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Characterization and lipid composition of the plasma membrane in grape leaves
~
Pergamon
0031-9422 (93) E0030-1
Phytochernistry, Vol. 35, No. 5, pp. 1249-1253, 1994 Copyright 9 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0031-9422/94 $6.00 + 0.00
CHARACTERIZATION AND LIPID COMPOSITION OF THE PLASMA MEMBRANE IN GRAPE LEAVES ALBERTO MAS,* JOSE NAVARRO-PEDREI~IO,~"DAVIDT. COOKE and CAROLYNS. JAMES Department of Agricultural Sciences, University of Bristol, AFRC Institute of Arable Crops Research, Long Ashton Research Station, Bristol BS18 9AF, U.K.
(Received in revisedform 14 September 1993)
KeyWord lndex--Vitis vinifera; Vitaceae; grape leaves; plasma membrane; sterols; phospholipids; membrane fluidity.
Abstract--Plasma membranes have been isolated from grape leaves using a two-phase aqueous polymer technique which differed from the usual procedure in order to counter difficulties posed by the fibrous nature and high starch content of this material. The lipid composition (sterols, phospholipids + sphingolipids and their fatty acids) of the purified plasma membrane was determined and compared with that of the microsomal fraction (100 000 9). The major differences between the membrane and microsomal fractions were increases in the proportions of stearic acid, phosphatidylcholine and, to a lesser extent, phosphatidylethanolamine and a decrease in linolenic acid. Phosphatidylglycerol and diphosphatidylglycerol were present in the microsomal fraction, but not in the plasma membrane. Unusually large amounts of cerebrosides (compared with some other plant species) were found in both membrane fractions. The lipid composition of the plasma membrane is discussed in the context of membrane fluidity.
INTRODUCTION The living cell represents a complex oflipids and proteins combined with many other components. Plant growth and development depends on an interaction between these factors and the external environment. It is now widely accepted that the plasma membrane represents a sensitive indicator of environmental changes [1, 21 and it is likely that the response of plants to their environment is regulated at the level of the plasma membrane. Although studies have been made of the plasma membrane from many plant species [I], some economically important crops have not been investigated. Of these, the grapevine (Vitis vinifera), is noteworthy and, to date, the isolation and characterization of the plasma membrane from the leaves has not been successfully achieved. Furthermore, detailed knowledge of the biochemistry and biophysics of the plasma membrane of this plant is still scarce and much information about this species centres largely around its responses to nutrient deficiencies and toxicities 1,-3-5]. The aim of our work was to isolate the plasma membranes from the leaves of grapevine and characterize them in terms of specific marker enzymes and their lipid
* Author to whom correspondence should be addressed at: Universitat Rovira i Virgili, Departament de Bioquimica i Biotecnologia, Facultat Quimica de Tarragona, 43005-Tarragona, Spain. I"Present address: Universitat de Alicante, Divisi6n. Agroquimiea, Facultat de Ciencias, 03080- Alicante, Spain.
composition. The usefulness of lipids as markers for membrane fractions is discussed along with the relationship between lipid composition and membrane fluidity in the grapevine leaves and compared with the available data from other plant plasma membranes. RESULTSAND DISCUSSION Grape leaves are very fibrous, which initially caused problems during the plasma membrane extraction procedure. This was overcome by performing the vacuum infiltration stage in three steps, so that a consistent homogenate was obtained. The microsomal pellet was associated with much starch which had to be removed before the plasma membrane could be purified. This was achieved by centrifuging the first upper-phase of the twophase system [6] for 12 min (instead of the usual 4 min). This procedure displaced the starch to the interface allowing the plasma membrane fraction to be removed for further purification. The yield of plasma membrane was about a third (0.3 mg ml- t protein) of that normally obtained from the leaves of other species, e.g. oat, rye and rice I-7, 8], although low yields of plasma membrane also have been reported for celery leaves [9]. The microsomal and plasma membrane ATPase (EC 3.6.1.35) activities (Table 1) indicated that the specific activity in the plasma membrane fraction was greater than that of the microsomal fraction. Furthermore, when compared with the microsomal fraction, the plasma membrane fraction showed greater potassium stimulation and vanadate and calcium inhibition, three charac-
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A. MAs et al.
Table 1. Specific activity of the Mg 2§ and (Mg 2§ - K+)-ATPase (EC 3.6.1.35) from the microsomal and plasma membrane fractions from grape leaves. The effects of calcium (100 mM) and vanadate (22 mM) inhibition are shown along with glucan synthase II activities Microsomal fraction (/~mol Pi mg- 1 protein min- 1)
Plasma membrane (#mol Pi mg- 1protein min - 1)
Mg 2§ -ATPase (Mg 2 § - K § Ca 2+-inhibited (Mg 2 § § VOW--inhibited (Mg 2 + - K §
0.046 _ 0.0007 (n = 9) 0.052+0.00l (n=9) 0.034+0.0012 (n = 5) 0.038 _ 0.0008 (n = 3)
0.31 +0.006 (n=9) 0.35 + 0.004 (n = 9) 0.16+0.001 (n=5) 0.23+0.001 (n=3)
Glucan synthase II
nmol UDPG mg- 1 protein min- 1 0.09 (n=2)
nmol UDPG mg- J protein min- 1 0.36 (n=2)
n = Number of determinations.
Table 2. Percentage phospholipid + sphingolipid and total phospholipid + sphingolipid and protein content (mg ml- 1) of microsomal and plasma membrane fractions of grape leaves (__+s.e.)
Cerebrosides Phosphatidylglycerol Diphosphatidylglycerol Phosphatidylethanolamine Phosphatidylcholine PE/PC Protein (mg m1-1)
Microsomal fraction
Plasma membrane
/tg mg - 1 protein
%
/~g mg- 1 protein
68.3_+3.6 1.6 _ 0.4 33.3 _ 1.3 8.6 _+0.3 11.2+0.6 0.76 + 0.02 4.57+0.18
55.5 _+1.3 100.0+ 21.0 1.4 __+0.4 nd* 27.1 _ 1.9 nd* 6.9 _ 0.1 35.3 _ 1.8 9.1+0.3 53.0+4.0* 0.71 + 0.26 0.21 +0.03*
% 53.4_+4.7 --17.6 _+4.6 30.3+5.1"
*Indicates significantly different from the microsomal fraction at the 95% level of confidence or better (n >/3). nd = Not detected.
teristic markers of plasma membrane [10-12]. A fourfold enrichment in glucan synthase II activity was found in the purified plasma membrane fraction, compared with the microsomal fraction (Table 1). This enzyme is also considered to be a specific marker of the plasma membrane [6]. Taken together, the enzyme marker assays appeared to confirm that the purified upper phase from the two-phase polymer system was enriched in plasma membrane. However, no marker enzyme assays were done to establish whether impurities such as mitochondrial membranes or thylakoids were present. Both these organelles can be characterized by their lipid content, as they possess lipids (namely, phosphatidylglycerol which is associated with the thylakoid and diphosphatidylglycerol or cardiolipin, found in the mitochondria) which are not normally present in the plasma membrane [13]. Phospholipid analysis indicated that neither of these lipids was present in the plasma membrane fraction, although they both appeared in the microsomal fraction (Table 2). The large a m o u n t of diphosphatidylglycerol (DPG) in the microsomes (27%) would imply that there were a considerable n u m b e r of mitochondria in this fraction, or that in this
plant species, D P G was also present in other organelles. F r o m these data, it is likely that contamination from mitochondria and thylakoids in the plasma membraneenriched fraction was minimal or absent. However, to measure thylakoid contamination, determinations of chlorophyllt, § were done. These showed that the microsomal fraction contained 433/~g chlorophyll m g - 1 protein (S.E. = _+62, n = 3) and the plasma membrane fraction, 68/~g chlorophyll m g - 1 protein (S.E. = _ 1.7, n = 3), indicating a greater than six-fold purification. The phospholipid profile of grape leaf membranes, as a whole, showed an unusually large a m o u n t of sphingolipid, the cerebrosides (over 50% of the total phospholipid + sphingolipid fraction), the greater amount (100/~g m g - 1 protein) being found in the plasma membrane (Table 2). This compares with 7% in etiolated mung bean hypocotyl plasma membranes [141 16% in barley shoot plasma membranes [15], 16% in the plasma membranes from shoots of Galium aparine (D. T. Cooke and J. M. Rooney, unpublished data) and 20% in etiolated maize shoot plasma membranes (D. T. Cooke and D. E. Evans, unpublished data). Cerebrosides are considered to be a major component of the plant
Plasma membrane of grape leaves plasma membrane and the tonoplast, and are thought to play a role in the modulation of water stress [13]. The plasma membrane was also enriched in phosphatidylcholine which represented about 30% of the total phospholipid+sphingolipid fraction (Table 2), a figure which is reasonably consistent with published data from other plant plasma membranes [1]. A trend towards an increase in the concentration of phosphatidylethanolamine was found in the plasma membrane (17%) compared with the microsomal fraction (7%). Again, these data agreed favourably with those obtained for the plasma membranes of other species [1]. The relative distribution of the phospholipid-bound fatty acids (Table 3) indicated that overall there was a great deal of similarity between the plasma membrane and the microsomal fraction. However, there were some subtle differences, in particular, the plasma membrane had a greater proportion of saturated fatty acids (an increase in 18:0 and a decrease in 18:3) than the microsomal fraction. This was reflected in a reduction in the plasma membrane bond-index, calculated from (%16:1 + % 18:1 + [2 x % 18: 2] + [3 x % 18: 3]), which is a guide to the degree of unsaturation. No differences were found between the sterol content of the plasma membrane and Table 3. Relative proportions (%) of fatty acids associated with the phospholipid acyl chains from the microsomal and plasma membrane fractions of grape leaves ( 4-s.e.)
16:0 16:1 18:0 18:1 18:2 18:3 Bond index 16/18
Microsomal fraction (%)
Plasma membrane (%)
34.6+_2.9 5.9+2.0 3.0+0.4 5.5 +0.6 26.3+0.8 26.54-0.8 143.6__+4.6 0.69+0.07
37.4_+4.7 6.94-2.1 5.4+__0.7* 9.2+ 1.6 23.7+ 1.9 17.44-0.6" 115.64-7.2 0.71 ___0.1
Bond-index, calculated from (% 16:1 + % 18:1 +[2 x %18:2] +[3 x %18:3]). *Indicates significantly different from the microsomal fraction at the 95% level of confidence or better (n t> 3).
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microsomal fraction and, although there was a trend towards an increase in sterol concentration (#g m g - t protein) in the plasma membrane fraction, the relative proportions of the sterols (cholesterol, campesterol, stigmasterol and sitosterol) remained constant (Table 4). The fluidity of the plasma membrane fraction was measured using steady-state fluorescence polarization, and the results compared with previously published data from other plasma membranes. The membrane orderparameter P, a measure of fluidity (where the greater the value of P, the less fluid the membrane) was found to be 0.294 + 0.005 (n = 6). This figure compares well with the values for oat and rye shoot plasma membranes, where P = 0.298 + 0.002 (n = 5) and 0.325 + 0.003 (n = 4), respectively [7]. In the plasma membrane fraction, it would appear that the lipid composition had little influence on the fluidity of the membrane, which was similar to that found in oat and rye. The large amount of cerebroside, which is considered to make the membranes more rigid (less fluid) [13], coupled with the similar effect imposed by increased saturation of the bound fatty acids, meant that, theoretically, the grape leaf plasma membrane order parameter (P) which is the reciprocal of fluidity, should have been high. In fact, it was no different from that reported for oat but more fluid (i.e. less ordered) than the rye plasma membranes. It is possible that the fluidity was moderated by sterol composition, in particular by the large amount of sitosterol present in the plasma membrane (Table 4) which, in theory, should result in a less ordered membrane as the 'cone of rotation' described by the C~7 acyl chain is greater for sitosterol than cholesterol and campesterol [16]. In contrast, recent work with soybean phosphatidylcholine bilayers [17] has shown that as little as 16 mol% sitosterol increases the lipid ordering of the membrane. In grapevine, sitosterol represented 32 mol% of the plasma membrane lipids and, thus, should have exerted an increased ordering effect on the membrane but, from the results, this did not appear to be the case. However, the lipid/protein ratio (calculated from [sterol + phospholipid + sphingolipid]/protein) in the plasma membrane was 1.18. This ratio is low and, at present, we have no explanation for this apparent anomaly, which may have been a contributing factor to there being no obvious relationship between fluidity and lipid composition. Nevertheless, it has already been pointed out [18] that the relationship between fluidity and membrane lipid
Table 4. Percentage sterol and total sterol content of microsomal and plasma membrane fractions of grape leaves ( + s.c.) (n >i3) Microsomal fraction
Plasma membrane
#g m g - 1
#g m g - t
protein Cholesterol Campesterol Stigmasterol Sitosterol
0.19+0.10 0.17+0.36 1.91+0.69 13.02 + 4.50
% 2.6+2.0 3.4+1.7 11.6+0.6 82.4 + 0.8
protein 0.91 +0.32 2.16+1.07 7.54+3.75 50.92 + 24.62
% 5.6+2.6 3.4+0.4 11.0+0.8 80.0 + 1.9
A. MAS et al.
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composition is not a simple one, a conclusion which our data would appear to confirm. EXPERIMENTAL
Plant culture. Grapevines (Vitis vinifera, L. cv Pinot Noir) were grown in pots containing John Innes No. 3 compost, under glass with supplementary light to simulate a minimum 16 hr day at 18~ day and 15~ night. When required, leaves were harvested from the mid-third region of extension growth and plasma membranes extracted immediately. Plasma membrane isolation and purification. Plasma membranes were isolated and purified using the aq. 2-phase phase polymer technique of ref. [6], with some minor modifications. All the following procedures were done at 4 ~ Leaves (~35 g) were finely chopped and vacuum infiltrated with 70 ml HEPES-sucrose buffer, (50 mM HEPES, 500 mM sucrose, 1 mM DTT, 5 mM ascorbic acid to pH 7.5 with NaOH) and insol. PVP (1 g). The buffer-satd material was subjected to 3 separate homogenizations using an Ultra-turrax homogenizer, with further vacuum infiltration and the addition of extra buffer between each homogenization to obtain a consistent extract. This was filtered through a 240/~m nylon cloth and the filtrate centrifuged at 10000 g for 15 min. The supernatant was centrifuged at 100000 g for 30 min to yield a microsomal pellet which was resuspended in potassium phosphate buffer (KPB) (4 ml), pH 7.8 (KPi 5 mM, sucrose 330 mM). The microsomal fr. (3 ml) was loaded on to 9 g of a 2-phase mixt. to yield a 12 g 2-phase system containing Dextran T-500 (6%, w/w), PEG 3350 (6% w/w), KC1 (3 mM), sucrose (330 raM) and KPB (5 mM). This was centrifuged at 4000 g for 12 min to remove the large amount of starch which would otherwise have contaminated the upper phase. The upper phase was then removed and the resulting plasma membrane-enriched ft. purified using a batch procedure [6]. The purified plasma membrane fr. was centrifuged at 100000 g for 30min, the pellet resuspended in PIPES-sucrose buffer (1 ml), (PIPES, 5 mM, sucrose 250 mM, to pH 6.5 with NaOH) and stored at - 2 0 ~ before use. For marker-enzyme assays, membranes were stored no longer than 48 hr and for lipid analysis, aliquots of suspended membranes were stored in CHC13-MeOH (1 : 2) at - 20~ Lipid analysis. In an Eppendorf tube, CHCI3-MeOH (0.75 ml) was added to resuspended membranes (0.5 ml) along with fl-cholestanol (20/A, 0.1 mgml-1), as an int. standard for sterol analysis. CHC13 (0.25 ml) was added, the mixt. shaken and centrifuged at 10000 g for 6 min. The CHC13 layer was retained, evapd to dryness under N 2 and made up to 100/zl with CHCI 3. For sterol analysis, 20 #1 of the CHCI 3 extract was placed in a glass vial (2 ml), evapd to dryness under N 2 and acetylated using pyridine (50/~1) and Ac20 (100/zl). After 2 hr the solvents were evapd under N2, EtOAc (20 #1) added and the sterols analysed by GC using an SE52-bonded capillary column coupled to FID, with H 2 as carrier (1 ml min-1) and a temp. prog. of 120-265 ~ at
10~ min-1. The inj. and detector temps were 250 and 320 ~, respectively. Bound fatty acids were determined by using 20-#1 portions of the CHC13 extract, evaporating to dryness under N2 and transmethylating with N a O M e - M e O H (0.5 N) (0.5 ml) and heating at 30 ~for 7 min. The resultant fatty acid Me esters were extracted with hexane (1 ml), evapd under N2, dissolved in EtOAc (20 pl) and analysed by GC using an RSL 500-bonded capillary column with FID and He as carrier (1 ml min- 1). The inj. and detector temps were 250 ~ and 300 ~, respectively. Phospholipids were analysed by HPLC, using the method described in ref. [19] with minor modifications. The CHCIa extract (20/zl) was injected into an Econosphere silica 3 ~ column (150 mm x 4.6 mm) and compounds detected with an evaporative light scattering detector. The detector conditions were as follows: N2 flow, 20 ml min- 1; detector response, 900 V; temp. 50~ A three-solvent gradient system was used, comprising: (A) hexane-dimethoxypropane (99:1), (B) isoPrOH-CHCI 3 (4:1) and (C) isoPrOH-H20 (1:1); the flow rate was 2 ml min- 1 throughout. The solvent programme was as follows: 100% A at time 0, and 1 min. At 5 min, A was 80% and B 20%. A, B and C were 42, 52 and 6%, respectively, at 5.1 min and at 15 min they were 35, 49 and 16%. At 20 min A was 42%, B 52% and C 6% and at 25 min A was 30% and B 70%. Finally, A was 100% at 30 min. Phospholipids were quantified by comparing peak areas with those of known standards. Glucan synthase II and A TPase assays, protein determinations and fluidity measurements. Glucan synthase II was estimated by aH-UDP-glucose linkage according to the method of ref. [20] with the modifications described in ref. [21]. ATPase (EC 3.6.1.35) activity was measured by hydrolysis of ATP and subsequent release of inorganic phosphate using the method described in ref. [22] as modified in ref. [21]. ATPase assay incubations were done at 37 ~ in the presence of Triton-X100 to overcome latency. KCI (50 raM), CaC12 (100 mM) or sodium vanadate (22 mM) were added specifically to stimulate or inhibit plasma membrane ATPase activity. Membrane fluidity was determined using steady-state fluorescence polarization with 1,6-diphenylhexatriene as the probe [7]. Protein was assayed by Coomassie-blue dye binding [23] with thyrogobulin as an int. standard. Chlorophyll analysis. Chlorophyllta § b~was determined by extracting appropriate amounts of microsomal and plasma membrane fraction with 80% Me2CO and measuring the absorbance at 652 nm. Data analysis. Data were analysed using unpaired 't'tests or 'analysis of variance' and each expt was repeated at least three times.
REFERENCES
1. Larsson, C., M~ller, I. M. and Widell, S. (1990) in The Plant Plasma Membrane, Structure, Function and Molecular Biology, p. 2. (Larsson, C. and Meller, I. M., eds). Springer, Berlin. ISBN 3-540-50836-8.
Plasma membrane of grape leaves 2. Shewfelt, R. L. (1992) in Plant Membranes: A Biophysical Approach to Structure, Development and Senescence, p. 192 et seq. Y. Y. Leshem. Kluwer Academic Publishers, Dordrecht. ISBN 0-7923-13534. 3. Chapman, H. D. (1966) Diagnostic Criteria for Plants and Soils. University of California, Division of Agricultural Sciences. 4. Serra, J. A., Perello, L. and Maquieira, A. (1981) Agrochimica 25, 156. 5. Rousos, P. A., Harrison, H. C. and Steffen, K. L. (1989) J. Am. Soc. Hort. Sci. 114, 149. 6. Larsson, C., Widell, S. and Kjellbom, P. (1987) Meth. Enzymol. 148, 558. 7. Cooke, D. T., Munkonge, F. M., Burden, R. S. and James, C. S. (1991) Biochim. Biophys. Acta 1061, 156. 8. Ros, R., Cooke, D. T., Martinez-Cortina, C. and Picazo, I. (1992) J. Exp. Bot. 43, 1475. 9. Rasheed, N. (1993) Doctoral Thesis, University of Liverpool. 10. Hodges, T. K. and Leonard, R. T. (1974) Meth. Enzymol. 32, 392. 11. Gallagher, S. R. and Leonard, R. T. (1982) Plant Physiol. 70, 1335.
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12. Berczi, A., Larsson, C., Widell, S. and Moiler, I. M. (1989) Physiol. Plant. 77, 12. 13. Leshem, Y. Y. (1992) in Plant Membranes: A Biophysical Approach to Structure, Development and Senescence, p. 27 et seq. Y. Y. Leshem. Kluwer Academic Publishers, Dordrecht. 14. Yoshida, S. and Uemura, M. (1986) Plant Physiol. 82, 807. 15. Rochester, C. P., Kjellbom, P. and Larsson, C. (1987) Physiol. Plant. 71, 257. 16. Douglas, T. J. (1985) Plant Cell Envir. 8, 687. 17. Schuler, I., Milon, A., Nakatani, Y., Ourisson, G., Albrecht, A.-M., Benveniste, P. and Hartmann, M.-A. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 6926. 18. Lee, A. G., Michelangeli, F. and East~ J. M. (1989) Biochem. Soc. Trans. 17, 962. 19. Christie, W. W. (1985) J. Lip. Res. 26, 507. 20. Ray, P. M. (1979) in Plant Organelles (Reid, E., ed.), p. 135. Ellis Horwood, Chichester. 21. Coupland, D., Cooke, D. T. and James, C. S. (1991) J. Exp. Bot. 42, 1065. 22. Ohnishi, T., Gall, R. S. and Mayer, M. L. (1975) Analyt. Biochem. 69, 261. 23. Bradford, M. (1976) Analyt. Biochem. 72, 248.
Pergamon
0031-9422 (93) E0030-1
Phytochernistry, Vol. 35, No. 5, pp. 1249-1253, 1994 Copyright 9 1994 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0031-9422/94 $6.00 + 0.00
CHARACTERIZATION AND LIPID COMPOSITION OF THE PLASMA MEMBRANE IN GRAPE LEAVES ALBERTO MAS,* JOSE NAVARRO-PEDREI~IO,~"DAVIDT. COOKE and CAROLYNS. JAMES Department of Agricultural Sciences, University of Bristol, AFRC Institute of Arable Crops Research, Long Ashton Research Station, Bristol BS18 9AF, U.K.
(Received in revisedform 14 September 1993)
KeyWord lndex--Vitis vinifera; Vitaceae; grape leaves; plasma membrane; sterols; phospholipids; membrane fluidity.
Abstract--Plasma membranes have been isolated from grape leaves using a two-phase aqueous polymer technique which differed from the usual procedure in order to counter difficulties posed by the fibrous nature and high starch content of this material. The lipid composition (sterols, phospholipids + sphingolipids and their fatty acids) of the purified plasma membrane was determined and compared with that of the microsomal fraction (100 000 9). The major differences between the membrane and microsomal fractions were increases in the proportions of stearic acid, phosphatidylcholine and, to a lesser extent, phosphatidylethanolamine and a decrease in linolenic acid. Phosphatidylglycerol and diphosphatidylglycerol were present in the microsomal fraction, but not in the plasma membrane. Unusually large amounts of cerebrosides (compared with some other plant species) were found in both membrane fractions. The lipid composition of the plasma membrane is discussed in the context of membrane fluidity.
INTRODUCTION The living cell represents a complex oflipids and proteins combined with many other components. Plant growth and development depends on an interaction between these factors and the external environment. It is now widely accepted that the plasma membrane represents a sensitive indicator of environmental changes [1, 21 and it is likely that the response of plants to their environment is regulated at the level of the plasma membrane. Although studies have been made of the plasma membrane from many plant species [I], some economically important crops have not been investigated. Of these, the grapevine (Vitis vinifera), is noteworthy and, to date, the isolation and characterization of the plasma membrane from the leaves has not been successfully achieved. Furthermore, detailed knowledge of the biochemistry and biophysics of the plasma membrane of this plant is still scarce and much information about this species centres largely around its responses to nutrient deficiencies and toxicities 1,-3-5]. The aim of our work was to isolate the plasma membranes from the leaves of grapevine and characterize them in terms of specific marker enzymes and their lipid
* Author to whom correspondence should be addressed at: Universitat Rovira i Virgili, Departament de Bioquimica i Biotecnologia, Facultat Quimica de Tarragona, 43005-Tarragona, Spain. I"Present address: Universitat de Alicante, Divisi6n. Agroquimiea, Facultat de Ciencias, 03080- Alicante, Spain.
composition. The usefulness of lipids as markers for membrane fractions is discussed along with the relationship between lipid composition and membrane fluidity in the grapevine leaves and compared with the available data from other plant plasma membranes. RESULTSAND DISCUSSION Grape leaves are very fibrous, which initially caused problems during the plasma membrane extraction procedure. This was overcome by performing the vacuum infiltration stage in three steps, so that a consistent homogenate was obtained. The microsomal pellet was associated with much starch which had to be removed before the plasma membrane could be purified. This was achieved by centrifuging the first upper-phase of the twophase system [6] for 12 min (instead of the usual 4 min). This procedure displaced the starch to the interface allowing the plasma membrane fraction to be removed for further purification. The yield of plasma membrane was about a third (0.3 mg ml- t protein) of that normally obtained from the leaves of other species, e.g. oat, rye and rice I-7, 8], although low yields of plasma membrane also have been reported for celery leaves [9]. The microsomal and plasma membrane ATPase (EC 3.6.1.35) activities (Table 1) indicated that the specific activity in the plasma membrane fraction was greater than that of the microsomal fraction. Furthermore, when compared with the microsomal fraction, the plasma membrane fraction showed greater potassium stimulation and vanadate and calcium inhibition, three charac-
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Table 1. Specific activity of the Mg 2§ and (Mg 2§ - K+)-ATPase (EC 3.6.1.35) from the microsomal and plasma membrane fractions from grape leaves. The effects of calcium (100 mM) and vanadate (22 mM) inhibition are shown along with glucan synthase II activities Microsomal fraction (/~mol Pi mg- 1 protein min- 1)
Plasma membrane (#mol Pi mg- 1protein min - 1)
Mg 2§ -ATPase (Mg 2 § - K § Ca 2+-inhibited (Mg 2 § § VOW--inhibited (Mg 2 + - K §
0.046 _ 0.0007 (n = 9) 0.052+0.00l (n=9) 0.034+0.0012 (n = 5) 0.038 _ 0.0008 (n = 3)
0.31 +0.006 (n=9) 0.35 + 0.004 (n = 9) 0.16+0.001 (n=5) 0.23+0.001 (n=3)
Glucan synthase II
nmol UDPG mg- 1 protein min- 1 0.09 (n=2)
nmol UDPG mg- J protein min- 1 0.36 (n=2)
n = Number of determinations.
Table 2. Percentage phospholipid + sphingolipid and total phospholipid + sphingolipid and protein content (mg ml- 1) of microsomal and plasma membrane fractions of grape leaves (__+s.e.)
Cerebrosides Phosphatidylglycerol Diphosphatidylglycerol Phosphatidylethanolamine Phosphatidylcholine PE/PC Protein (mg m1-1)
Microsomal fraction
Plasma membrane
/tg mg - 1 protein
%
/~g mg- 1 protein
68.3_+3.6 1.6 _ 0.4 33.3 _ 1.3 8.6 _+0.3 11.2+0.6 0.76 + 0.02 4.57+0.18
55.5 _+1.3 100.0+ 21.0 1.4 __+0.4 nd* 27.1 _ 1.9 nd* 6.9 _ 0.1 35.3 _ 1.8 9.1+0.3 53.0+4.0* 0.71 + 0.26 0.21 +0.03*
% 53.4_+4.7 --17.6 _+4.6 30.3+5.1"
*Indicates significantly different from the microsomal fraction at the 95% level of confidence or better (n >/3). nd = Not detected.
teristic markers of plasma membrane [10-12]. A fourfold enrichment in glucan synthase II activity was found in the purified plasma membrane fraction, compared with the microsomal fraction (Table 1). This enzyme is also considered to be a specific marker of the plasma membrane [6]. Taken together, the enzyme marker assays appeared to confirm that the purified upper phase from the two-phase polymer system was enriched in plasma membrane. However, no marker enzyme assays were done to establish whether impurities such as mitochondrial membranes or thylakoids were present. Both these organelles can be characterized by their lipid content, as they possess lipids (namely, phosphatidylglycerol which is associated with the thylakoid and diphosphatidylglycerol or cardiolipin, found in the mitochondria) which are not normally present in the plasma membrane [13]. Phospholipid analysis indicated that neither of these lipids was present in the plasma membrane fraction, although they both appeared in the microsomal fraction (Table 2). The large a m o u n t of diphosphatidylglycerol (DPG) in the microsomes (27%) would imply that there were a considerable n u m b e r of mitochondria in this fraction, or that in this
plant species, D P G was also present in other organelles. F r o m these data, it is likely that contamination from mitochondria and thylakoids in the plasma membraneenriched fraction was minimal or absent. However, to measure thylakoid contamination, determinations of chlorophyllt, § were done. These showed that the microsomal fraction contained 433/~g chlorophyll m g - 1 protein (S.E. = _+62, n = 3) and the plasma membrane fraction, 68/~g chlorophyll m g - 1 protein (S.E. = _ 1.7, n = 3), indicating a greater than six-fold purification. The phospholipid profile of grape leaf membranes, as a whole, showed an unusually large a m o u n t of sphingolipid, the cerebrosides (over 50% of the total phospholipid + sphingolipid fraction), the greater amount (100/~g m g - 1 protein) being found in the plasma membrane (Table 2). This compares with 7% in etiolated mung bean hypocotyl plasma membranes [141 16% in barley shoot plasma membranes [15], 16% in the plasma membranes from shoots of Galium aparine (D. T. Cooke and J. M. Rooney, unpublished data) and 20% in etiolated maize shoot plasma membranes (D. T. Cooke and D. E. Evans, unpublished data). Cerebrosides are considered to be a major component of the plant
Plasma membrane of grape leaves plasma membrane and the tonoplast, and are thought to play a role in the modulation of water stress [13]. The plasma membrane was also enriched in phosphatidylcholine which represented about 30% of the total phospholipid+sphingolipid fraction (Table 2), a figure which is reasonably consistent with published data from other plant plasma membranes [1]. A trend towards an increase in the concentration of phosphatidylethanolamine was found in the plasma membrane (17%) compared with the microsomal fraction (7%). Again, these data agreed favourably with those obtained for the plasma membranes of other species [1]. The relative distribution of the phospholipid-bound fatty acids (Table 3) indicated that overall there was a great deal of similarity between the plasma membrane and the microsomal fraction. However, there were some subtle differences, in particular, the plasma membrane had a greater proportion of saturated fatty acids (an increase in 18:0 and a decrease in 18:3) than the microsomal fraction. This was reflected in a reduction in the plasma membrane bond-index, calculated from (%16:1 + % 18:1 + [2 x % 18: 2] + [3 x % 18: 3]), which is a guide to the degree of unsaturation. No differences were found between the sterol content of the plasma membrane and Table 3. Relative proportions (%) of fatty acids associated with the phospholipid acyl chains from the microsomal and plasma membrane fractions of grape leaves ( 4-s.e.)
16:0 16:1 18:0 18:1 18:2 18:3 Bond index 16/18
Microsomal fraction (%)
Plasma membrane (%)
34.6+_2.9 5.9+2.0 3.0+0.4 5.5 +0.6 26.3+0.8 26.54-0.8 143.6__+4.6 0.69+0.07
37.4_+4.7 6.94-2.1 5.4+__0.7* 9.2+ 1.6 23.7+ 1.9 17.44-0.6" 115.64-7.2 0.71 ___0.1
Bond-index, calculated from (% 16:1 + % 18:1 +[2 x %18:2] +[3 x %18:3]). *Indicates significantly different from the microsomal fraction at the 95% level of confidence or better (n t> 3).
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microsomal fraction and, although there was a trend towards an increase in sterol concentration (#g m g - t protein) in the plasma membrane fraction, the relative proportions of the sterols (cholesterol, campesterol, stigmasterol and sitosterol) remained constant (Table 4). The fluidity of the plasma membrane fraction was measured using steady-state fluorescence polarization, and the results compared with previously published data from other plasma membranes. The membrane orderparameter P, a measure of fluidity (where the greater the value of P, the less fluid the membrane) was found to be 0.294 + 0.005 (n = 6). This figure compares well with the values for oat and rye shoot plasma membranes, where P = 0.298 + 0.002 (n = 5) and 0.325 + 0.003 (n = 4), respectively [7]. In the plasma membrane fraction, it would appear that the lipid composition had little influence on the fluidity of the membrane, which was similar to that found in oat and rye. The large amount of cerebroside, which is considered to make the membranes more rigid (less fluid) [13], coupled with the similar effect imposed by increased saturation of the bound fatty acids, meant that, theoretically, the grape leaf plasma membrane order parameter (P) which is the reciprocal of fluidity, should have been high. In fact, it was no different from that reported for oat but more fluid (i.e. less ordered) than the rye plasma membranes. It is possible that the fluidity was moderated by sterol composition, in particular by the large amount of sitosterol present in the plasma membrane (Table 4) which, in theory, should result in a less ordered membrane as the 'cone of rotation' described by the C~7 acyl chain is greater for sitosterol than cholesterol and campesterol [16]. In contrast, recent work with soybean phosphatidylcholine bilayers [17] has shown that as little as 16 mol% sitosterol increases the lipid ordering of the membrane. In grapevine, sitosterol represented 32 mol% of the plasma membrane lipids and, thus, should have exerted an increased ordering effect on the membrane but, from the results, this did not appear to be the case. However, the lipid/protein ratio (calculated from [sterol + phospholipid + sphingolipid]/protein) in the plasma membrane was 1.18. This ratio is low and, at present, we have no explanation for this apparent anomaly, which may have been a contributing factor to there being no obvious relationship between fluidity and lipid composition. Nevertheless, it has already been pointed out [18] that the relationship between fluidity and membrane lipid
Table 4. Percentage sterol and total sterol content of microsomal and plasma membrane fractions of grape leaves ( + s.c.) (n >i3) Microsomal fraction
Plasma membrane
#g m g - 1
#g m g - t
protein Cholesterol Campesterol Stigmasterol Sitosterol
0.19+0.10 0.17+0.36 1.91+0.69 13.02 + 4.50
% 2.6+2.0 3.4+1.7 11.6+0.6 82.4 + 0.8
protein 0.91 +0.32 2.16+1.07 7.54+3.75 50.92 + 24.62
% 5.6+2.6 3.4+0.4 11.0+0.8 80.0 + 1.9
A. MAS et al.
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composition is not a simple one, a conclusion which our data would appear to confirm. EXPERIMENTAL
Plant culture. Grapevines (Vitis vinifera, L. cv Pinot Noir) were grown in pots containing John Innes No. 3 compost, under glass with supplementary light to simulate a minimum 16 hr day at 18~ day and 15~ night. When required, leaves were harvested from the mid-third region of extension growth and plasma membranes extracted immediately. Plasma membrane isolation and purification. Plasma membranes were isolated and purified using the aq. 2-phase phase polymer technique of ref. [6], with some minor modifications. All the following procedures were done at 4 ~ Leaves (~35 g) were finely chopped and vacuum infiltrated with 70 ml HEPES-sucrose buffer, (50 mM HEPES, 500 mM sucrose, 1 mM DTT, 5 mM ascorbic acid to pH 7.5 with NaOH) and insol. PVP (1 g). The buffer-satd material was subjected to 3 separate homogenizations using an Ultra-turrax homogenizer, with further vacuum infiltration and the addition of extra buffer between each homogenization to obtain a consistent extract. This was filtered through a 240/~m nylon cloth and the filtrate centrifuged at 10000 g for 15 min. The supernatant was centrifuged at 100000 g for 30 min to yield a microsomal pellet which was resuspended in potassium phosphate buffer (KPB) (4 ml), pH 7.8 (KPi 5 mM, sucrose 330 mM). The microsomal fr. (3 ml) was loaded on to 9 g of a 2-phase mixt. to yield a 12 g 2-phase system containing Dextran T-500 (6%, w/w), PEG 3350 (6% w/w), KC1 (3 mM), sucrose (330 raM) and KPB (5 mM). This was centrifuged at 4000 g for 12 min to remove the large amount of starch which would otherwise have contaminated the upper phase. The upper phase was then removed and the resulting plasma membrane-enriched ft. purified using a batch procedure [6]. The purified plasma membrane fr. was centrifuged at 100000 g for 30min, the pellet resuspended in PIPES-sucrose buffer (1 ml), (PIPES, 5 mM, sucrose 250 mM, to pH 6.5 with NaOH) and stored at - 2 0 ~ before use. For marker-enzyme assays, membranes were stored no longer than 48 hr and for lipid analysis, aliquots of suspended membranes were stored in CHC13-MeOH (1 : 2) at - 20~ Lipid analysis. In an Eppendorf tube, CHCI3-MeOH (0.75 ml) was added to resuspended membranes (0.5 ml) along with fl-cholestanol (20/A, 0.1 mgml-1), as an int. standard for sterol analysis. CHC13 (0.25 ml) was added, the mixt. shaken and centrifuged at 10000 g for 6 min. The CHC13 layer was retained, evapd to dryness under N 2 and made up to 100/zl with CHCI 3. For sterol analysis, 20 #1 of the CHCI 3 extract was placed in a glass vial (2 ml), evapd to dryness under N 2 and acetylated using pyridine (50/~1) and Ac20 (100/zl). After 2 hr the solvents were evapd under N2, EtOAc (20 #1) added and the sterols analysed by GC using an SE52-bonded capillary column coupled to FID, with H 2 as carrier (1 ml min-1) and a temp. prog. of 120-265 ~ at
10~ min-1. The inj. and detector temps were 250 and 320 ~, respectively. Bound fatty acids were determined by using 20-#1 portions of the CHC13 extract, evaporating to dryness under N2 and transmethylating with N a O M e - M e O H (0.5 N) (0.5 ml) and heating at 30 ~for 7 min. The resultant fatty acid Me esters were extracted with hexane (1 ml), evapd under N2, dissolved in EtOAc (20 pl) and analysed by GC using an RSL 500-bonded capillary column with FID and He as carrier (1 ml min- 1). The inj. and detector temps were 250 ~ and 300 ~, respectively. Phospholipids were analysed by HPLC, using the method described in ref. [19] with minor modifications. The CHCIa extract (20/zl) was injected into an Econosphere silica 3 ~ column (150 mm x 4.6 mm) and compounds detected with an evaporative light scattering detector. The detector conditions were as follows: N2 flow, 20 ml min- 1; detector response, 900 V; temp. 50~ A three-solvent gradient system was used, comprising: (A) hexane-dimethoxypropane (99:1), (B) isoPrOH-CHCI 3 (4:1) and (C) isoPrOH-H20 (1:1); the flow rate was 2 ml min- 1 throughout. The solvent programme was as follows: 100% A at time 0, and 1 min. At 5 min, A was 80% and B 20%. A, B and C were 42, 52 and 6%, respectively, at 5.1 min and at 15 min they were 35, 49 and 16%. At 20 min A was 42%, B 52% and C 6% and at 25 min A was 30% and B 70%. Finally, A was 100% at 30 min. Phospholipids were quantified by comparing peak areas with those of known standards. Glucan synthase II and A TPase assays, protein determinations and fluidity measurements. Glucan synthase II was estimated by aH-UDP-glucose linkage according to the method of ref. [20] with the modifications described in ref. [21]. ATPase (EC 3.6.1.35) activity was measured by hydrolysis of ATP and subsequent release of inorganic phosphate using the method described in ref. [22] as modified in ref. [21]. ATPase assay incubations were done at 37 ~ in the presence of Triton-X100 to overcome latency. KCI (50 raM), CaC12 (100 mM) or sodium vanadate (22 mM) were added specifically to stimulate or inhibit plasma membrane ATPase activity. Membrane fluidity was determined using steady-state fluorescence polarization with 1,6-diphenylhexatriene as the probe [7]. Protein was assayed by Coomassie-blue dye binding [23] with thyrogobulin as an int. standard. Chlorophyll analysis. Chlorophyllta § b~was determined by extracting appropriate amounts of microsomal and plasma membrane fraction with 80% Me2CO and measuring the absorbance at 652 nm. Data analysis. Data were analysed using unpaired 't'tests or 'analysis of variance' and each expt was repeated at least three times.
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