ~

80

I

60

\

B

g

-'~T

~T

Aj

100

T

! ~ '"~

,

o

.

0___.

°

\

0

/.,

\. / '0 <>-" '0

40

3

e". .,

20

o+--+--+--+--+--+--+--+--+--+--+--+--+--+~

3

pH

o+-----+-----+-----+-----~ 10 20 o 15

Fig. 2: Changes in lipase activities in A) fat layer fraction (0--0) and B) particulate fraction (e--e) as a function of pH. Buffers used: citrate buffer pH: 4-6.5 and glycine-NaOH pH: 7 -9.

Days

Fig. 1: Changes in lipid content of Citrus cotyledons during germination. (0--0): attached cotyledons; (e--e): detached cotyledons. SD are given for n: 8.

until the second day after the start of imbibition. A rapid lipolysis occurred from day 2 to day 16 and during this period, about 58 % of total lipids were digested.

and 50mM sodium cacodylate, pH 7.2, grinding medium (Trelease et a!., 1971). After addition of a further 4 mL of grinding medium the crude homogenate was strained through 4 layers of muslin and centrifuged at 19,000 x g for 30 min. The middle aqueous layer, which was used for the enzyme assay, was carefully removed from between the floating fat and the pellet with a pipette. All steps were carried out between 0 - 4 dc. The glyoxylate phenylhydrazine method (Ford et ai., 1976) was used for estimation of isocitrate lyase activity.

Intracellular location of lipase activities The crude extract from cotyledons was separated into fat layer, supernatant and particulate fractions, and were analyzed for lipase activity at two different pH values (5 and 8). In Citrus cotyledons two separate lipases could be distinguished, one lipase with an acidic pH optimum whose activity was found principally in the fat layer and a second lipase found predominantly in the particulate fraction. The relationship between pH and the activities of the two enzymes is shown in Fig. 2. The enzyme in the fat layer shows optimal activity at pH 5.0 and in the particulate fraction shows an optimum at pH 7.5 - 8.0. The crude homogenate of the cotyledons was subjected to sucrose gradient centrifugation. The glyoxysomal fraction was located at a density of 1.25 g-l cm- J (indicated by the marker enzyme isocitrate lyase), while the lipid bodies floated on top of the gradient. Most of the acid lipase activity (78 %) was recovered in the fat layer fraction, whereas less than 25 % of the alkaline lipase activity was detected in this fraction. Within the gradient, the alkaline lipase activity exhibited a pronounced peak, corresponding to the glyoxy-

Carbohydrate analysis For carbohydrate analysis, 200 mg of lyophilized material was extracted three times with 20 mL boiling ethanol (80 %) and the homogenate centrifuged for 10 min at 4000 x g. Soluble sugars (glucose and sucrose) in the supernatant were determined by HPLC (Hewlett Packard 1084 B) using a Polygosil60-NH (10 IJ.m) capillary column. Acetonitrile (82.5% in water with a flow of 2.5 mL min-I) was used as the eluent.

Results

C'banges in lipid content of cotyledons during germination The rate of lipid breakdown is illustrated in Fig. 1. Lipid mobilization in cotyledons from intact seeds did not begin

1.12

.?;o

1.2>

1.11

.?;o

20

.,'>

~u

" 0

"

.S 0 -'" 0

0

""'0

15

Fig. 3: Enzyme distribution after separation of crude extract from Citrus cotyledons on a sucrose density gradient (after 8 days from the start of germination) a) Alkaline lipase activity and b) Isocitrate lyase activity.

~

15

~

~

E

'0

10

0

5


10

15

Fraction

10

.!!!

...I'.,.. v· - -. . .

.,"> 0

1.2>

1.11

0

0

"' g.

1.12

20

"

.~

0

~

\ 20

25


30

.••/\..\

0

10

15

Fraction

20

25

30

4

P. GARCiA-AGUSTIN, M. J. BENACHES-GASTALDO, and E. PRIMo-MILLO In contrast, maximum lipase activity at pH 8 was found in the particulate fraction. Alkaline lipase activity increased to a peak at day 16 (Fig. 5). The lowest lipase activities at either pHs 5 or 8 were found in the supernatant fraction and both declined slowly during germination. Both acid and alkaline lipase activities were present in the dry seeds (Figs. 4 and 5).

c

'E c

o

"z," o o

i1: "o

Tbe effect of the embryonic axis on lipid breakdown in cotyledons

E c

Days

Fig. 4: Changes in the acid lipase activity in Citrus cotyledons during germination. Activity in fat layer (6--6); particulate (0--0) and supernatant (0--0), at pH: 5.0. SD are given for n: 8. 10,----------------------,

c

o

"z," o o

'-

i1: "o

E c

10

20

30

40

Days

Fig.5: Changes in the alkaline lipase activity in Citrus cotyledons during germination. Activity in particulate (0--0), fat layer (6--6) and supernatant (0--0) at pH: 8.0. SD are given for n:8. somal fraction that showed the highest isocitrate lyase activity (Fig. 3). Two other minor peaks of alkaline lipase activity were also found at densities of 1.12 and 1.18g- 1 cm- 3, probably corresponding to the mitochondria and membrane fractions (Lord et al., 1972). These activities could be produced by contaminating glyoxysomes, since some residual isocitrate lyase activity was also detected in both fractions (Fig. 3).

Fig. 1 shows that the rate of lipid breakdown was lower in excised cotyledons when compared with tissue obtained from intact seedlings. However, substantial hydrolysis of lipid occurs in the absence of the axis. At 16 days from the start of imbibition, cotyledons that had been excised from the axis had consumed 24 % less lipid when compared with cotyledons from intact seedlings.

Tbe effect of the embryonic axis and honnones on enzyme activities in cotyledons Acid and alkaline lipase activities in excised cotyledons were lower than in the cotyledons from intact seedlings. At 15 days after the onset of germination, acid lipase activity from the fat layer and alkaline lipase activity from the particulate fraction were reduced about 30 % and about 45 %, respectively, when comparing detached cotyledons with intact ones (Tables 1 and 2). When excised cotyledons were incubated in 10- 5 M GA3 or 10- 4 M kinetin, both lipase activities were restored almost to the same level as in cotyledons from the whole seedling. When intact cotyledons were incubated in GA3 or kinetin, no significant increases in both lipase activities were observed (Tables 1 and 2). Examination of isocitrate lyase activity during germination indicated that the presence of the axis was a necessary Table 1: Effects of hormones on the acid lipase of isolated C limon cotyledons (nmol FFA/cotyledon min) (Fraction: fat layer). An ANOVA was done in each experiment. Different letters designate significant differences found among values of the same column resulting from a contrast test p:s:; 0.05. All analytical values are the mean of four different experiments.

Attached cotyledons

Changes in lipase activities during germination Lipase activities were determined during germination at pHs 5 and 8 in the fat layer, supernatant and particulate fractions separated from the crude extract of cotyledons during germination. When lipase was assayed at pH 5, the activity increased markedly in the fat layer up to 16 days after the start of imbibition and then decreased. Acid lipase activity in the particulate fraction was lower than in the fat layer, also reaching a peak at day 16 (Fig. 4).

Detached cotyledons

Days Control GA3 10- 5 GA3 10- 4 GA3 10- 3 Kin 10- 5 Kin 10- 4 Kin 10- 3

5 3.7a 3.9a 4.1 a 3.6a 3.6a 3.8a 3.7 a

10 4.5a 4.8a 4.6a 4.4 a 4.3a 4.3a 4.5a

15 7.4 a 7.8 a 7.7 a 7.3a 7.2a 7.5 a 7.0a

Control G A3 10- 5 GA3 10- 4 GA3 10- 3 Kin 10- 5 Kin 10- 4 Kin 10- 3

2.1 b 3.5a 3.5 a 3.4a 2.4b 3.6a 3.4a

2.3b 4.3a 4.1 a 4.1 a 2.7b 4.2a 4.0a

5.2b 7.1 a 7.0a 6.8a 5.4 b 6.9a 6.7 a

Lipid mobilization in Citrus cotyledons during germination Table 2: Effects of hormones on the alkaline lipase of isolated C. li· mon cotyledons (nmol FFAIcotyledon min) (Fraction: Particulate). An ANOVA was done in each experiment. Different letters designate significant differences found among values of the same column resulting from a contrast test p:!S; 0.05. All analytical values are the mean of four different experiments.

Attached cotyledons

Detached cotyledons

Days Control GA3 10- 5 GA3 10- 4 GA3 10- 3 Kin 10- 5 Kin 10- 4 Kin 10- 3

5 3.4a 3.3a 3.6a 3.2a 3.0a 3.2a 3.1 a

10 6.1 a 6.3a 6.2a 5.9a 5.8 a 6.2a 5.7 a

15 8.5a 8.9a 8.6a 8.2 a 8.4a 8.1 a 8.0a

Control GA3 10- 5 GA3 10- 4 GA3 10- 3 Kin 10- 5 Kin 10- 4 Kin 10- 3

1.8b 3.2a 3.1 a 2.9a 1.9b 3.1 a 3.0a

3.2b 6.0a 6.2a 5.8 a 3.8b 5.6a 5.6a

4.7b 7.8a 7.9a 7.5a 4.9b 7.3a 7.7 a

of the embryonic axis the amount of sucrose and glucose increased up to the 8th and 12th days of germination, respectively, and then decreased. This was most probably due to translocation to the developing embryo. In excised cotyledons sucrose and glucose contents were lower than in intact tissue but their levels increased during incubation (Fig. 7).

Discussion The data show that two different lipases, one with optimal activity at pH 5 (acid lipase) and another with pH optimum between 7.5 - 8.0 (alkaline lipase), seem to be present in the cotyledons of Citrus during the period of germination and early seedling growth. Two lipases appear also in the endosperm of castor bean seeds (Muto and Beevers, 1974) but in other oil seeds the acid lipase is absent and only the alkaline lipase activity increased during germination (Huang and Moreau, 1978). In Citrus cotyledons both acid and alkaline lipase activities showed a parallel pattern of evolution, increasing to a peak

Table 3: Effects of hormones on the isocitrate lyase activity of isolated C. limon cotyledons at 8 days after imbibition (!lmol glyoxylatel cotyledon min). An ANOVA was done in each experiment. Differ· ent letters designate significant differences found among values of the same column resulting from a contrast test p:!S; 0.05. All analytical values are the mean of four different experiments. Control GA3 10- 5 GAJ 10- 4 GAJ 10- 3 Kin 10- 5 Kin 10- 4 Kin 10- 3

Attached cotyledons 4.4 a 4.7 a 4.5a 4.3a 4.3a 4.5 a 4.1 a

c:

'E c:

o

"0 Q)

~ o o

'., -0

Detached cotyledons 1.9b 3.1 c 2.9c 2.9c 2.0b 2.3 b 2.1 b

prerequIsite for maximum enzyme development, which showed a peak at 8 days from the start of germination. However, development of isocitrate lyase activity was partially inhibited in excised cotyledons (Fig. 6). Incubations of excised cotyledons with GA3 (10- 5 M) as optimum concentration stimulated the development of isocitrate lyase activity. At this concentration a peak of maximum activity was found at 8 days of germination (Fig. 6). However, the maximum activity reached by excised cotyledons incubated in GA3 (10- 5 M) was lower than that in cotyledons from intact seedlings (Fig. 6). Isocitrate lyase activity was not significantly affected by incubating intact cotyledons in GA3 or Kinetin solutions (Table3).

The effect of the embryonic axis on soluble sugar content in cotyledons The sucrose and glucose contents during germination and early seedling growth are illustrated in Fig. 7. In the presence

5

~ o

b

'"

(5

E

" 5

10

15

Days

Fig. 6: a) Changes in isocitrate lyase activity in Citrus cotyledons during germination. (0--0) attached cotyledons; (e--e) de· tached cotyledons and (6--6) detached cotyledons with GA3 (10- 5 M). SD are given for n : 8.

80

a

/o~?

0

~

60

~ co 0

"~"

4ll

0

/

" '-

'"

E

"-

V~

/e

20

a

~o

b

a

10

15

20

a

10

15

20

Days

Fig. 7: Changes in soluble sugar content of Citrus cotyledons during germination. a) (0--0) sucrose in attached and (e--e) de· tached cotyledons. b) (0--0) glucose in attached and (e--e) detached cotyledons. SD are given for n: 8.

6

P. GARCiA-AGUSTIN, M.

J. BENACHES-GASTALDO, and E. PRIMo-MILLO

by the 16th day of germination, concomitant with the decrease in total lipids. Ho~ever, in castor bean endosperm, the acid lipase was most active at an early stage of germination when storage lipids were not being utilized. The activity of the alkaline lipase increased later when fat was being utilized and declined as the lipids were depleted (Muto and Beevers, 1974). In Citrus cotyledons, most acid lipase activity was found in the floating fat layer obtained from crude extracts. The acid lipase of castor bean seeds was also localized in the membrane of the lipid bodies (Ory et aI., 1968; Muto and Beevers, 1974). Other plants such as rape and mustard (Lin and Huang, 1983) showed lipase activity in the lipid bodies of the cotyledons. The alkaline lipase activity associated with glyoxysomes detected in Citrus cotyledons has been also reported in several other oil seeds (Muto and Beevers, 1974; Huang and Moreau, 1978; Lin et aI., 1982). In general, this lipolytic activity increased during germination, as has been observed in Citrus cotyledons. With respect to the control of lipid mobilization in Citrus cotyledons, it has been shown that maximum lipolysis occurs in the presence of the axis. If the axis is removed, an important breakdown of lipids occurs, but less than when the axis is present. A similar situation was reported by Slack et al. (1977) in cucumber cotyledons. In this case the axis and the testa controlled the rate of lipid mobilization. Examination of lipase activities in Citrus cotyledons during germination indicated that the presence of the axis is necessary for maximum enzyme development. In excised cotyledons both acid and alkaline lipase activities were markedly reduced. Isocitrate lyase, one of the key enzymes of the glyoxylate cycle, increased its activity in intact cotyledons of Citrus during germination. However, removal of the axis partially inhibited enzyme development in the cotyledons. Penner and Ashton (1967) reported that isocitrate lyase formation in squash cotyledons was enhanced by the axis. However, Ford et al. (1976) pointed out that declining enzyme activity in excised squash and cucumber cotyledons may simply be the result of decreased oxygen supply to partially submerged seed parts. However, this cannot explain the data presented here, as precautions were taken to ensure that isolated cotyledons had equal access to oxygen and water as cotyledons attached to the axis. In the megagametophytic tissue of pine seeds the development of isocitrate lyase activity during germination was inhibited in the total absence of the embryo (Bilderback, 1974). In contrast, the developing embryo appeared to have no influence on the levels of isocitrate lyase in the endosperm of castor bean (Huang and Beevers, 1974; Marriot and Northcote, 1975 a, b), soybean cotyledons and cucumis cotyledons (Slack et al., 1977). Axial control of enzyme activity could be accomplished by two possible mechanisms, i.e. (1) source-sink effects and (2) hormonal control. In the first case, the axis might control food mobilization by acting mainly as a sink, and regulation of enzyme activity within the cotyledons could be based upon an enzyme/product feed-back inhibition relationship (Chapman and Davies, 1983). The source-sink hypothesis is supported by the observation that glucose and sucrose, the

end products of the oxidation of fatty acids, inhibited the isocitrate lyase activity in excised cotyledons of cucumber and castor bean (Lado et aI., 1968; Tarpley and Choinski, 1986). The inhibition of isocitrate lyase activity by sucrose in excised castor bean cotyledons was overcome when half seed, i.e. cotyledon with endosperm, were incubated in sucrose (Tarpley and Choinski, 1986) and it is suggested that the endosperm may be acting as a sink for sugars. A criticism of the source sink control hypothesis has been the observation that some lipolysis occurred in cucumber cotyledons even in the absence of the embryonic axis (Davies and Chapman, 1979 a). This effect is probably a consequence of the formation of a secondary sink in which excess sugars are used to form starch. Excised cucumber cotyledons accumulate starch and probably represent a mechanism in the tissue that regulates the concentration of sucrose and glucose (Chapman and Galleschi, 1985). The inhibition of isocitrate lyase activity observed when Citrus cotyledons were detached from the embryo cannot be explained by the source-sink model, as glucose and sucrose levels were lower in excised cotyledons than in intact ones. An alternative to the source-sink hypothesis is the hormonal-control model. In this case, the hormones produced by the embryonic axis would be transported to specific target cells in the seed, inducing the secretion of the reserve-mobilizing enzymes. The conditions required to demonstrate the hormonal control of the mobilization of seed reserves are: a) hydrolytic enzyme activities should remain at lower levels in axis-less-seeds, and b) the effect of the embryonic axis should be replaceable by plant growth regulators. In Citrus cotyledons lipase activities were reduced to some extent in the total absence of the embryo. Also, development of isocitrate lyase activity was partially inhibited in excised cotyledons. Incubations of excised cotyledons in GA3 or Kinetin solutions were effective in maintaining the development of lipase activities at similar levels than in intact cotyledons. Gibberellic acid (10- 5 M) produced a stimulation of isocitrate lyase activity in excised cotyledons, but did not reach the activity level developed by intact cotyledons. In castor bean endosperm, exogenously applied GA3 stimulates isocitrate lyase activity (Marriot and Northcote, 1975 a, b, 1977; Gonzalez and Delsol, 1981; Choinski, 1982). In addition, AMO-1618 inhibited the development of this activity when applied to dry seeds (Choinski, 1982). However Martin and Northcote (1982) demonstrated that the stimulation of isocitrate lyase activity by the application of GA3 to germinating castor bean seeds was not specific and may be due to the action of GA3 in increasing the overall production of rRNA and mRNA, which accelerates the rate of total protein synthesis during germination. The application of AMO1618 retards the development of isocitrate lyase activity but also retards protein synthesis as a whole. In previous work we observed that protein degradation occurs at a high rate in cotyledons of germinating Citrus seeds (Garda-Agustin and Primo-Millo, 1990). The removal of the embryonic axis accelerated protein breakdown in excised cotyledons (GardaAgustin et al., 1991). It is therefore possible that the effect of exogenously applied hormones on stimulation of lipases and isocitrate lyase in excised Citrus cotyledon is non-specific

Lipid mobilization in Citrus cotyledons during germination

and hormones would act regulating the rate of protein synthesis and degradation in the tissue as suggested by Martin and Northcote (1982). The data presented here indicate that lipolysis in Citrus cotyledons seems to be only partially dependent on the axis and is not entirely consistent with the source-sink or the hormonal-control models for reserve mobilization in seeds.

References BILDERBACK, D. E.: The regulatory role of the embryo on the development of isocitrate lyase activity during germination of Ponderosa Pine seeds. Physio!. Plant. 31, 200-203 (1974). BRADDOCK, R. J. and J. W. KESTERSON: Citrus seed oils. Florida Agr. Expt. Station, Gainesville. Tech. Bull. No. 756. 30 pp. (1973 a). CHAPMAN, J. M. and H. W. DAVIES: Control of the breakdown of food reserves in germinating dicotyledonous seeds: a reassessment. Annals of Botany 52, 593-595 (1983). CHAPMAN, J. M. and L. GALLESCHI: The control of food mobilization in seeds of Cucumis sativus L. VI. The production of starch. Annals of Botany 55, 29-34 (1985). CHOINSKI, J. S. Jr.: Effect of AMO-1618 on glyoxysomal enzyme activity and sterol synthesis in castor bean endosperm. J. Plant Growth. Regu!. 1,227 -242 (1982). DAVIES, H. V. andJ. M. CHAPMAN: The control of food mobilization in seeds of Cucumis sativus L. I. The influence of embryonic axis and testa on protein and lipid degradation. Planta 146, 579-84 (1979 a). DAVIES, H. V. and J. M. CHAPMAN: The control of food mobilization in seeds Cucumis sativus L. II. The role of the embryonic axis. Planta 146, 585-90 (1979 b). DUNCOMBE, W. G.: The colorimetric determination of long-chain fatty acids in the 0.05-0.5lJ.mole range (Abstr.). Biochem. J. 83, 6 (1959). FORD, M. J., P. SLACK, M. BLACK, and J. M. CHAPMAN: A reexamination of the reputed control of cotyledonary metabolism by the axis. Planta 132, 205-208 (1976). GARciA-AGUSTIN, P., M. J. BENACHES, and E. PRIMO-MILLO: Control by the embryo axis of the breakdown of storage proteins in cotyledons of germinating seeds of (Citrus limon (L.). Burm f. Journal of the Science of food and agriculture (1991), in press. GARciA-AGUSTIN, P. and E. PRIMo-MILLO: Ultrastructural and biochemical changes in cotyledon reserve tissues during germination of Citrus seeds. J. expo Bot. 40 (212),383 -390 (1989). - - Changes in some nitrogenous components during the germination of Citrus seeds. Scientia Horticulturae 43, 69 - 81 (1990). GONZALEZ, E. and M. A. DELSOL: Induction of glyconeogenic enzymes by gibberellin A in endosperm of castor bean seedlings. Plant Physio!. 67, 550-554 (1981). HUANG, A. H. c.: Enzymes of glycerol metabolism in the storage tissues of fatty seedlings. Plant Physio!. 55, 555 - 558 (1975 a). HUANG, A. H. C. and H. BEEVERS: Developmental changes in endosperm of germinating castor bean independent of the embryonic axis. Plant Physio!. 54, 277 -279 (1974). HUANG, A. H. C. and R. A. MOREAU: Lipases in the storage tissues of peanut and other oil seeds during germination. Planta 141, 111-116 (1978). HUTTON, D. and P. K. STUMPF: Characterization of the {3-oxidation systems from maturing and germinating castor bean seeds. Plant Physio!. 44, 508-516 (1969).

7

JACKS, T. J., L. Y. YATSU, and A. M. ALTSCHUL: Isolation and characterization of peanut spherosomes. Plant Physio!' 42, 585-597 (1967). LADO, P., M. SCHWENDIMANN, and E. MAli: Repression of isocitrate lyase synthesis in seeds germinated in the presence of glucose. Biochim. Biophys. Acta 157, 140-148 (1968). LIN, Y. H. and A. H. C. HUANG: Lipase in lipid bodies of cotyledons of rape and mustard seedlings. Archives of Biochemistry and Biophysics 225, 360-369 (1983). LIN, Y. H., R. A. MOREAU, and A. H. C. HUANG: Involvement of glyoxysomal lipase in the hydrolysis of storage triacylglycerols in the cotyledons of soybean seedlings. Plant Physio!' 70, 108-112 (1982). LIN, Y. H., L. T. WIMER, and A. H. C. HUANG: Lipase in the lipid bodies of corn scuttella during seedling growth. Plant Physio!. 73,460-463 (1983). LORD, J. M., T. KAGAWA, and H. BEEVERS: Intracellular distribution of enzymes of the cytidine diphosphate choline pathway in castor bean endosperm. Proc. Nat!. Acad. Sci. U.S.A. 69, 2429-2432 (1972). MAESHIMA, M. and H. BEEVERS: Purification and properties of glyoxysomal lipase from castor bean. Plant Physio!. 79, 489-493 (1985). MARRIOT, K. M. and D. H. NORTHCOTE: The breakdown of lipid reserves in the endosperm of germinating castor beans. Biochem. J. 148,139-144 (1975 a). -

- Induction of enzyme activity in the endosperm of germinating castor beans. Biochem. J. 152, 65-70 (1975 b).

-

- The influence of abscisic acid, adenosine 3'5' cyclic phosphate and gibberellic acid on the induction of isocitrate lyase activity in endosperm of germinating castor bean seeds. J. Exp. Bot. 28, 219-224 (1977).

MARTIN, C. and D. H. NORTHCOTE: The action of exogenous gibberellic acid on isocitrate lyase mRNA in germinating castor bean seeds. Plant a 154, 174-183 (1982). -

- The action of exogenous gibberellic acid on polysome formation and translation of mRNA in germinating castor bean seeds. Planta 158, 16-26 (1983).

MUTO, S. and H. BEEVERS: Lipase activities in castor bean endosperm during germination. Plant Physio!. 54, 23 - 28 (1974). ORY, R. L.: Acid lipase of the castor bean. Lipids 4, 177 -185 (1969). ORY, R. L., ST. ANGELO, and A. M. ALTSCHUL: The acid lipase of the castor bean. Properties and substrate specificity: J. Lipid Research 3,99-105 (1962). ORY, R. L., L. Y. YATSU, and H. W. KIRCHER: Association of lipase activity with the spherosomes of Ricinus communis. Arch. Biochern. Biophys. 264, 255-264 (1968). PENNER, D. and F. M. ASHTON: Hormonal control of isocitrate lyase synthesis. Biochim. Biophys. Acta (Arnst.) 148, 481-485 (1967). SLACK, P. T., M. BLACK, and J. M. CHAPMAN: The control of lipid mobilization in Cucumis cotyledons. Journal of Exp. Bot. 104, 569-77 (1977). TARPLEY, L. and J. S. CHOINSKI, Jr.: The effect of the embryo axis and exogenous sucrose on lipolysis and glyosysomal enzyme development in Ricinus communis (castor bean) endosperm and cotyledons. Physio!. Plant. 68, 419-425 (1986). TRELEASE, R. M., W. M. BECKER, P. J. GRUBER, and E. H. NEWCOMB: Microbodies (glyoxysomes and peroxisomes) in cucumber cotyledons. Plant Physio!. 48, 461-475 (1971). YATSU, L. Y. and T. J. JACKS: Spherosome membranes. Half unitmembranes. Plant Physio!' 49, 937 -943 (1972).