Plant Physiol. (1976) 57, 375-381

Studies on Lipid Synthesis and Degradation in Developing Soybean Cotyledons1 Received for publication September 3, 1975 and in revised form November 10, 1975

RICHARD F. WILSON2 AND ROBERT W. RINNE United States Department of Agriculture, Agricultural Research Service, Department of Agronomy, University of Illinois at Urbana, Champaign, Illinois 61801 ABSTRACT The metabolic activity of individual lipid dasses found in developing soybean cotyledons (Glycine max.) is estimated by determining the degradation rate of the compound under given conditions. Pulse-labeling and dual substrate labeling are used to evaluate this parameter. These studies indicate first order decay kinetics for phosphatidic acid, pbosphatidylinositol, phosphatidyicholine, phosphatidylethanolamine, N-acylphosphatidylethanolamine, diglyceride, and zero order kinetics for triglyceride in cotyledons var. "Harosoy 63" at 30 days after flowering. Decay coefficients for acyl groups and lipid-glycerol moieties within specific lipid classes from either method are comparble. Half-life (t ,2) calculations from the decay coefficients indicate extremely rapid turnover rates (0.08 to 3.4 hours at 25 C) and suggest similar turnover rates of acyl groups and lipid-glycerol in diglyceride and all phospholipids except N-acylphosphatidylethanolamine where acyl groups are replaced independent of the glycerol moiety. These experiments reveal not only different metabolic activity between lpid components of soybean cotyledons, but also describe a new method for measuring Upid turnover in plants.

The rate of phospholipid synthesis in developing soybean cotyledons is considerable. Since only small changes in total phospholipid content are noted during the growing season (8), the rate of phospholipid degradation also must be high. The degradation rate of lipids may be determined by several methods. The most commonly used procedure is pulse labeling. Kagawa et al. (6) and Dybing and Craig (5) have used pulse labeling to monitor phospholipid degradation activity in castor bean endosperm and developing flax seed. We have employed this procedure to determine the degradation rates of individual lipid classes in developing soybean cotyledons. Decay coefficients were calculated for both lipid-fatty acid and lipid-glycerol in several lipid classes from the descending slope of lipid class specific activity. The values obtained were in close agreement with the results of a less cumbersome technique, dual substrate labeling. The dual substrate-labeling technique was modified for the study of lipid-fatty acid and lipid-glycerol turnover from the procedure of Dehlinger and Schimke (4). It has potential application in studies of cellular component degradation in both plant and animal tissues. We have compared its use ' Cooperative investigations of the Agricultural Research Service, United States Department of Agriculture, and Illinois Agricultural Experiment Station. 2 This research represents partial fulfillment of the Ph.D. requirements of R. F. W.

375

to pulse labeling in the approximation of lipid turnover in developing soybean cotyledons.

MATERIALS AND METHODS

Sampling Plant Material. Soybean (Glycine max. L. Merr. var. "Harosoy 63") was grown at the University of Illinois South Agronomy Farm, Urbana, during the 1974 growing season. Greenhouse grown soybeans var. "Steele" were used when field grown beans were not available. Developing seeds used in these studies were harvested from plant nodes 6 to 9 (cotyledons were attached to node 1) 30 DAF.3 All of the material was kept in the pod on ice and used within 1 hr of harvest. Excised cotyledon halves (500 mg fresh weight) were used in each experiment. Incorporation of Labeled Substrate. Acetate-2'4C and glycerol-2'4C were incorporated at 15 and 25 C. Incubations were carried out for various intervals in 0.1 M MES buffer (pH 5) at a final volume of 1.5 ml. Termination and subsequent handling of the tissue in all studies were as described (8). Rates of incorporation for each lipid class were calculated from the slope of regression analysis. Pulse labeling of acetate-2'4C and glycerol-2'4C were conducted at 15 and 25 C. These experiments were initiated with a 15 min incubation of the 14C substrate in 0.1 M MES buffer, pH 5, at a final volume of 1.5 ml. After the pulse, the radioactive incubation media were removed, and the tissue was washed twice with 2 ml of distilled H20. The tissue then was incubated in MES, pH 5, containing either 2.5 ,umoles sodium acetate or 25 ,umoles glycerol at a final volume of 1.5 ml. The reactions were terminated at various intervals after the '4C pulse. Degradation rates were calculated from the slope of the decay curve (2). Dual label isotope incorporation was performed at 15 and 25 C with acetate-2'4C and acetate-23H, or glycerol-2'4C and glycerol-23H. In each set, the tissue was exposed to the 14C substrate for 2 hr in 0.1 M MES, pH 5, at a volume of 1.5 ml. After 2 hr, the 3H-labeled substrate was introduced So the incubation without removal of 14C. The reaction was continued for 3 to 15 min at a new volume of 1.6 ml, then it was terminated, and the ratio of 3H/'4C dpm in each lipid class was determined. Lipid extraction, separation of lipid classes, and lipid analysis procedures were identical to those described (9). Radioactivity was determined by liquid scintillation with 10 ml of modified Bray's scintillation fluid, using the channel ratio method of quench correction for materials containing single isotopes and external standardization with an external source for samples with 3 Abbreviations: PA: phosphatidic acid; PI: phosphatidylinositol; PC: phosphatidylcholine; PE: phosphatidylethanolamine; PG: phosphatidylglycerol; DPG: diphosphatidylglycerol; NPE: N-acylphosphatidylethanolamine; DG: diglyceride; TG: triglyceride; DAF: days after flowering;

Plant Physiol. Vol. 57, 1976

WILSON AND RINNE

376

molecule is labeled by glycerol-2'4C, nearly the same order of lipid class radioactivity is established with acetate-2'4C. After 2 hr of acetate-214C labeling, the phospholipids in decreasing order of incorporation are NPE, PC, TG, DG, PI, PE, PG + DPG, and PA. Rates of acetate and glycerol incorporation (pmoles/hr/mg RESULTS fresh weight) at 25 C for the described fractions of Harosoy 63 Incorporation of acetate-214C and glycerol-2'4C at 25 C into at 30 DAF have been calculated in Table II. Linear regression CO2, water-soluble, insoluble, and total lipid fractions by soy- coefficients of 0.9 or greater were found in all cases. Incorporabean cotyledons of "Harosoy 63" at 30 DAF are compared in tion of glycerol-2'4C into lipid was observed also at 15 C (Table Table I. The observed labeling patterns of acetate-2'4C are III). The rate of glycerol incorporation by individual lipid at similar to those reported (8). After 5 min of incubation with 15 C was approximately half the rate observed for glycerol at acetate, the total lipid fraction contains 73.8% of the incorpo- 25 C. rated radioactivity. Glycerol-214C also enters the lipid fraction Because the synthetic rates reported for the individual lipid rapidly. After 5 min of incubation, 17.3% of the incorporated classes also were affected by the degradation rate of the species, glycerol carbon appears in the lipid-glycerol and increases to the degradation rate of the various lipid classes was defined at 15 75.7% of the total incorporated dpm at 4 hr. Table II illustrates and 25 C, using two different techniques, "pulse chase" and 3H, the radioactivity from acetate and glycerol incorporated into the '4C-substrate incorporation. In pulse chase studies with acetatevarious lipid fractions. Although a different portion of the lipid 2'4C, peak specific radioactivities (dpm/,umole) in lipid classes of

dual isotopes. All experiments were conducted at least twice and the results were representative of those replications. Computations and statistical analyses were performed with a Wang 600 programmable calculator.

Table I. Incorporation of Acetate-2 14C and Glycerol-2 14C by Developing Soybean Cotyledons Excised cotyledons 500 mg fresh wt) var. "Harosoy 63" at 30 DAF were incubated with either acetate-2'4C (0.5 pACi, 10 nmoles) or glycerol-214C (1 ,uCi, 0.1 ,tmole) at 25 C in 0.1 M MES buffer, pH 5, in a final volume of 1.5 ml. The rate of incorporation was calculated from the slope of regression analysis of data for each lipid class.

Time of Incorporation

Fraction

5

15

30

60

120

240

Rate of

min

min

min

min

min

min

Incorporation

dpm

x 10 3

pmoles/hr/mg fresh weight

Acetate-2

C a

c02

0.1

0.1

0.3

0.6

3.2

...

Solids

5.3

11.0

18.7

31.1

44.3

...

0.45

Soluble

19.8

42.1

54.3

69.2

89.1

...

0.67

Total Lipid

70.8

200.6

331.9

557.8

742.0

...

6.67

96.0

253.9

405.2

658.6

878.7

...

co2

0.0

0.1

0.4

1.4

7.1

14.1

0.46

Solids

0.4

1.1

2.4

9.1

27.1

85.4

2.05

Soluble

64.0

102.9

69.9

93.2

120.0

180.4

2.74

Total Lipid

13.5

37.6

66.0

149.9

353.2

887.8

22.50

77.9

141.7

148.7

253.6

507.4

1147.7

0.03

Total Water

Total Glycerol-214C

Total Water

Total a

Not

determined.

377

SYNTHESIS AND DEGRADATION OF LIPIDS

Plant Physiol. Vol. 57, 1976

Table II. Incorporation of Acetate-2"4C and Glycerol-2'4C into Lipid Classes by Developing Soybean Cotyledons as described in Table I. media Reaction Time of Incorporation

Lipid Class

5

15

30

min

min

min dpm

x

60

120

240

min

min

min

Rate of

Incorporation| pmoles/hr/mg

10

fresh

Acetate-2

weight

C

Phosphatidic acid

0.3

0.6

0.3

0.2

0.1

...a

0.13

Phosphatidyliinositol

0.3

0.9

1.0

3.8

5.6

...

0.70

Phosphatidylcholiine

2.0

5.9

8.8

14.9

17.3

...

1.20

PhosphatidyIethanoIam ifne

0.1

0.6

2.7

2.9

5.7

...

0.36

Phosphatidylglycerol/Cardiolipin

0.3

0.5

1.2

1.3

0.7

...

0.42

N-acylphosphatidylethanoI8amifne

1.9

6.0

16.8

18.8

24.0

...

1.62

Diglyceride

0.9

2.3

5.0

4.5

5.7

...

1.78

11.5

...

1.11

70.6

...

...

1.91

0.43

0.5

0.8

1.1

3.3

6.3

17.6

36.9

49.7

Phosphatidic acid

0.18

0.17

0.06

0.36

0.37

Phosphatidylinositol

0.07

0.27

0.56

1.01

1.58

9.06

0.92 4.89

Triglyceride Total

Glycerol-2 14C

Phosphatidylcholine

0.21

0.69

1.31

3.01

10.20

20.68

Phosphatidylethanolamine

0.03

0.14

0.22

0.42

2.75

4.89

1.22

Phosphatidylglycerol/Cardiolipin

0.03

0.05

0.08

0.34

0.95

1.99

0.47

N-acylphosphatidylethanolamine

0.29

1.19

2.69

6.30

23.85

31.21

8.34

Diglyceride

0.37

0.82

1.24

2.10

5.93

8.26

2.00

Triglyceride

0.07

0.10

0.25

0.70

3.87

8.06

1.25

3.43

6.41

14.24

49.50

86.06

Total a

1.97 ...

Not determined.

Harosoy 63 cotyledons were recorded at zero time for PA and DG, between 15 and 30 min for PC, between 30 min and 1 hr for PI and NPE, and at 12 hr for PE and TG (Table IV). When glycerol-2'4C was replaced with cold glycerol, peak specific radioactivities at 25 C were found at zero time for PA and DG, at 30 min for PE, at 1 hr for PI and PC, between 1 and 2 hr for NPE, and at 12 hr for TG (Table V). Although the specific radioactivities of lipid classes from pulse-labeling glycerol at 15 C were reduced, peak activities for PA, PI, PC, and TG occurred at the same time interval as at 25 C. DG, however, reached its highest level after 15 min, NPE at 1 hr, and PE at 4 hr (Table V). Hyperbolic curves were derived from the plot of the velocity of specific radioactivity decrease (ASA/At) with time at both temperatures for each lipid class, except TG which obeyed zero order kinetics. The decay rate (k) of each lipid species displaying first order kinetics was calculated from formula 1 (2): k = 2.3(M) log (SA2/SA)/t2 -tl

(1)

where k = the decay rate (,umoles/hr/500 mg fresh weight); M = the concentration of the lipid species present; SA, = the specific radioactivity at ti; and SA2 = the specific radioactivity at t2. The degradation of TG was interpreted by formula 2 (2):

= M(ASA/At). (2) acetate-23H/acetate-2'4C of ratio the In dual labeling studies, dpm and glycerol-23H/glycerol-2'4C dpm at 25 and 15 C was calculated from each lipid class. In Table VI the ratios developed by dual labeling were compared with degradation values from pulse-labeling experiments. Although the ratios from dual labeling carry no units or distinction of polarity, in most cases the absolute values were not different from pulse decay coefficients. This observation held true for two different temperature conditions. It was assumed that the ratio of the dual label provided not only a relative value, but also a quantitative degradation value which was defined by the kinetics of the molecule in question. Degradation rates of fatty acids within a lipid class, measured by dual acetate experiments, were ranked NPE DG > PI ' PC 2 PA > PE > TG (Table VI). Lipid class glycerol degradation determined by dual glycerol experiments at 25 C indicated the degradation of PA was > DG > PC = PI > PE > NPE > TG. The degradation rates of the lipid classes also were expressed in terms of t1/2 (Table VII), which is given by formula 3 (2): (3) t1/2 = 0.693(M)/k. Half-life (t12) of a specific type of phospholipid molecule is the time. required by the tissue to utilize or degrade one-half of the endogenous concentration found in a given tissue mass.

k

Reaction media

Plant Physiol. Vol. 57, 1976

WILSON AND RINNE

378 as

Table III. Incorporation Rate of Glycerol-2'4C into Lipid Classes of Developing Soybean Cotyledons described in Table I, at 15 C, using var. "Steele."

Time of Incorporation

LiDid Class

30

60

120

240

Rate of

min

min

min

min

Incorporat ion

dpm

x

104

pmo I

es/hr/mg

fresh weight

Phosphatidic acid

0.93

0.95

I

.23

1.79

0.23

Phosphatidyl inositol

0.40 0.80

I

.35

2.55

0.54

Phosphatidylchol ine

1.44 3.90

6.36

10.87

2.36

Phosphat i dy I ethano I am i ne

0.07 0.19

0.48

0.84

0.20

Phosphatidylglycerol/Cardiol i pin

0.19 0.50

0.84

.65

0.40

N-acy I phos phat i dy ethanol amine

2.32

7.50

13.04 25.66

5.89

Diglyceride

2.27

3.03

3.68

4.87

0.65

Triglyceride

0.19 0.63

1.62

4.92

I

I

.25

Table IV. Specific Radioactivities of Lipid Classes of Developing Soybeans Pulse-labeled with Acetate-214C Excised cotyledons (500 mg fresh wt) var. "Harosoy 63" at 30 DAF were pulsed with acetate-214C (0.5 uCi, 10 nmoles) in 0.1 M MES buffer, pH 5, at 25 C for 15 min in a final volume of 1.5 ml. The incubation media were removed, cotyledons were washed with distilled H20, and then incubated in 0.1 M MES buffer, pH 5, containing 2.5 ,umoles of sodium acetate at 25 C in a final volume of 1.5 ml.

0

LiPid Class

min

15

30

60

120

180

240

720

min

min

min

min

min

min

min

dprn x 10

Ip/womle/500 0.9

0.1

0.0

0.0

0.0

3,4 4.7

3.8

3.6

3.4

2.3

7.2

7.0

5.1

5.2

5.2

4.1

1.3

1.4

1.5

1.6

1.9

2.2

3.0

1.6

2.4

2.7 2.5

2.4

2.3

2.3

1.2

22.1

10.5

10.2

9.6

9.5

8.6

6.4

...

0.1

0.13

0.17

0.25

0.28

0.54

Phosphatidic acid

1.9

Phosphatidyl inositol

2.6

...

Phosphatidylcholine

5.4

7.3

Phosphatidylethanolamine

1.2

1.0

mg fresh weight

1.5 a

N-acy I phos phat i dy I -

ethanolamine

Diglyceride

Triglyceride

0.06

0.09

Not analyzed. where E = the tissue concentration of a component, units/mass; DISCUSSION = a zero order rate constant of synthesis, units/time/mass; kd = be and The relation of synthesis expressed by kS degradation may a first order rate constant for degradation given as time'/mass; a simple formula, 4 (1): and assuming no change in total tissue mass occurs during dt. = dE/dt kS kdE (4) Although the rate of synthesis of lipid molecules is dependent on

Thipeforelation 4fs(nthesis

Plant Physiol. Vol. 57, 1976

379

SYNTHESIS AND DEGRADATION OF LIPIDS

Table V. Specific Radioactivities of Lipid Classes of Developing Soybeans Pulse-labeled with Glycerol-2'4C Excised cotyledons (500 mg fresh wt) var. "Harosoy 63" and "Steele" at 30 DAF were pulsed with glycerol-214C (1 ,uCi, 0.1 mole) in 0.1 M MES buffer, pH 5, at 25 and 15 C, respectively, for 15 min in a final volume of 1.5 ml. The incubation media were removed, cotyledons were washed with distilled H20, and then incubated at the respective temperatures in 0.1 M MES buffer, pH 5, containing 25 umoles of glycerol in a final volume of 1.5

0 Lipid Class

15

30

60

120

180

240

720

min min

min

min

min

min

min

min

4/mole/500

dpm x 10

mg fresh weight

Harosoy 63 (25 C) Phosphatidic acid

2.70

1.90

1.60

1.30

1.10 0.80 0.50

Phosphatidylinositol

0.63

0.77

0.84 0.94 0.87

0.74 0.59 0.55

Phosphatidylcholine

0.95

1.15

Phosphatidylethanolamine 0.28

0.37

0.56 0.41

0.78

0.85

a .

.

.

1.50

1.31

1.28 0.99 0.79 0.43

0.41

0.44

N-acy I phosphati dy I -

ethanolamine

0.40

0.89 0.89 0.74 0.75

0.73

Diglyceride

4.17 2.89

1.95 2.04 2.13

1.69 2.10

1.54

Triglyceride

0.01

0.02

0.02

0.03

0.06

0.08

0.18

Phosphatidic acid

3.98

2.17

1.47

1.04 0.75 0.22

0.10

0.09

Phosphatidylinositol

0.08

0.15

0.22

0.26

0.16

0.16 0.21

0.24

Phosphatidylcholine

0.24 0.85

1.12

1.60

1.19

1.01

0.08

0.09 0.16

0.13

0.15 0.18 0.17

0.15

0.49

0.57

0.77

0.70

0.59

0.69

0.47

Diglyceride

0.53

0.80

0.78

0.73

0.51

0.37

0.27

0.22

Triglyceride

0.003 0.005 0.01

0.02

0.02

0.03

0.03

0.06

0.05

Steele (15 C)

Phosphatidylethanolamine 0.03

1.24 0.64

N-acylphosphatidylethanolamine

a

Not analyzed.

several factors, the absolute rate of degradation always will be a function of E. In a steady state, i.e. dEldt = 0, k, = kdE and E = k,lkd. Therefore, the amount of lipid is a function of both rate of synthesis and rate of degradation. Several techniques may be utilized to measure these parameters for individual lipid classes. Curstadt and Sjovall (3) employed GC-mass spectral analysis of biliary PC species labeled by (1,12H2) ethyl alcohol to distinguish fatty acid and lipid-glycerol turnover of the PC pool in rat. Pulse labeling procedures have been used to define lipid precursor-product relationships in plant tissues. Dybing and Craig (5) chose acetate-1 14C and malonate-'4C to demonstrate the relation of polar lipids and diglycerides in TG biosynthesis by developing flax embryos. Roughan (7) reported turnover of pumpkin glycerolipids pulsed with either '4CO2 or acetate-I 14C. Kagawa et al. (6) have calculated the t1/2 of PC in mitochondria and glyoxysomes of excised castor bean endosperm cotyledon halves, at 50

and 10 hr respectively, using pulse chase experiments with choline-'4C.

In developing soybean cotyledons, acetate and glycerol are preferentially used to label separate portions of the lipid molecule, fatty acid, and glycerol, respectively. Phospholipids pulsed with acetate display decay kinetics similar to the experiments of Roughan (7). The decay coefficients (,umoles/hr.mg fresh weight) of lipid classes labeled at 25 C by acetate or glycerol indicate rapid degradation of PA, DG, intermediate rates for PC, PI, PE, and lowest values for TG. At a lower temperature, 15 C, the lipid synthetic rate was cut nearly 2-fold, and the t112 of all lipids except PA and extended nearly 2-fold. Although there was little difference between the t1/2 of lipid-glycerol and fatty acids of PA, PI, PC, PE, and DG, fatty acids of NPE were metabolized more rapidly than the glycerol moiety. Dual label studies at both temperatures verified these trends.

WILSON AND RINNE

380

Plant Physiol. Vol. 57, 1976

Table VI. Comparison of Pulse Labeling Degradation Rates and Dual Labeling 3H/14C Ratios of Lipid Classes in Developing Soybean Cotyledons Pulse labeling degradation rates were calculated from the slope of the decay curve of the respective data given in Tables IV and V. In dual label experiments, 3H/14C ratios were determined from the dpm incorporated into each lipid class using either acetate-2'4C (0.5 ,uCi, 10 nmoles) and acetate-23H (5 ,uCi, 10 nmoles) or glycerol-2'4C (1 ACi, 0.1 umole) and glycerol-23H (20 uCi, 0.1 iLmole). Cotyledons (0.5 g fresh wt) var. "Harosoy 63" (30 DAF) incubated at 25 C or var. "Steele" (30 DAF) incubated at 15 C were exposed to '4C substrate for 2 hr in 0.1 M MES, pH 5, at a volume of 1.5 ml. After 2-hr incubation, the 3H-labeled substrate was introduced to the reaction without removal of 14C. The incubation was continued for 3 to 15 min at a new volume of 1.6 ml, then terminated.

Steele

Harosoy 63

Pulse

Glycerol

Glycerol

Acetate 3

H/ 14CC Pu Ise

Lipid Class

ratea ratio

Phosphatidic acid

-2.18

Phosphatidylinositol Phosphatidylcholine

3

H/I 14CC Pulse

3

H/ 147C

rate

ratio

rate

ratio

1.06

-3.43

3.31

-5.01

5.83

-1.54

1.53

-1.13

1.07

-0.25

0.27

-1.04

1.00

-1.45

1.40

-0.74

0.50

Phosphatidylethanolamine +0.83

0.83

+0.55

0.59

+0.31

0.35

-1.66

1.99

-0.50

0.47

-0.87

0.54

Diglyceride

-1.53

1.60

-2.61

2.33

-1.73

2.00

Triglyceride

+0.70

0.55

+0.31

0.20

+0.27

0.29

N-acy I phosphat i dy I -

ethanolamine

a

4moIes/hr/500

mg

fresh weight.

Table VII. Half-Life of Lipid Classes in Developing Soybean Cotyledons Values are calculated from data in Table VI.

Steele

Harosoy 63

Fatty

Lipid Class

acid

Glycerol

Glycerol

t, (hr)

Phosphatidic acid

0.08

0.10

0.02

Phosphatidylinositol

0.37

0.48

1.75

Phosphatidylcholine

0.57

0.46

0.71

Phosphatidylethanolamine

0.53

0.52

0.94

N-acylphosphatidylethanolamine

1.41

3.40

4.27

Diglyceride

0.09

0.08

0.29

The dual labeling procedure, originated by Arias et al. (1) to determine protein turnover of rat liver endoplasmic reticulum, has not been applied previously to plant tissue. The technique was modified here to measure lipid turnover in developing soybean cotyledons. 3H and 14C isotopic species of acetate and glycerol having different specific radioactivity were administered for different periods of time in the same tissue. Soybean cotyle-

dons (30 DAF) were incubated with 14C substrate (low specific radioactivity) for 2 hr, followed by short exposure of 3H substrate (high specific radioactivity). The shortest period of 3H incubation was selected that gave approximately equal 3H dpm incorporated as 14C dpm in the total lipid fraction. About 10 to 20 umoles of 14C substrate was incorporated per ,umole of 3H substrate, therefore, the probability of 14C product metabolism

Plant Physiol. Vol. 57, 1976

SYNTHESIS AND DEGRADATION OF LIPIDS

was high. The period of 3H exposure was kept short because a lipid class which was degraded rapidly also had to be synthesized rapidly and would have higher specific radioactivity following a short period of 3H substrate incorporation. The ratio of acetate3H/acetate-'4C and glycerol-3H/glycerol-14C dpm incorporated by a specific lipid class determined the metabolic activity of the compound. Lipids exhibiting high turnover had high 3H/14C rates, while those having low turnover had low rates. With the exception of PA in cotyledons exposed to acetate at 25 C, the 3H/'4C ratios derived for each lipid class were similar to degradation rates calculated by pulse labeling with the same substrates. The degradation rate for PA from acetate-'4C in both soybean lines was higher than the 3H/'4C acetate ratio at 25 C. When the 3H-acetate incubation period was shortened from 15 to 3 min using Harosoy 63 cotyledons (30 DAF), the 3H/'4C ratio of PA became 1.93. This indicated that fatty acids on PA turned over too rapidly to measure accurately with 15 min 3H exposure, yet with proper modification the technique gave accurate estimation of even very rapidly degraded lipids. Soybean cotyledons (30 DAF) incubated with either labeled acetate or glycerol at pH 5 demonstrate extremely high lipid synthetic activities. As a group, phospholipids receive a large portion of the label in relation to neutral lipids. The apparent synthetic rates of these lipid classes do not distinguish differences in metabolic activity between classes; however, metabolic activities can be determined through definition of degradation rates for each class. In these expenments degradation rates for individual phospholipids and neutral lipids are calculated by two methods, pulse chase and 3H/'4C substrate labeling. The absolute values for turnover of acyl groups and lipid-glycerol are in agreement from both techniques using different soybean lines at two different temperatures. Decay of all phospholipid and DG classes displays

first order kinetics. Half life (t12) calculations from the decay coefficients indicate extremely rapid turnover rates and suggest similar t112 of acyl groups and lipid-glycerol in DG and all phospholipids except NPE where acyl groups are replaced independent of the NPE-glycerol moiety. These experiments reveal not only different metabolic activities between lipid components, but also describe a new method for measuring these parameters in plants. The dual label technique has been shown to give accurate estimation of lipid degradation of soybean lipids in developing cotyledons. This method has the advantage of rapid analysis of lipid degradation in a single tissue sample (whereas several samples are required for pulse labeling) and of fewer laboratory technique requirements. The method has been used to determine protein as well as lipid degradation rates and has high potential application in studies of degradation of other cell components. LITERATURE CITED 1. ARIAs, 1. M., D. DoyLE, AND R. T. SCHIYE. 1969. Studies on the synthesis and degradation of proteins of the endoplasmic reticulum of rat liver. J. Biol. Chem. 244: 3303-3315. 2. ARONOFF, S. 1961. Techniques of Radiobiochemistry. Iowa State University Press, Ames. p. 87. 3. CURSTADT, T. AND J. SJOVALL. 1974. Biosynthetic pathways and turnover of individual biliary phosphatidylcholines during metabolism of (1,1-2H2) ethanol in rat. Biochim. Biophys. Acta 369: 173-195. 4. DEHLINGER, P. J. AND R. T. SCHIsKE. 1971. Size distribution of membrane proteins of rat liver and their relative rates of degradation. J. Biol. Chem. 246: 2574-2583. 5. DYING, C. D. AND B. M. CRAIG. 1969. Fatty asid biosynthesis and incorporation into lipid classes in seeds and seed tissues of flax. Lipids 5: 422-429. 6. KAGAWA, T., J. M. LowD, AND H. BEEVERS. 1973. The origin and turnover of organelle membranes in castor bean endosperm. Plant Physiol. 51: 61-65. 7. ROUGHAN, P. G. 1970. Turnover of the glycerolipids of pumpkin leaves. Biochem. J. 117: 1-8. 8. WILSON, R. F. AND R. W. RINNE. 1974. Phospholipids in the developing soybean seed. Plant Physiol. 54: 744-747. 9. WILSON, R. F. AND R. W. RINNE. 1976. Effect of freezing and cold storage on phospholipids in developing soybean cotyledons. Plant Physiol. 57: 270-273.