Planta

Planta 149, 4 6 " 7 1 (1980)

9 by Springer-Verlag 1980

Phospholipid Composition and Fatty Acid Desaturation in the Roots of Rye During Acclimatization of Low Temperature Positional Analysis of Fatty Acids D.T. Clarkson, K.C. Hall, and J.K.M. Roberts* Agricultural Research Council Letcombe Laboratory, Wantage OX12 9JT, U.K.

Abstract. When the roots of rye plants grown at 20 ~ C were cooled to 8 ~ C the concentration of phospholipid in them more than doubled over a 7 d period in comparison with that in roots remaining at 20 ~ C. The relative abundance of lecithin (PC) declined while that of phosphatidyl ethanolamine (PE) increased; this change was completed after 2 d cooling. Labelling with 32p suggested that turnover of phospholipids may be inhibited by low temperature. Acyl lipids contained an increased proportion of linolenic acid (18:3) and reduced proportion of linoleic acid (18:2) when roots were cooled at 8 ~ C for 7 d. The ratio of these acids is a relatively more sensitive indicator of desaturation than is the double bond index. Cooling brought about no change in the abundance of the principal saturated acid, palmitic (16:0). In the first 3 days of cooling PC and PE desaturated markedly while there was no change in galactosyl and neutral lipids. Desaturation did not appear to be greatly sensitive to the concentration of dissolved 02 and was only partly inhibited in 8~ C solutions where the oxygen concentration was lowered to 0.5-2.0%. Positiona l analysis of acyl chains in PC and PE showed that more than 90% of all 16:0 is associated with position I while 65% of the 1 8 : 2 + 1 8 : 3 is associated with position II. When roots are cooled the abundance of 18 : 3 increases in both chains but the relative distribution of saturated and unsaturated fatty acids remains constant in positions I and II. At both 20~ and 8 ~ C there is a high probability that a saturated chain in position I will be paired with the polyunsaturated one in position II. Key words: Acclimatization - Fatty acid desaturation - Phospholipids - Secale. * P r e s e n t a d d r e s s : Biological Science Department, Stanford University, Stanford Ca. 94305, USA

PC=Lecithin; PE=phosphatidyl ethanolamine; TLC = thinlayer chromatography; BHT = butylated hydroxytoluene Abbreviations:

0032-0935/80/0149/0464/$01.60

Introduction Changes in the composition of the fatty acid components of membrane phospholipids are recognized as important in the acclimatization of all types of organisms to temperature (Simon 1974; Kuiper 1974; Martin et al. 1976) even though such changes may not be obligatory in all organisms (Cronan and Gelmann 1975). The composition may alter by variations in the chain length and in the number of double bonds in the chain, apparently to maintain an approximately constant fluidity in the hydrocarbon domaine of membranes over the range of environmental temperature in which the organism can live. This is probably one of several factors in maintaining membrane permeability (Kuiper 1974) and in regulating the activity of membrane-associated enzymes (Raison 1973; Cronan and Gelmann 1975). There is physiological evidence that membrane transport processes in the roots of plants can become adapted to environmental temperature. Water movement through roots of Phaseolus vulgar& (Kuiper 1964), Glycine max and Brassica oleracea (Markhart et al. 1979) and ion transport in Hordeum vuIgare and Secale cereale (Clarkson et al. 1974; Clarkson 1976) at any given temperature seem to be greatly increased in roots which have been acclimatized at cool temperatures. Studies of Arrhenius plots of the effect of temperature on ion movement into the xylem and the volume flow of sap exuding from detached root systems of barley and rye indicated that the relationship between temperature and material fluxes showed a very marked increase in sensitivity below a certain temperature (Clarkson 1976). This varied according to the temperature at which the roots had been grown. These breaks in the Arrhenius plots depended on the preceding growth temperature, and thus conformed with a commonplace, but rarely tested, idea that they are related to phase transitions

D.T. Clarkson et al. : Phospholipid and Fatty Acid Changes in Secale Roots i n t h e h y d r o c a r b o n d o m a i n e s o f t h e cell m e m b r a n e s ( L y o n s et al. 1 9 6 4 ; R a i s o n a n d C h a p m a n 1976). We now examine the influence of growth temperature on the phospholipid species present in roots, and the extent of desaturation of their fatty acids, paying particular attention to the time course of any changes which occur in relation to the changes in rates of ion transport and hydraulic conductivity ( C l a r k s o n et al. 1 9 7 4 ; C l a r k s o n 1976). W e a l s o describe, for the first time in root material, the separate c o m p o s i t i o n o f t h e acyl c h a i n s o n t h e f i r s t a n d s e c o n d carbon atoms of lecithin (PC) and phosphatidyl etha n o l a m i n e ( P E ) i n a n a t t e m p t t o see w h e t h e r b o t h chains are equally affected by desaturase activity when roots are cooled.

Materials and Methods (a) Plant Material and Culture Conditions. Rye (Secale cereale, cv. lovatzpatoni) seedlings were germinated on trays lined by damp filter paper at 20~ and transferred when 24 h old to a water culture system (for details see Clarkson 1976) where they grew for 11-13 d with roots and shoots at 20 ~ C. At the beginning of the experimental period plants were placed in fresh culture solution maintained either at 20~ or at 8~ C; the shoots in both cases being at 20 ~ C. In most experiments the solutions were aerated with oil-free compressed air, but in one series of experiments nitrogen was bubbled through the solution. (b) Labelling with 32p and Phosphate Analysis. In the first experiment, described in the Results, plants were treated at 8~ C and 20 ~ C for 8 d in culture solution containing 10 mmol m -3 phosphate labelled with 56.10 -* Bq 32p 1-1. Radioactivity and total phosphate in phospholipids separated by TLC (see below) were estimated after elution, drying and digestion in a small volume of concentrated perchloric acid: nitric acid mixture (2:3). 3zp was counted on planchettes by a low background end window counter (Beckman) and total P was estimated, after the removal of silica, by an automated procedure based on the phosphomolybdate blue test. (e) Lipid Extraction. Roots were rinsed in distilled water, the surplus water was removed and they were then placed in cold mortars containing liquid nitrogen. The brittle frozen plant material could be quickly ground to a fine powder in the presence of the liquid N2. When the N2 had evaporated the plant material was ground in a 2 : 1 mixture by volume of chloroform/methanol, containing 45 gg ml-1 nupercaine (phospholipase A inhibitor see Moreau et al. 1974) and cooled to < - 2 0 ~ C, but subsequently allowed to warm up to 10~ C. All subsequent steps for separating lipids from aqueous-methanol-soluble components were as described in Folch et al. (1957). Semi-Purified lipids were made up to a standard volume in chloroform and stored under N2 at - 2 5 ~ C. (d) Thin Layer Chromatography. Aliquots of lipid extract containing an estimated 2 4 mg of lipid were evaporated to a small volume under a stream of N2 and spotted onto plates of Silica Gel G (Anachem) in a Nz atmosphere. The lipids were separated by twodimensional chromatography. Solvent 1 contained chloroform/ methanol/ammonia (7N) (65 : 30:4 plus 50 ~g ml 1 BHT; solvent 2 was chloroform/methanoI/acetic acid/water (170:25 : 25 : 3 plus 50 lag m l - i BHT). The plates were dried after development in

465

a vacuum oven at 35~ C. When phospholipids were labelled with 32p they could be located by placing the dried plate against prepacked X-ray film (Kodirex) in a sealed box with a Nz atmosphere for 2 4 h. Otherwise, the plates were lightly sprayed with 0.05% dichlorofluorescein in dry methanol and viewed under UV light.

(e) Positional analysis of Fatty Acids. In some experiments phospholipids separated TLC were scraped from the plates and drawn, by a slight vacuum, into a short glass column made from a pasteur pipette. The silica gel was then treated with 5 ml chloroform followed by 5 ml chloroform/methanol (1 : 1), the eluate was evaporated to dryness under a stream of nitrogen gas and taken up in 1 ml of dry, peroxide-free diethyl ether containing 5% methanol, transferred to a 15 ram. 125 mm test tube and treated with 20 gl of an aqueous solution of rattlesnake, Crotalus adamanteus, venom (1 mg venom m1-1 of 100mol m -3 Tris buffer, pH 8, plus 10 10 mmol m - 3 CaC12). The hydrolysis was allowed to proceed overnight at 20 ~ C; the ether was then evaporated in a stream of nitrogen and the dried residue taken up in approximately 50 tal chloroform/methanol (1 : 1) and applied to the starting line of Silica gel H (Anachem) plates. The plates were developed in chloroform/methanol/ammonia (7N) (230:90:15) and the spots, corresponding to the hydrolysis products, lysophospholipid and free fatty acids from the second carbon atom (position II), were detected by UV light after lightly spraying the vacuum-dried plates with 0.05% dichlorofluorescein. The free fatty acids, running near the solvent front, were eluted from the silica gel with chloroform and the lysophospholipids, left near the starting line, were eluted with chloroform/ methanol (1 : t). (f) Preparation of Methyl Exters of Fatty Acids. Samples of phospholipids and free fatty acids were evaporated to dryness under vacuum in 50 ml round-bottom flasks. Hydrolysis of the lipids and methylation of fatty acids using boron trifluoride followed the procedure of van Wijngaarden (1967). (g) Gas Chromatography. Methyl esters in 1 2 gl cyclohexane or N-heptane were injected onto the top of a 2.5 m. 3.2 mm stainless steel column of a cyanosilicone (SP 2340 - Supelco), initially at 170~ C for 5 min. and thereafter increasing to 200 ~ C at the rate of 2 ~ C min- 1. Peak areas were integrated using either Infotronics CRS 304 or Sigma 10 Data Station (Perkin Elmer). Purified methyl esters and mixtures of known composition were used to tentatively identify peaks. Selected samples were analysed further by GC/mass spectrometry to confirm the identity of the major peaks (We are indebted to Dr. H. Anderson and colleagues at The Macauley Institute for Soil Research for this service).

Results Lipid Composition o f the Roots. T h e t o t a l l i p i d extracted from roots which had been cooled somewhat exceeded that from 20~ controls but there was a much more striking effect on the amount of phospholipid which more than doubled after 7 days treatment ( T a b l e 1). M o r e l a b e l l e d p h o s p h o l i p i d w a s o b t a i n e d f r o m c o o l e d r o o t s t h a n f r o m t h o s e g r o w n a t 20 ~ C. There were increases in the amounts of three p h o s p h o l i p i d s ( T a b l e 1). T h e c o n c e n t r a t i o n o f P C d o u b l e d d u r i n g 7 d a y s a t 8 ~ C ; t h i s w a s less t h a n t h e r a t e o f i n c r e a s e o f t o t a l p h o s p h o l i p i d . P E increas~ ed in concentration more than three-fold while PG i n c r e a s e d a t t h e s a m e r a t e as t h e t o t a l lipid. T h u s

466

D.T. Clarkson et al, : Phospholipid and Fatty Acid Changes in Secale Roots

Table 1. Yields of total and labelled lipid phosphorus in various phospholipids in roots of rye grown at 20 ~ C and 8 ~ C Lipid species

Total phosphorus

Labelled phosphorus

nmol g 1 root flesh weight (% distribution) 20 ~ C control

8~ C 2d

8~ C 7d

20 ~ C control

80 C 7d

Lecithin (PC) Phosphatidylethanolamine (PE) Phosphatidylglycerol (PG) Phosphatidylinsitol (PI) Phosphatidic acid (PA) Spot 9 - diphosphatidylglycerol? (DPG) Spot 1 - phosphatidylserine? (PS)

235 (60) 90 (23) 23 (6) 20 (5) 13 (3) 13 (3) nd -

309 (51) 222 (37) 46 (8) 7 (1) 10 (2) 3(<1) 6(
495 (51) 319 (33) 65 (7) 36 (4) 22 (2) 16 (2) 11 (1)

179 (63) 43 (15) 19 (7) 23 (9) 9 (3) 4 (1) 5 (2)

234 (52) 118 (28) 54 (12) 29 (6) 8 (1) 3(<1) 3(<1)

Total lipid P

392

603

964

-

Total labelled P specific activity of labelled P (mmol m 3 32p/mmol m - 3 p)

-

-

-

281 (0.72)

Total lipid (mg g - 1 root fresh weight)

2.40

4.02

449 (0.47)

3.81

Table 2. Comparison of the fatty acid composition of major phospholipids from roots of rye grown for 8d at 20 ~ C or 8 ~ C Fatty acid

Percentage composition Growth temperature 20 ~ C

Palmitic Palmitoleic Stearic Oleic Linoleic Linotenic Others Bond index a

16:0 16 : 1 18 : 0 18 : 1 18 : 2 18 : 3

8~ C

PC

PE

PG

PI

PC

PE

PG

PI

26.0 nd tr 1.8 48.1 23.3 0,8

20.3 1.8 tr 1.2 51.1 24.4 1.2

42.3 4.2 19.7 .2.8 8.0 18.1 4.9 a

43.6 tr 1.0 2.3 28.7 18.8 5.6"

24.4 nd 0.4 3.3 33.0 38.8 0.1

21.4 4.5 0.3 2.9 31.2 38.7 0.9

42.4 1.6 2.0 3.3 15.7 30.0 5.0"

37.2 0.5 2.0 5.3 23.7 30.4 0.9

159

178

79

117

186

186

129

144

includes peaks tentatively identified as 14:0 and 17:0

the proportion of P C declined while that of PE increased and PG remained constant. The change in relative proportions of PC and PE occurred in the first 2 days of cooling. It should be noted that phosphatidic acid was a very minor component at all times and this is taken to indicate minimal phospholipid hydrolysis by phospholipase D (Mazliak 1973).

Fatty Acid Composition of Phospholipids. The principal fatty acid components of the major phospholipids (Table 2) from roots grown for 8 days at 20 ~ C and 8~ C were palmitic (16:0), linoleic (18:2) and linolenic (18: 3), with stearic (18:0) being an important component in PG at 20 ~ C. Growth at lower temperatures increased the double bond index in every case, the

effect being least marked with PE, which was the most unsaturated at 20 ~ C, and most marked in PI and PG. In each phospholipid the principal change was in the relative proportion of 18:2 and 18:3. This is illustrated in Fig. 1, where it can be seen that during cooling the increase in 18:3, in both PC and PE, was about equivalent to the decrease in 18:2, the abundance of 16:0 was unchanged. A fuller analysis o f the ratio of 18:2/18:3 (Table 3) shows that in PC and PE, where the ratio was high in 20~ grown roots, 3 days at 8 ~ had a very marked effect on the ratio (see also Fig. 1). In the galactosyl and neutral lipids, however, the shift in the ratio became marked only between the fourth and eighth days of cooling (Table 3). Thus the desaturase system had a more

D.T. Clarkson et al. : Phospholipid and Fatty Acid Changes in Secale Roots

467

Positional Analysis of Fatty Acids from PC and PE. 5O

~',

PC .....

. i

PE

40 ",,

*S

. . . .

.

~

18:3

"-...o 18:2

30 E 16:0

13_

20

0

2

4

Time at 8~

6

8

(days)

Fig. 1. Changes in the proportions of palmitic (16 : 0), linoleic (18: 2) and linolenic (18:3) acid associated with lecithin (PC) and phosphatidyl-ethanolamine (PE) extracted from roots of rye grown at 8~ C after an initial period at 20~ C

i m m e d i a t e effect o n the d e s a t u r a t i o n of the phospholipids.

Influence of Dissolved Oxygen on Desaturation. The influence of the c o n c e n t r a t i o n of dissolved oxygen in d e s a t u r a t i o n was e x a m i n e d to see if the process could be p r e v e n t e d by cooling roots in the presence of low c o n c e n t r a t i o n s o f dissolved oxygen. U s i n g b o t h d o u b l e b o n d index a n d 1 8 : 2 / 1 8 : 3 ratio as criteria o f d e s a t u r a t i o n it is clear (Table 4) that cooling roots at a lower oxygen t e n s i o n caused some d e s a t u r a t i o n of the fatty acids b u t it was a p p r e c i a b l y less t h a n that f o u n d at n o r m a l levels o f dissolved oxygen.

Table 3. Ratio of linoleic to linolenic acids (I8:2/18 : 3) in various lipids from rye roots after transfer of the roots from 20~ C to 8~ C

Lipid

PC PE PG PI PA MGDG DGDG Combined neutral lipids Total lipids

I n b o t h lipids there was a very m a r k e d difference in the c o m p o s i t i o n of the acyl chains in the I a n d II positions (Table 5). T h e acyl chains attached at p o s i t i o n I c o n t a i n e d a high p r o p o r t i o n o f 16:0 with far fewer d o u b l e b o n d s t h a n in the chains attached to p o s i t i o n II. W h e n roots were cooled at 8 ~ the following sequence of events occurred in b o t h chains in b o t h lipids. There was a t r a n s i e n t increase in the a b u n d a n c e of oleic acid (18 : 1) which reached a peak after day 2 a n d then declined to a level n o t significantly different from the control. There was a t e n d e n c y for 16:0 to become less a b u n d a n t in p o s i t i o n I. There was a m a r k e d a n d significant ( P < 0 , 0 1 ) increase in 18:3 in all chains b u t an equivalent decrease in the a b u n d a n c e of 18:2 was seen clearly only in p o s i t i o n II. I n general the 1 8 : 2 / 1 8 : 3 ratio was a m o r e sensitive i n d i c a t o r of the effect of cooling t h a n the d o u b l e b o n d index. C o o l i n g roots at 8 ~ C quickly activated the desaturase system (Table 5). I n a further experiment, roots which h a d been cooled for 2 d were replaced in solution at 20 ~ C for 2 m o r e days before extraction a n d positional analysis of fatty acids. D u r i n g the 2 days when the roots were r e t u r n e d to 20 ~ C it is evident that d e s a t u r a t i o n of acyl chains in b o t h positions continued (Table 6) rather t h a n reversed a l t h o u g h it was n o t as great as in roots kept at 8 ~ C for 4 days.

Discussion

Lipid Composition of the Roots. I n view of our interest in ion t r a n s p o r t we w o u l d have preferred to m a k e specific analysis of the p l a s m a m e m b r a n e of rye roots, since this is p r o b a b l y the rate limiting m e m b r a n e . We have n o t a t t e m p t e d m e m b r a n e f r a c t i o n a t i o n since Table 4. Effect of low oxygen concentration on desaturation of lecithin (PC) and phosphatidylethanolamine (PE) treated at 8~ C for 3d Concentration of Bond dissolvedO2~ indexb (%)

Ratio I8 :2/18 : 3

Duration of 8~ C treatment (d)

Lipid Temperanalysed ature (oc)

0

3

4

8

PC

2.06 2.09 0.44 1.53 2.45 1.00 0.92 1.39 1.58

1.27 1,43 0.98 -1.61 1.48

0.91 1.20 1.03 0.95 1.09 1.38 1.05 0.95 1.30

0.85 0.81 0.50 0.78 0.73 0.92 0.45 0.64 0.88

20 8 8

21 2l 0.5-2

159 176 170

1.96 1.25 1.75

PE

20 8 8

21 21 0.5-2

141 174 165

2.32 1.33 1.97

Ratio 18:2/18:3

The percentage by volume of oxygen in the equilibrium gas phase b Calculated as the sum of the products of (% composition of given fatty acid • number of double bonds)

D.T. Clarkson et al. : Phospholipid and Fatty Acid Changes in Secale Roots

468

Table 5. Positional analysis of acyl chains from position I and II of lecithin (PC) and phosphatidylethanolamine (PE) from roots of rye cooled at 8 ~ C Fatty Acid % composition_+ S.E.

18:2/18:3

Bond Index

16:0

18:0

18:1

18:2

18:3

42, 8 _+ 1.9 39.8-+0.7 38.9 -+ 1.4 39.0_+2.1

1,2 _+0.2 2.0-+0.6 2.1 -+ 0.6 1.1 _+0.2

3.6 + 0.7 7.6-+ 1.3 6.3 _+0.5 4.5+0.3

34,7 _+ 1.1 31.6_+0.2 32.4-+ I. 1 31.0_+ 1.5

16.3 _+0.7 17.8-+1.1 19.6 -+ 1.1 23.3_+ 0.9

2.12 1.78 1.65 1.33

122 124 130 136

4.0 _+ 1.1 3.4 _+0.7 3.5_+0.7 4.3_+1.7

1.3 -+ 0.2 0.9 _+0.2 1.2_+0.3 0.8-+0.3

4.9 -+ 0.8 8.5 _+0.9 7.1 _+0.4 5.3_+0.2

57.9 _+ 1.4 51.2 + 1.2 48.0_+2.1 42.2_+0.1

30.3 _+0.8 35.5 _+ 1.6 39.4_+2.8 46.5_+2.4

1.91 1.44 1.22 0.91

216 217 221 229

46.4 -+ 1.5 40.8-+3.0 42.3-+2.7 37.0_+ 0.7

1.7 _+0.3 3.2+_ 1.9 1.9_+0.4 1.1 _+0.3

4.1 + 0.9 7.4+_2.7 5.2_+0.7 4.2 _+0.3

34.2 _+ 1.5 27.7+ 1.5 30.3_+2.2 32.4_+ 1.0

14.0 _+0.8 16.6_+ 1.0 19.1 _+0.9 24.2_+ 0.1

2.44 1.67 1.58 1.34

117 113 123 141

5.1_+0.7 9.2-+2.0 9.8 -+ 2.0 4.5-+2.4

1.7_+0.3 0.9_+0.2 1.6-+ 0.5 1.5-+0.5

2.1_+0.3 3.3_+0.1 3.3 _+0.5 3.0+0.5

60.3_+1.14 51.3_+2.1 48.1 _+2.0 44.5-+1.7

28.3_+1.1 31.2_+1.7 35.9 + 1.4 42.1 _+2.3

2.13 1.65 1.34 1.05

208 199 207 218

a) Position I PC

PE

control 2d 4d 7d Position II control 2d 4d 7d b) Position I

controI 2d 4d 7d Position II control 2d 4d 7d

it seems that it is still not possible to prepare pure plasma membranes from plant cells in the quantities necessary for analysis. Thus, different presumptive plasma membrane markers often sediment differently on density gradient centrifugation (see Quail 1979). Using a "plasma membrane enriched" fraction may add to, rather than diminish, interpretive problems if the proportion of contaminants of different origin varies from occasion to occasion. Without this information however it is not possible to interpret the increase in the relative abundance of PE in the total

Table 6. Influence of a transfer from cool to warm conditions on the ratio of linoleic (18:2) and linolenic (18 : 3) acid in roots of rye; analysis of acyl chains in positions I and II of lecithin (PC) and phosphatidylethanolamine (PE) Lipid species

Treatment

18:2/18:3 ratio Position I Po

PC

20 ~ C control 8 ~ C 2d 8 ~ C 2 d + 2 0 ~ C 2d 8 ~ C 4d

2.35 2.31 1.98 1.58

2.23 1.46 1.30 1.10

PE

20 ~ C control 8 ~ C 2d 8 ~ C 2 d + 2 0 ~ C 2d 8 ~ C 4d

3.15 2.74 2.05 1.50

2.80 2.23 1.87 1.24

lipid extract from roots - an increase in the relative abundance of a given type of membrane is one likely explanation. The substantial increase in the amount of phospholipid and a somewhat smaller increase in the total lipid concentration of roots when they have been cooled for 2-7 days at 8 ~ C suggest that there may be a considerable expansion of total membrane area which is relatively enriched in phospholipids in comparison with roots grown at 20 ~ C. This suggests that the expansion may be of intracellular membranes since they are known to contain relatively more phospholipid than the plasma membrane (Keenan et al. 1973; Mazliak 1977). Thus the endoplasmic reticulum and cytoplasmic vesicles might be probable areas for expansion; in some circumstances the ER is known to increase markedly when growth is slowed down by low temperature (Kimball and Salisbury 1973) and oxygen deficiency (Pomeroy and Andrews 1978). The phospholipid content of plants is generally known to increase in cold conditions (Redshaw and Zalik 1968; Kuiper 1970; de la Roche etal. 1972; Kedrowski and Chapin 1978) and it has been claimed that phospholipid biosynthesis is stimulated during cold hardening (Willemot 1975). In the present work incorporation of 32p at 20 ~ C and 8 ~ C showed that more labelled phospholipid accumulated in cooled roots than in warm ones. From a number of points of view it is unlikely that synthesis is faster at the lower temperature and the result is

D.T. Clarkson et al. : Phospholipid and Fatty Acid Changes in Secale Roots probably explained by slower turnover o f phospholipid at 8 ~ C. The values for the specific activity of labelled phosphate in phospholipid (Table 1) are compatible with this suggestion. More evidence on this matter is required. While rye roots are growing at 8 ~ C their relative growth rate is less than 0.03 d -1. In the 2-7 day period at this temperature the root system could not have increased its weight by more than 25% - it is most unlikely that all of the increased yield of lipid or polyunsaturated fatty acids (see below) could have been restricted to the tissue that formed while the roots were being cooled. We conclude that the lipid composition and concentration of pre-existing cells is likely to change when roots are cooled.

Desaturation of Fatty Acids. Phospholipid species in rye roots may differ markedly from one another in the relative abundance of the common fatty acids and in rate at which the desaturase enzyme system produces 18:3 when roots are cooled (Tables 2 and 3). Desaturation is best measured, therefore, on separated lipid species rather than on whole lipid extracts. Desaturation of PC and PE changes greatly 3 to 4 days before it can be unequivocally detected in whole lipid composition and concentration of pre-existing galactosyl and neutral lipid fractions. It is clear that species differ in this respect. A comparison of permafrost and hot spring populations of Carex aquatilis in Alaska revealed no difference in the desaturation of neutral lipids from roots but a significant difference in phospholipids (Kedrowski and Chapin 1978). Exactly the opposite result was found in roots of Brassica napus grown at low temperature and at 25~ (Smolenska and Kuiper 1977). In general, however, linolenic acid has been found to increase in many species when they are grown in the cold and in some instances, as in the present work, this increase appears to be at the expense of 18 : 2 (de la Roche et al. 1972; Farkas et al. 1975; Smolenska and Kuiper 1977). This change is thought to increase the membrane fluidity by decreasing the scope for orderly packing of acyl chains in the membrane interior (Chapman 1975). Direct evidence for this is mostly from model or microbial systems (eg. James and Branton 1973) and there are relatively few examples from the cells of higher plants where crucial physical observations have been made (eg. Lyons et al. 1964). Our own work (Clarkson 1976) suggested that membrane properties might adapt rapidly when rye roots are transferred from 20 ~ C to 8 ~ C. This suggestion was made to explain a marked shift in the break point of an Arrhenius plot of volume flow in the xylem of detached roots (Jr) at different temperatures. The present work shows that the desaturation of fatty

469

acids associated with the principal phospholipids increased markedly in this period of time and thus may be consistent with the shift in the break in the Arrhenius plot to a lower value. The evidence is, however, wholly circumstantial. Desaturase enzyme systems in a number of species depend critically on O2 as a cofactor (Stumpf 1976). It has been suggested that desaturation of fatty acids in response to reduced temperature is merely a reflection of the increased solubility of oxygen. In bulb scale tissue from Narcissus it was shown that an increase in dissolved oxygen concentrations at 20~ could promote as much desaturation as cooling the tissue at 10~ (Harris and James 1969). This elegant hypothesis does not seem to apply to the roots of rye. When dissolved O2 was reduced to 0.5~2.0% in solutions at 8 ~ C, desaturation of PC and PE was appreciable although not as great as in these lipids from roots grown in aerated solutions. Thus oxygen is unlikely to become a limiting factor in desaturase activity until it was depleted to a very low level (see also Martin et al. 1976). A similar conclusion can be reached from a study of lipid desaturation on submerged anaerobic rice plants (Vartapetian et al. 1978).

Positional Analysis and Pairing of Acyl Chains. Our work extends the reports of the structure of plant phospholipids (Debuch 1957; Mudd et al. 1969; Devor and Mudd 1973) to include those in roots. The composition of the acyl chains in position I and II in both PC and PE is strikingly different; more than 90% of the palmitic acid is associated with position I and about 65% of the total linoleic and linolenic acids with position II. Even though the acyl chains in both positions desaturate as the temperature is lowered, these relative distributions are maintained. This process is, therefore, quite different for that reported by Miller et al. (1976).for temperature acclimatization in goldfish. In this organism some unsaturated fatty acids were found associated exclusively with position II, eg. 18:1 and 20 : 1, in warm acclimatized fish, but appeared to switch to position I when fish were adapted to 6 ~ C water temperature. Given the distribution of 16 : 0 and 18:2 and 18 : 3 in PC and PE from rye roots, a high proportion of these lipids will probably have a saturated, and therefore relatively stiff, chain in position I paired with a polyunsaturated, and therefore relatively pliant, one in position II. This was demonstrated specifically for PC from cauliflower inflorescences by Devor and Mudd (1973) who analysed the fatty acid composition of diglycerides prepared by the action of phospholipase C. They found that 37% of all the diglycerides had 16:0 paired with 18 : 3, and that diglycerids containing only 16:0 were absent. The func-

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D.T. Clarkson et al. : Phospholipid and Fatty Acid Changes in Secale Roots

tional significance o f this pairing o f a stiff and a pliant chain remains obscure.

Relationship with Ion Transport and Hydraulic Conductivity. While the time course of the induction of e n h a n c e d ion transport (Clarkson 1976) and of fatty acid desaturation by cooling have certain features in common, two pieces of evidence suggest that they are not causally related. First, desaturation continues to increase over a 7 day period of cooling whereas enhancement of ion transport was greatest after 2-3 days (Clarkson 1976). Second, enhancement of ion transport quickly disappeared (in 24-48 h) after roots were returned from a cool solution to a warm one (Clarkson 1976), whereas once desaturation was set in motion by cooling it increased to some extent during the 48 h after roots were returned to 20 ~ C (Table 6). The increased rate of ion transport seems more likely to depend on increased numbers of porters which form at lower temperatures (Clarkson 1976), rather than directly on increased fluidity in the membrane interior. M e m b r a n e fluidity m a y have a more direct influence on the permeability of roots to water. R o o t resistance to water flow can increase sharply below a critical temperature, but this critical tempeature can vary depending on how far the roots have been cold acclimatized (Kuiper 1964; M a r k h a r t et al. 1979). The work of M a r k h a r t et al. (1979) is particularly valuable in this respect because they used hydrostatic pressure to drive water through excised root systems fast enough to minimize the influence of solute transport to the xylem on the driving force for water movement. The slope of an Arrhenius plot of water flow versus temperature in soybeans changed markedly at 14 ~ C for roots grown at 28 ~ C day/24 ~ C night; this transition temperature was lowered to 8.7 ~ C in roots grown at 17 ~ C d a y / l l ~ C night. M e m b r a n e lipids became desaturated in roots grown at the lower temperature regime (Markhart, personal communication). I f desaturation, and the consequent increase in fluidity, occurs in the same parts of the m e m b r a n e as those through which water passes, increased water permeability m a y result from an increased frequency of structural transitions, creating transient pores through which water can move.

We are grateful for the analysis of fatty acid esters by mass spectrometry to Dr. H. Anderson & Colleagues of The Macauley Institute for Soil Research, and to N. Miller from ARC Institute of Animal Physiology for his advice concerning positional analysis. We also thank A. Rees, I.W. Harper, Janice Sawyer and our colleagues in the Chemistry and Electronics Section of the Letcombe Laboratory for invaluable practical assistance.

References Chapman, D. (1975) Phase transitions and fluidity characteristics of lipids and cell membranes. Q. Rev. Biophys. 8, 185-235 Clarkson, D.T. (1976) The influence of temperature on the exudation of xylem sap from detached root systems of rye (Secale cereale) and barley (Hordeum vulgare). Planta 132, 297-304 Clarkson, D.T., Shone, M.G.T., Wood, A.V. (1974) The effect of pretreatment temperature on the exudation of xylem sap by detached barley root systems. Planta 121, 81-92 Cronan, J.E., Gelmann, E.P. (1975) Physical properties of membrane lipids: biological relevance and regulation. Bacteriol. Rev. 39, 232-256 Debuch, H. (1957) l~ber die enzymatische Spaltung des Lecithins aus Sojabohnen. Hoppe-Seyler's Z. Physiol. Chem. 306, 279286 de la Roche, I.A., Andrews, C.J., Pomeroy, M.K., Weinberger, P., Kates, M. (1972) Lipid changes in winter wheat seedlings (Triticum aestivum L.) at temperatures inducing cold hardiness. Can. J. Bot. 50, 2401-2409 Devor, K.A., Mudd, J.B. (1973) Structural analysis of phosphatidylcholine of plant tissue. J. Lipid Res. 12, 396M02 Farkas, T., D6ri-Hadlaczky, E., Belea, A. (1975) Effect of Temperature upon Linolenic acid level in wheat and rye seedlings. Lipids 10, 331-334 Folch, J., Lees, M., Sloane, S.G.H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 49%509 Harris, P., James, A.T. (1969) The effect of low temperatures on fatty acid biosynthesis in plants. Biochem. J. 112, 325-330 James, R., Branton, D. (1973) Lipid and temperature-dependent structural changes in Acholeplasma laidlawii cell membranes. Biochim. Biophys. Acta 323, 378-390 Kedrowski, R.A., Chapin, F.S. III. (1978) Lipid properties of Carex aquatilis from hot spring and permafrost-dominated sites in Alaska: implications for nutrient requirements. Physiol. Plant. 44, 231-237 Keenan, R.W., Leonard, R.T., Hodges, T.K. (1973) Lipid composition of plasma membranes from Arena sativa roots. Cytobios 7, 103-112 Kimball, S.L., Salisbury, F.B. (1973) Ultrastructural changes of plants exposed to low temperatures. Am. J. Bot. 60, 1028-1033 Kuiper, P.J.C. (1964) Water uptake of higher plants as affected by root temperature. Meded. Landbouwhogesch., Wageningen 63-4, 1-11 Kuiper, P.J.C. (1970) Lipids in alfalfa leaves in relation to cold hardiness. Plant Physiol. 45, 684-686 Kuiper, P.J.C. (1974) Role of lipids in water and ion transport. In: Recent advances in chemistry and biochemistry of plant lipid. Proc. Phytochem. Soc. 12, 359-386 Lyons, J.M., Wheaton, T.A., Pratt, H.K. (1964) Relationship between the physical nature of mitochondrial membranes and chilling sensitivity in plants. Plant Physiol. 39, 262-268 Markhart, A.H. III., Fiscus, E.L., Naylor, A.W., Kramer, P.J. (1979) Effect of temperature on water and ion transport in soybean and broccoli systems. Plant Physiol. 64, 83-87 Martin, C.E., Hiramitsu, K., Kitajima, Y., Nozawa, Y., Skriver, L., Thompson, G.A., Jr. (1976) Molecular control of membrane properties during temperature acclimation. Fatty acid desaturase regulation of membrane fluidity in acclimating Tetrahymena cells. Biochemistry 15, 5218-5227 Mazliak, P. (1973) Lipid metabolism in plants. Annu. Rev. Plant Physiol. 24, 287-310 Mazliak, P. (1977) Glyco- and phospholipids of biomembranes in higher plants. In : Lipids and lipid polymers in higher plants, pp. 48-74 Tevini, M., Lichtenthaler, H.K., eds. Springer, Berlin Heidelberg New York

D.T. Clarkson et al. : Phospholipid and Fatty Acid Changes in Secale Roots Miller, N.G.A,, Hill, M.W., Smith, M.W. (1976) Positional and species analysis of membrane phospholipids extracted from goldfish adapted to different environmental temperatures. Biochim. Biophys. Acta 455, 644 654 Moreau, F., Dupont, J., Lance, C. (1974) Phospholipid and fatty acid composition of outer and inner membranes of plant mitochondria. Biochim. Biophys. Acta 345, 29d~304 Mud& J.B., van Vliet, H.H., van Deenan, L.L. (1969) Biosynthesis of galactolipids by enzyme preparations from spinach leaves. J. Lipid Res. 10, 623-630 Pomeroy, M.K., Andrews, C.J. (1978) Metabolic and nltrastructnral changes in winter wheat during ice encasement under field conditions. Plant Physiol. 61, 806-811 Quail, P.H. (1979) Plant cell fractionation. Annu. Rev. Plant Physiol. 30, 425-484 Raison, J.K. (1973) Temperature-induced phase changes in membrane lipids and their influence on metabolic regulation. Symp. Soc. Exp. Biol. 27, 485 Raison, J.K., Chapman, E.A. (1976) Membrane phase changes in chilling-sensitive Vigna radiata and their significance to growth. Aust. J. Plant Physiol. 3, 291-299

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Redshaw, E.S., Zalik, S. (1968) Changes in Lipids of cereal seedlings during vernalization. Can. J. Biochem. 46, 1093 1097 Simon, E.W. (1974) Phosphotipids and Plant Membrane Permeability. New Phytol. 73, 377-420 Smolenska, G., Kuiper, P.J.C. (1977) Effect of low temperature upon lipid and fatty acid composition of roots and leaves of winter rape. Physiol. Plant. 41, 29-35 Stumpf, P.K. (1976) Lipid metabolism. In: Plant biochemistry, 3rd ed., pp. 427-461, Bonner, J., Varner, J.E. eds. Academic Press, New York Vartapetian, B.B., Mazliak, P., Lance, C. (1978) Lipid biosynthesis in rice coleoptiles grown in the presence or in the absence of oxygen. Plant Sci. Lett. 13, 321 328 Wijngaarden, D. van (1967) Modified rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Anal. Chem 39, 848-849 Willemot, C. (1975) Stimulation of phospholipid biosynthesis during frost hardening of winter wheat. Plant Physiol. 55, 356-359

Received 1 March; accepted 1 April 1980