Plant Physiol. (1983) 73, 1028-10320032-0889/83/73/1028/05/$00.50/0

Some Enzymic Activities in the Germinating Oil Palm (Elaeisguineensis) Seedling'

Received for publication May 24, 1983 and in revised form August 22, 1983

KHAIK CHEANG 002 AND PAUL K. STUMPFDepartment ofBiochemistry and Biophysics, University ofCalifornia, Davis, California 95616

ABSTRACT

In germinating oil palm (Elacis guiweesuis var D x P) seedling, anactive lipase was present in the shoot but absent from both the kermeland the haustorium. It has an optimum pH of 6.2 and a smaller peak atpH 8.6. The shoot lipase was active against a number of mono-, di-, andtriacylglycerols as well as the endogenous lipids present in the shoot,haustorium, and kernel. Activity against related substrates were in theorder: trilaurin > dilaurin > monolaurin but monopalmitin > dipalmitin> tripaimitin. The level of the enzyme in the seedling was highest at arelatively early stage of growth (18-21 days) and also higher in dark-grown seedlings. Glyoxylate bypass enzymes (malate synthetase andisocitrate lyase), glutamate-oxaloacetate transaminase, phosphoenolpyr-uvate carboxykinase and lauroyl-coenzyme A oxidase were located in thehaustorium. The levels of the enzymes paralleled seedling developmentand were slightly higher in light-grown seedlings. Fatty acyl-coenzyme Asynthetase activity was very low and was found in both the shoot andhaustorium.

The germination of the oil palm seed is a slow process. Inmodern nurseries, the seeds are first heated to 38C to 40C for40 d and then soaked in water to raise the moisture content (6,15). This procedure mimics the natural conditions in West Africawhere the seed germinates with the onset of the rains followinga prolonged dry season (14). In the ungerminated seed, the oilpalm embryo lies embedded at one end of the solid fatty endo-sperm (kernel). A thin testa covers the endosperm and the wholestructure is enclosed by a hard endocarp (shell). Above theembryo lies the germ pore where the shell is much thinner. Thegerm pore is covered by a fiber plug (Fig. 1).On germination, the embryo forces its way out through the

germ pore. The emergent embryo forms a button of tissue whichquickly develops a plumule (shoot) and a radicle (root). At thesame time, the other end of the embryo enlarges to form acotyledonary structure called the haustorium (2). The hausto-rium is a spongy structure and is convoluted along the long axisof the nut. As the seedling grows, the haustorium enlarges whilethe surrounding endosperm is broken down and absorbed. Even-tually, after about 3 months, the haustorium fills the entire cavityin the nut. Although the first leaf appears between 20 and 40 d,it does not become fully expanded until later and the seedlingremains partly dependent upon the supply of reserve endosperm

'Supported in part by National Science Foundation Grant PCM 79-03976.

2 Permanent address: Department of Biochemistry, University of Ma-laya, Kuala Lumpur, Malaysia.

material (mainly TG3) until about 60 d (4).To study how the storage lipid material in the kernel is utilized

during germination and seedling development, we have exam-ined some enzymes involved in the metabolic process, namelytwo glyoxylate bypass enzymes (malate synthetase and isocitratelyase), a gluconeogenic enzyme (PEP CK), a transaminase(GOT), and three lipid-utilization enzymes (lipase, acyl-CoAsynthetase, and acyl-CoA oxidase). The distribution of theseenzymes in the seedling and their activities during seedlinggrowth were studied.

MATERIALS AND METHODSPlant Material. Heat-treated seeds of Elaeis guineensis var D

x P were provided by Dr. A. C. Soh, Highlands Research Unit,Kelang, Selangor, Malaysia. The seeds were germinated andgrown as described (12). Extracts of shoot,4 haustorium, andkernel were prepared by grinding the tissue from 3 to 5 seedlingsin approximately 8 ml 0.01 M Tricine-NaOH buffer (pH 7.5) ina mortar. The extract, filtered through two layers of Miracloth,was used as the source of enzymes. All procedures were carriedout at 4°C.

Chemicals. '4C-Labeled lipid substrates were purchased fromsources described elsewhere (12). Fatty acyl-CoAs, 1,3-dilaurin,and monolaurin were obtained from Sigma.Enzyme Assays. Malate synthetase, isocitrate lyase, PEP CK,

and GOT were assayed according to Cooper and Beevers (3).Acyl-CoA oxidase was assayed according to Gerhardt (5) withthe omission of NaN3. Acyl-CoA synthetase was assayed in areaction mixture made up of 25 mm Tricine-NaOH buffer (pH7.8), 2 mM ['4C]fatty acid (8000 cpm), 2 mm CoA, 10 mM ATP,10 mM MgCI2, and approximately 0.5 mg enzyme protein in atotal volume of 0.25 ml. The reaction was terminated after 0,20, 40, and 60 min at room temperature and ["C]acyl-CoAformed was separated according to Mancha et al. (9) and countedin a Beckman LS-230 liquid scintillation counter with 10 mlPCS:xylene (2:1, v/v) counting mixture. Lipase activity wasassayed by two separate procedures. [14C]Triacylglycerol andother substrates were prepared by emulsifying a suspension ofthe substrate (50 Mmol in 1 ml 2% Triton X-100) for 1 to 2 minwith a Sonicator Cell Disruptor, ModelW 185F (Ultrasonics Inc.,Plainview, New York). Samples were counted to check that theemulsion was uniform. The reaction mixture contained in 1 mlof solution: 75 mm Mes (pH 6.2), 5 mm CaCI2, 10 mm TGemulsion (7000 cpm), and approximately 0.5 mg enzyme pro-tein. Samples (200 Ml) were removed after 0 and 30 min at roomtemperature and assayed according to Kaplan (7). The secondlipase assay was used for nonradioactive substrates and to follow

3 Abbreviations: PEP CK, phosphoenolpyruvate carboxykinase; GOT,glutamateoxaloacetate transaminase; TG, triacylglycerol; DG, diacylgly-cerol; MG, monoacylglycerol; ACC, amino cyclopropane carboxylic acid.

' Refers to the root and shoot system.1028

METABOLIC ACTIVITIES OF OIL PALM SEEDLING

N *

0-Ar-#

en

S

c

FIG. 1. Morphology of oil palm seedling. A, FreeB, 7-d-old seedling; C, longitudinal section of seed

longitudinal section of 3-week-old seedling; ar, advof testa; e, embryo; en, endosperm; f, fiber pluhaustorium; pl, plumule; r, radicle; s, shell.

hydrolysis of endogenous lipids in the extrfreshly prepared extracts were used. Samplesmoved from the reaction mixture at 0, 20, 4(assayed according to Nixon and Chan (I 1).glassware were used and the activity was catime course of each incubation. A standard cunmol palmitic acid was carried out with each eactivity by the Nixon-Chan assay was exprefatty acid released per unit time. All other enz,expressed as amount of substrate utilized per

Analytical Procedures. Protein was estimamethod. Spectrophotometric measurementsBeckman DU recording spectrophotometer. Iing was carried out as previously described (I1

RESULTS

Enzyme Activities during Seedling Growththe development of the external shoot and rcinternal haustorium after seed germination. Efrom the shoot, haustorium, and kernel and Eenzymic activities. The activities of malate sylyase, and GOT in the haustorium extract ar2B. Other enzymic activities are shown in Talin this experiment were germinated from aseeds and showed slower growth than those in Iit is evident that the patterns of enzyme develexperiments were similar and activity levelsmeasured in both experiments were aboutsynthetase, isocitrate lyase, and GOT levels wehigher than those ofPEP CK and acyl-CoA oxwas found in the shoot extract where the leseedling was about the same as those of i

isocitrate lyase, and GOT.Enzyme Activities in Shoot and Kernel I

enzymic activities found in haustorium extrneither absent or very low in shoot and kenmalate synthetase levels were only 3, 6, and 2 1

in shoot extracts and 4, 1, and 0 nmol/min,

extracts at 14, 21, and 28 d of growth, respectively. The corre-sponding levels in haustorium extracts were 199, 392, and 645

pl nmol/min/seedling (Fig. 2B). For isocitrate lyase at the samestages of growth, shoot extracts contained 1, 2, and 4 nmol/min/seedling, kernel extracts 2, 3, and 0 nmol/min/seedling, andhaustorium extracts 90, 223, and 339 nmol/min/seedling, re-

t r spectively (Fig. 2B). GOT, PEP CK, and acyl-CoA oxidaseB) activities were not detectable in shoot and kernel extracts at all

stages of growth examined. In haustorium extracts, acyl-CoAh oxidase activity was only observed with short chain substrates

(decanoyl-CoA and lauroyl-CoA). No activity was obtained withmyristoyl-CoA, palmitoyl-CoA, or stearoyl-CoA.

en Fatty Acyl-CoA Synthetase. Fatty acyl-CoA synthetase activitywas very low in the developing seedling but was detectable bythe assay method used. Nevertheless, in each incubation samples

S were taken at four different times (0, 20, 40, and 60 min) andthe activity was calculated from the time plot obtained. Both

r D shoot and haustorium extracts contained comparable levels ofactivity in the seedling (Table II) which were 20 to 500 timeslower than the other activities shown in Table I. The enzymewas most active with oleic acid and lauric acid; myristic andpalmitic acids were poor substrates.

shly germinated seed; Lipase Activity in Developing Seedling. As shown in Table I,through embryo; D, an active triacylglycerol lipase was present in shoot extracts. Thisentitious root;c, ', lipase activity in shoot extracts appeared on germination andg; g, germ pore; h, reached a peak after 18 to 21 d. Under the conditions of the first

' assay, the reaction proceeded linearly for at least 80 min with asaturating TG substrate concentration of about 20 mm (Fig. 3).

act. In this assay, The pH curve shows an optimum value of 6.2 with a smallers (200 ul) were re- peak at 8.6 (Fig. 3). Using this assay, little or no activity was), and 60 min and found in haustorium or kernel extracts compared with the shootOnly acid-washed extract (experiment 1, Table III). There were no inhibitors eitherIculated from the in haustorium or in kernel extracts since the shoot extractirve using 0 to 120 remained equally active in their presence. Lack of lipase activityexperiment. Lipase in either extract was also not related to a requirement for assed as amount of cofactor or activator in the other extract since both extractsyme activities were incubated together still showed no activity.unit time. These points were also illustrated in experiment 2 (Table III)ited by the Lowry in which hydrolysis of endogenous lipid in the extracts werewere made on a followed. The results showed that in fresh haustorium extracts,Radioactive count- some endogenous lipids apparently underwent hydrolysis. How-2). ever, this was lower than when compared with the hydrolysis of

endogenous lipids in the shoot extract. In 1 h, only 0.8% of thetotal saponifiable fatty acids in the haustorium extract was re-leased, whereas the fatty acids released in the shoot extract

I. Figure 2A shows represented its total saponifiable fatty acid content. The hydrol-)ot system and the ysis of endogenous lipids was largely eliminated by boiling theIxtracts were made extract for 5 min. Incubation of shoot extract with haustoriumassayed for various and/or kernel extracts showed that the lipase in the shoot extractrnthetase, isocitrate was able to hydrolyze the endogenous lipids in these extracts.re shown in Figure More hydrolysis was seen with the haustorium lipids, althoughble I. The seedlings more endogenous lipids were present in the kernel extract.different batch of Lipase activity in haustorium and kernel extracts was alsoFigure 2. However, investigated at other pH values ranging between 4.0 to 8.6 andlopment in the two from seedlings grown in the presence and absence of light fromof those enzymes 3 to 43 d after germination. In all cases, no measurable activitythe same. Malate was detected using the radioactive assay. There was also no,re five to ten times activity when 3 mm cAMP, AMP, ADP, ATP, GTP, or CoASHidase. Active lipase were added to the incubation mixture. Finally, extracts of tissuesvel of activity per previously exposed to IAA, ACC, kinetin, or gibberellin did notmalate synthetase, show significant shifts in lipase activities.

Table IV shows the activity of the shoot extract on variousExtracts. The five substrates. It is interesting to note that while the enzyme wasacts (Table I) were more active with trilaurin than dilaurin or monolaurin, it had anel extracts. Thus, greater activity with monopalmitin and dipalmitin than withnmol/min/seedling tripalmitin. Haustorium extracts had no activity against all these/seedling in kernel substances at pH 6.2 and pH 8.6.

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E

to

Plant Physiol. Vol. 73, 1983

600

to

400 0

0Eo_

Days after Sprouting

FIG. 2. Growth of seedling and enzyme activity in haustorium. Seedlings were grown in the dark and haustorium extracts were assayed for malatesynthetase (x), isocitrate lyase (A), and GOT (0). Fresh weight of tissue per seedling: shoot (0), haustorium (0).

Table I. Enzyme Activities in Oil Palm Seedlings Grown in the Presence and Absence ofLightIn this experiment, the seedlings showed slower growth than in Figure 2.

Haustorium Extract Shoot extract

Time after Malate synthe- s Acyl-CoA oxiL-Sprouting tase Isocitrate lyase GOT PEPCK dase Lipase

Light Dark Light Dark Light Dark Light Dark Light Dark Light Dark

d nmol/min/seedling13 57 84 11 29 137 179 12 12 NDb ND 128 27418 168 91 37 40 230 153 54 34 11 14 333 29221 118 205 44 103 98 206 10 21 7 17 236 57426 228 170 69 81 231 258 21 19 16 12 199 41930 462 357 138 116 703 545 66 47 26 26 77 41234 616 541 142 157 774 470 130 21 27 25 33 10937 752 570 208 192 687 407 160 91 51 43 34 11343 902 309 237 88 1040 324 188 55 26 36 93 273

'Substrate was lauric acid.b Not determined.

DISCUSSIONIn considering how the developing oil palm seedling utilizes

the reserve triacylglycerols from its endosperm, the role of alipase is ofcritical importance. In other germinating oil seeds, anactive lipase is found in the storage tissue itself, e.g. castor beanendosperm (10) or soybean cotyledons (3). The oil palm seedlingis unusual in developing a special structure called the haustorium(Fig. 1) which is involved in the utilization of storage material.One would therefore expect an active lipase to be located withinthis structure. However, the evidence represented in this studyclearly shows the loalization of an active lipase(s) in the shootand root system (Table III). The shoot extract was active againstexogenous lipid substrates (Table IV) as well as endogenous lipids

present in the shoot, haustorium, or kernel (Table III). The majoractivity in the extract was an acid lipase with pH optimum at6.2 but a minor component with optimum activity at pH 8.6was also present (Fg. 3). In contmst, haustorium and kernelextracts were completely inactive against exogenous lipid sub-strates under a wide variety ofconditions. A very small hydrolysisofthe endogenous lipids in haustorium extract was observed butit amounted to only 0.8% of the endogenous fatty acids in 1 hof incubation. Incubation of the extract in the presence ofpossible activators, e.g. cAMP or other nucleotides and planthormones, did not reveal any increased lipase activity (12).

In the absence of a lipase in either the haustorium or kernel,it would be worthwhile to consider whether the shoot lipase has

METABOLIC ACTIVITIES OF OIL PALM SEEDLING

Table II. Fatty Acyl-CoA Synthetase Activity in Extracts of37-DaySeedling Grown in the Dark

Fatty CoA Synthetase Activity

Shoot Haustorium Kernelnmol/h * seedling

C1rfatty acid 15.7 24.4 0

Fatty Acid Substrate

C12 C14 C16 Ct8:'Haustorium extract 24.4 3.7 6.1 65.3

E0

E

p H

FiG. 3. pH Curve of shoot lipase activity and the effect of substrateconcentration.

any physiological role in the total seedling. The specificity of thelipase did not give any clue to its possible role in the seedling. Itwas active against a number of simple triacylglycerols but itsactivity toward the diacyl or monoacyl forms was different forC12 and C16 fatty acyl compounds (Table IV). The level of theenzyme was consistently higher in dark-grown seedlings (TableI) in which presumably more reserve material was required to bemobilized. On the other hand, this level was highest at 18 to 21d and decreased thereafter (Table I), whereas the five haustorialenzymes all increased continuously up to 43 d. However, thegreatest difficulty in attributing a role in lipid mobilization inthe seedling to this lipase was its location in the shoot. It isdifficult to imagine how it could be involved in hydrolyzingkernel lipids in vivo without any movement ofthe lipid from thekernel to the shoot. Although we have shown that the haustoriumcan absorb triacylglycerols without prior hydrolysis, there is noevidence for its translocation to the shoot ( 12) where presumablythe fatty acids would be converted to hexose phosphates.A number ofenzymes are involved in converting reserve lipids

to sucrose and water-soluble substances during seed germination

Table III. Lipase Activity in Seedling ExtractsLipase assay was carried out at pH 6.2 according to Kaplan (experi-

ment 1) or to Nixon and Chan (experiment 2). Extracts were preparedfrom 25-day seedlings grown in the dark.

Tri-['4C]LaurinProtein Hydrolyzedmg nmol/h

Experiment IHaustorium (0.11) 0Shoot (0.18) 1090Kernel (0.24) 13Shoot (0.18) + haustorium (0.11) 1114Shoot (0.18) + kernel (0.24) 1069Haustorium (0.11) + kernel (0.24) 0

FA Released fromEndogeneous Lipids

nmol/hExperiment 2Haustorium' (0.42) 17.5 (2.8)bShoota (0.66) 39.8 (3.4)bKernel' (0.83) 3.5 (O)rShoot (0.33) + haustorium (0.21) 122.2Shoot (0.33) + kernel (0.41) 48.5Shoot (0.33) + haustorium (0.21) +

kernel (0.41) 128.1Haustorium (0.21) + kernel (0.41) 7.4

' Total saponifiable fatty acids in extracts used were: haustorium, 2194nmol; shoot, 31.8 nmol; kernel, 2803 nmol.

b Extract boiled 5 min.

Table IV. Specificity ofShoot LipaseLipase assay was carried out at pH 6.2 according to Kaplan (experi-

ment 1) or to Nixon and Chan (experiment 2).

['4C]Substrate ['4C]Substrate Hydrolyzed

itmol/mg protein*hExperiment I

Tridecanoin 4.14 (9.61)Trilaurin 5.76 (5.55)8Trimyristin 1.15 (1.34)8Tripalmitin 3.84 (3.20)8Dipalmitin 11.17 (9.32)8Monopalmitin 12.43 (10.13r

FA Released

itmol/mg protein - hExperiment 2No substrate 0.06Trilaurin 3.921,3-Dilaurin 2.64Monolaurin 0.85

a Assayed at pH 8.6.

(1). Some of these enzymes were assayed and appeared to belargely located in the haustorium (Table I) since shoot and kernelextracts contained very much lower levels of activity. The levelsofthe three most active enzymes assayed, namely malate synthe-tase, isocitrate lyase, and glutamate-oxaloacetate transaminasefollowed closely the development ofthe seedling (Fig. 2). Growthin the presence of light did not have a significant effect on thelevels of these enzymes in the seedling. Active photosynthesisdoes not occur before about 60 d (4) and reserve material wasstill being utilized. In fact, the light-grown seedlings showedslightly higher levels of enzyme activity in the haustorium in

most cases (Table I). Fatty acyl-CoA synthetase activity was

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Plant Physiol. Vol. 73, 1983

surprisingly low in the seedling and was found in both the shootand the haustorium (Table II). This might be partly due tothioesterase activity. Short-chain fatty acid substrates were morereadily activated, but good activity was also seen with oleic acid.This is in line with the observation that haustorium slices wereunable to esterify or metabolize myristic and palmitic acids whencompared with lauric acid (12).

In conclusion, this paper has presented evidence that an activelipase is locWalied only in the shoots ofthe germinating seed, thatthe kernel is essentially devoid of enzyme activity that relate tolipid utilization, and that the haustorium, devoid of lipase activ-ity, nevertheless contains the necesary enzymes for the conver-sion of fatty acids via the glyoxylate bypass to hexose phosphate.The interaction between the kernel, haustorium, and shoot re-mains a puzzle which may, in part, be revealed by carefulcytological analyses of possible vascular systems that may beinvolved in transporting lipids from the kernel to the shoot andthen back to the haustorium for final processing. In fact, anearlier report by Rab6chault and Cas (13) showed the presenceofsuch a vascular system in the haustorium. Transverse sectionsof the haustorium showed a layer of epidermal cells coveringlarge internal parenchymal cells. All around the periphery be-neath the epidermal layer were smaller procambium cells ar-ranged into bundles ('faisceaux procambiaux') which werealigned longitudinally along the haustorium.

Acknowledgments-We are indebted to Dr. A. C. Soh for the preparation andsupply of heat-treated oil palm seeds.

LITERATURE CITED

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2. BOATMAN SG, WM CROMBLE 1958 Fat metabolism in the West African oilpalm (Elaeis guineensis): Part 2. Fatty acid metabolism in the developingseedling. J. Exp Biol 9: 52-74

3. COOPER TG, H BEEVERS. 1969 Mitochondria and glyoxysomes from castorbean endosperm: enzyme constituents and catalytic components. J BiolChem 244: 3507-3513

4. CORLEY RHV 1976 Germination and seed growth. In RHV Corley, JJ Hardon,BJ Woods, eds, Developments in Crop Science (1) Oil Palm Research.Elsevier Science Publishing Co, Amsterdam, pp 23-25

5. GERHARDT B 1981 Enzymatic activities ofthe j-oxidation pathway in spinachleaf peroxisomes. FEBS Lett 126: 71-73

6. HUSSEY G 1958 An analysis of the factors controlling the germination of theseed of the oil palm, Elaeis guineensis (Jacq.). Ann Bot 22: 259-284

7. KAPLAN A 1970 A simple radioactive assay for triglyceride lipase. AnalBiochem 33: 218-225

8. Deleted in proof.9. MANCHA M, GB STOKES, PK STUMPF 1975 Fat metabolism in higher plants.

The determination of acyl-acyl carrier protein and acyl coenzyme A in acomplex lipid mixture. Anal Biochem 68: 600-608

10. MUTO S, H BEEVERS 1974 Lipase activities in castor bean endosperm duringgermination. Plant Physiol 54: 23-28

11. NixoN M, SHP CHAN 1979 A simple and sensitive colorimetric method forthe determination of long-chain free fatty acids in subcellular organelles.Anal Biochem 97: 403-409

12. OO KC, PK STUMPF 1983 Metabolism of the germinating oil palm (Elaeisguineensis) seedling. In vivo studies. Plant Physiol 73: 1033-1037

13. RABECHAULT H, S CAS 1974 Recherches sur la culture in vitro des embryonsde palmier a huile (Elaeis guineensis Jacq. var dura Becc.). Oleagineux 29:73-78

14. REEs AR 1962 High-temperature pre-treatment and the germination of theseed of the oil palm, Elaeis guineensis (Jacq.). Ann Bot 26: 569-581

15. TURNER PD, RA GILLBANKS 1974 Oil palm cultivation and management.Incorpd Soc Planters, Kuala Lumpur, pp 36-37

1032 00 AND STUMPF