Pergnmon 0031-9422(94)EOO25-N Phytcxhemisrry, Vol. 36. No. 4, pp. 899-902 1994 Copynghr % 1994 Elvvicr Sdcna Ltd

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TRIACYLGLYCEROL SYNTHESIS IN DEVELOPING KERNELS OF GROUNDNUT AS INFLUENCED BY ALIPHATIC ALCOHOLS

PUSHP SHARMA and C. P. MALIK*

Department of Botany, Punjab Agricultural University, Ludhiana-141004, India

(Receir;ed in revised form 10 Nocember 1993)

Key Word Index-Armhis hypogaea; Leguminosae; triacylglycerol; aliphatic alcohols; glycerol; cotyledons; oil.

Abstract-In developing groundnut (Arachis hypogaea) kernels, the most active period of lipid, chiefly triacylglycerol (TG) accumulation is between 15 and 35 days after podding (DAP). Between 5 and 15 DAP starch content was enhanced but declined thereafter up to maturity. With progressive development of kernels both phospholipids and free fatty acids decreased, whereas total glycerides continuously increased. At 15 DAP, [U-*4C]fructose was incorporated into TG at a high rate. The incorporation of [1-*4CJacetate into TG increased progressively during the active period of lipid accumulation. The rate of incorporation of the latter into TG was about three-fold greater than that of fructose. Addition of glycerol (0.5 mM) in the incubation medium stimulated lipid accumulation and is attributed to the limited availability of glycerol and other metabohtes, especially during the active period of lipid accumulation in seed [ 15-35 DAP]. Foliar spray treatments with a mixture of aliphatic alcohols enhanced incorporation of [U-‘4C]fructose and [l-“C]acetate into TG of the developing kernels. The regulatory mechanisms involved in the stimulation of lipid accumulation are discussed.

INTRODUCI’ION

From the leaves, the photosynthate is transported as sucrose to the seed compartment where it is converted to the final storage reserve, e.g. triacylglycerol (TG) in groundnut seeds. TG is deposited in oleosomes of the groundnut cotyledon and it is important to understand the dynamics of the process through which the develop- ing oil seeds mobilize the flow of sucrose carbon to TG. Aliphatic alcohols enhance oil concentration in ground- nut [l], but the precise role of these PGRs in TG biosynthesis has not been well characterized. With a view to understanding this, we undertook investigations on the developing groundnut kernels. The enzymes concerned with TG synthesis exist in seeds but the seeds channel the flow of carbon chiefly to the storage product, i.e. oil during seed maturity. The period between 15 and 30 days after podding (DAP) constitutes the phase of rapid lipid accumulation. We describe the effect of aliphatic alcohols and glycerol on the incorporation of the carbon skeleton labelled precursors-fructose, acetate and NaHCO,-by developing groundnut kernels.

RESULTS AND DlSCUSSlON

Initially the fruit wall had very high contents of metab- olites which temporarily acted as a first sink, these were

*Author to whom correspondence should be addressed.

transported to the enlarging seed (Table 1). The transfer of sucrose from pod wall to seed has been demonstrated [2]. With the enlargement of the seed, the fruit wall gradually thinned and its inner tissue degenerated. Thus, with age, the pod matured and most of its soluble proteins and soluble sugars underwent reduction.

The contents of soluble sugars and proteins declined abruptly in the seeds during 15 to 30 DAP, whereas oil biosynthesis, which was low initially, became rapid dur- ing 15 to 35 DAP and continued until maturity (Table 2). Initially the accumulation of TG was slow in the develop- ing kernels, but was most rapid between 15 and 35 DAP. At maturity, the increase was negligible. Between 5 and 15 DAP, starch content enhanced but thereafter declined up to maturity. Soluble sugars and proteins decreased ab- ruptly and gradually between 5 and 15 DAP. At 5 DAP, total glycerides were maximum (78.8%) compared with free fatty acids (FFA 10%) and phospholipids (5.6% Table 3). With progressive development of seeds, both phospholipids and FFA decreased, whereas total gly- cerides steadily increased.

Three distinct phases of lipid accumulation are dis- tinguishable in the developing groundnut seeds: 5 to 15 DAP, 15 to 35 DAP, and 35 DAP to maturity. During the initial phase very little lipid is formed, though in the middle phase there is rapid accumulation of lipid. On a dry weight basis as much as 28-45% of the newly synthesized material was lipid, though the rate of protein synthesis was low. During 35 DAP to maturity, lipid

899

900 P. SHARMA and C. P. MALIK

Table 1. Distribution of various metabolites (mgg-’ fr. wt) in fruit wall and seed at

different days after podding (DAP)

Fruit DAP Seed DAP

Component 10 30 50 10 30 50

Soluble sugar Starch

(WS)

Proteins

(SS)

Lipids (dry wt)

14.6 25.4 14.9 9.2 24.4 15.3 5.9 I.2 ND 9.4 32.6 14.8

30.4 976 11.2 50.6 55.8 75.4

2.4 1.8 1.9 2.6 7.2 10.8

3.6 3.0 0.6 24.8 124.0 285.0

WS: water soluble, SS: salt soluble.

Table 2. Distribution of various metabolites (% of dry wt) at from [1-‘4C]acetate was incorporated into TG compared different periods of podding in M-13 with 31.9% with hexose (Table 4).

Days after podding

Metabolites 5 15 25 35 M

Soluble sugars 12 4 2.5 1.8 1.8

Starch 16 27 15.0 12.0 10.0

Proteins 55 38 33.0 30.0 28.0

Oil 15 28 33.8 45.0 47.6

Table 3. The relative proportions of phospholipids, free fatty

acids and total glycerides in the oil (% in oil)

Days after podding

Oil component 5 15 25 35 M

Phospholipids 5.6 3.4 2.2 0.5 0.4

Free fatty acids 10.0 7.0 5.9 4.8 3.8 Total glycerides 78.8 82.0 84.0 92.4 94.2

synthesis continued but at a comparatively reduced rate. It demonstrates that the period between 15 to 35 DAP was the active period of lipid synthesis.

The total incorporation of [U-‘4C]fructose, [ l-t4C]- acetate and NaH14C0, into TG was studied in the seed. Using three concentrations (1, 5 and 10 @i) the rates of incorporation were linear up to 10 PCi with the fructose and acetate but not NaH”C0,. At 20 and 30 DAP, incorporation of [U-‘4C]fructose was higher than [l- “‘Clacetate or NaH14C0, into aqueous extract. The incorporation was high at 30 DAP, though incorporation of NaHt4C0, was minimum. The incorporation of fruc- tose and acetate into TG increased with the concentra- tion of the two precursors, and the stage of development of seed was critical in determining their utilization with good efficiency for TG synthesis. By contrast, the incorp- oration of NaHt’CO, did not increase proportionally with their concentration. At 20 DAP, 88.4% of the label

An increase in oil (1.9%) was observed in mature kernels sampled from plants given two foliar sprays of aliphatic alcohols. The rate of [U-t4C]fructose incorp- oration into TG increased at 20 DAP but decreased appreciably at 30 DAP. By contrast, incorporation of [l- “C] acetate into lipids was stimulated at both stages of seed development. Stimulation of incorporation of label from NaH14C0, into lipids was also observed. Addition of glycerol (0.5 mM) in the incubation medium decreased the incorporation of hexoses but greatly stimulated the incorporation of acetate. The effect was most marked at 30 DAP.

It is widely accepted that sucrose is the major compon- ent of translocated photosynthate and that it is not hydrolysed extracellularly [3]. Our initial studies re- vealed that both radiolabelled hexoses (glucose, fructose) were utilized efficiently for TG synthesis at all phases. A comparison of the incorporation of [U-‘4C]fructose and [1-14C]acetate into lipid indicated a developmental pat- tern concomitant with TG synthesis. However, incorp- oration of acetate was several-fold more than fructose, when the incorporation of two precursors at two stages of development was compared. Thus, the incorporation of [U-“C]fructose into TG at 15 DAP was lower than that of Cl-t4C]acetate whereas the reverse was observed at 35 DAP. The incorporation of [U-‘4C]fructose was five-fold higher than [1-‘4C]acetate. The stimulation of TG bio- synthesis with the addition of glycerol in the incubation medium could be attributed to the limited availability of glycerol and other desired metabolites in the control tissue especially during the active period of lipid accumu- lation in seeds (15-35 DAP). Evidently, glycerol enters the metabolic pathway through glycerol kinase and this then stimulates TG synthesis. Similar observations were made in almond [4]. The stimulation of label from [U-“C]fructose into TG indicates that its glycolytic metabolite, DHAP, provides glycerol for harnessing fatty acids by acylation, besides providing acetate for fatty acid synthesis.

The observed increase in [l-“Clacetate incorporation into TG at 35 DAP with added glycerol suggests its restricted availability along with other metabolites in the

Triacylglycerol synthesis in groundnut 901

Table 4. Incorporation of [U-W]fructose. [l-‘*C]acetate and NaH”C0, at 20 and 30 DAP

Rate of incorporation into lipids and aqueous extract

(CPM hr-’ 500 mg-‘)

Praursor

DAP

20 30

L % A % L % A %

1. [U-“C]Fructose ($i) 1 5

10

10 + glycerol

AA

2 [1-‘4C]Acetate (&i)

1

5

10 lO+glycerol

AA

3. NaH”C0, (&i)

1

5 10

10 + glycerol

AA

8260 - 12600

19100 - 22600 27900 (31.9) 59600 29100 (25.8) 83700

34100 (32.5) 70700

7720 -

34500 -

86900 (88.4) 379c00 (90.5)

53ooo (93.4)

1100

2850

11400

430 - 777

1030 - 6200 630 (76.5) 195

45200 (79.6) 7550 678 (82.6) 143

- (68.1) (74.2)

(67.5)

- - (11.6)

(9.5)

(6.6)

(23.6)

(20.4) (17.5)

6590

34300

35400 37300

52000

7980

9120

16400

95300

346 694

152

682

312

- - (33.5) (19.1)

(20.4)

- (0.81)

(91.9)

(0.92)

- - (12.1)

(55.5)

(63.8)

12800

42300

70200

158000

204OoO

8810

4840

3750 8420

1900

288

560

1090

546 177

- (67.5) (80.9)

(79.7)

- (99.2)

(8.1) (99.1)

-

(87.9)

(44.5) (66.2)

AA: Cotyledons sampled from plants given foliar spray of aliphatic alcohols. Glycerol -0.5 mM.

seed tissues during the late stages of development. This deficiency appears to be compensated by the exogenous supply of glycerol causing enhanced fatty acid biosyn- thesis.

Since we failed to observe the activity of glycerol- phosphate dehydrogenase at both stages (unpublished data), G-3-P appears to be formed exclusively through the glycerokinase reaction for TG biosynthesis.

The decreased incorporation of NaH14C0, into lipid with the increase of label might be due to channelling of carbon to other metabolic paths. However, the stimula- tion by addition of glycerol indicates that synthesis of fatty acids is increased, and glycerol acylates most of the fatty acid to form triglycerides at early stages (20 DAP) of seed development. However, at 30 DAP, the precursors seem to have been amply available and glycerol did not further enhance the synthesis of lipids. Similar observa- tions have also been reported in higher plants [S].

Seed yield in groundnut is strongly influenced by the availability of assimilates during its development [ 1, 43, which would be affected by assimilates partitioning dur- ing this period. Aliphatic alcohols considerably increased total dry matter mainly in the reproductive parts at the expense of stem growth and leaf. Thus, increase in oil percentage in seed kernels with aliphatic alcohols appears to be a consequence of increased availability of assimilates and their enhanced diversion towards ultimate sinks. Higher chain alcohols appear to facilitate transport and transposition of sugar or oligosaccharides [6]. Their role

in improving physiological efficiency of the plant has also been demonstrated [7, 83.

EXPERIMENTAL

[1-14C] Acetate (sp. act. 55.5 mCimmol_‘), [U-14C] fructose (sp. act. 240 mCi mmol- ‘) and NaH”CO, (sp. act. 51.9 mCimmol_‘) were obtained from the Isotope Division of Bhabha Atomic Research Centre, Bombay. Groundnut seeds Arachis hypogaea L. Var M-13 were obtained from the Department of Plant Breeding, Punjab Agricultural University, Ludhiana. There were two treat- ments: control, aliphatic alcohols [l pgml- ‘1 given as foliar spray at 15 and 30 DAS. The mixture of aliphatic alcohols had the following composition [%I: C-24 tetra- cosanol: 7-10; C-26 hexacosanol: 12-16; C-28 octacos- anol: 15-20: C-30 triacontanol: 24-30, C-32 dotriacon- tanol: 1 l-14; and tetratriacontanol: 4-5 dissolved in Triton X-100.

Incorporation of labelled precursors into cotyledon slices. Pods at desired stages were sampled and put in ice in a plastic beaker. Seeds were gently dissected out and cut into slices about 0.5 mm thickness. Tissue (500 mg) was taken in 1 ml of KPi buffers [200 mM, pH 7.43 and were preincubated for lo-20min at 30” with constant agitation. Subsequently the desired amounts of labelled precursors (as given above) were added. After incubation for I hr the medium was drained off and the cotyledon slices were repeatedly washed with 1 ml each of cold H,O.

902 P. SHARMA and C. P. MALIK

Lipids were extracted when the slices were boiled in 2 ml MeOH followed by addition of 1 ml CHCI, followed by addition of 1 ml CHCI, and 0.5 ml H,O [4].

Rudioactioity counting. Samples were counted in a Meenakshi Packard Beta liquid Scintillation Counter for i4C using 0.5% PPO in dioxane as scintillation fluid. The rates of incorporation into triacylglycerols and aqueous fraction were separately analysed. For the estimation of total soluble sugars [9], starch [lo], proteins [ 1 I]. lipids [12], free fatty acids [I33 and total glycerides [14] standard methods were followed.

Acknowledgements-The authors gratefully acknowledge the financial grant made by the USDA office of the International Cooperation and Department authorised by PL-480. Special thanks are due to Dr. R. L. Kalra for radioactivity analysis.

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