Permeability and membrane lipid metabolism of Phaseolus vulgaris infected with Uromyces phaseoli II. Changes in lipid concentration and 32P incorporation into phospholipids

Physiological

Plant PatholoQ

(1974)

4, 1 l-23

Permeability and membrane lipid metabolism of Phaseolus vulgaris infected with Uromyces phaseolit II. Changes in lipid concentration into phospholipids H. H.

HOPPE

and R.

HEITEFUSS

Institut fitr Pfanzenfiathologti und PJEanzenschutz, 34 Gdttingen, Federal Republic of Germany (Accepted for publication

and 32P incorporation

Georg August

Universitiit,

June 1973)

Phospholipids and glycolipids of rust-infected leaf halves and uninfected halves of the same leaves were quantitatively determined and compared with the lipids of healthy primary leaves of bean plants. The phosphatidyl-choline, phosphatidyl-ethanolamine, phosphatidylinositol and sulphoquinovosyl diglyceride (SL) content of the infected halves was the same as in the control plants. The infected tissue showed a decrease in monogalactosyl diglyceride (MGG), digalactosyl diglyceride (DGG) and phosphatidyl-glycerol (PG) and an increase in phosphatidyl-serine (PS) and phosphatidic acid content. These differences were localized mainly in the region of the pustule and not in the surrounding mycelium-free tissue. The amount of all phospholipids and glycolipids in the uninfected leaf halves was slightly lower than in the controls. The ungerminated and germinated uredospores of the parasite contained no MGG, DGG, SL or PG. The infected half leaves showed an increase in seP incorporation into all phospholipids especially into PS. The differences in “sP-incorporation between the phospholipids of the uninfected half leaves and the controls were only small. These results were discussed in relation to permeability changes of rust-infected bean tissue.

INTRODUCTION Rust-infected plant tissue shows an increase in membrane permeability [32-341. The barrier properties of the membranes are reduced resulting in a stimulation of electrolyte, amino acid and sugar leakage from the tissue of bean leaves infected with Uromycesphaseoli [lo]. Since phospholipids and glycolipids are important functional constituents of biological membranes [25j we investigated the metabolism of these lipids in relation to permeability during pathogenesis using infected half leaves as described in the previous paper [IO]. Heitefuss & Fuchs [8] found a higher incorporation of 32P into phosphor-$ choline, a constituent of the phospholipid, phosphatidyl-choline, in rust-infected wheat than in healthy plants. Lfidecke & t The following abbreviations will be used in this paper: cts/min = counts per minute; days p.i. = days after inoculation; I = infected leaf half, H = uninfected half of the same leaf; C = control leaf; DPG = diphosphatidyl-glycerol; PA = phosphatidic acid; PCH, PE, PG, PI, PS = phosphatidylcholine, -ethanolamine, -glycerol, -inositol and -serine, respectively; DGG = digalactosyl diglyceride; MGG = monogalactosyl diglyceride; SL = sulpholipid (sulphoquinovosyl diglyceride) ; PX = unidentified phospholipid.

12

H. H. Hoppe

and

R. Heitefuss

Beiss [18] reported changes in the relative amount of phospholipids and the appearance of lysophosphatidylcholine in diseased plant tissue. Stimulation of phospholipase activity of bean plants after infection with Sclerotium sclerotiorum and Thielaviojsis basicola was observed by Lumsden & Bateman [19] and Lumsden [20]. These authors discussed the significance of their results with respect to changes of membrane permeability. MATERIALS

AND

METHODS

The materials used in this study, the cultivation already been described in a previous paper [IO]. Chlorophyll

Chlorophyll Quantitative

and inoculation

of plants have

determination

was quantitatively determination

assayed by a method described by Ziegler & Egle [39].

ofphosphol$ids

and glycol$kls

Lipids were extracted from bean leaves following the method described by Beiss [2] which was slightly modified by us. Five grams of leaf material were homogenized in 110 ml boiling solvent mixture consisting of 30 ml CHCl,, 60 ml MeOH and 20 ml Ha0 (1 : 2 : 0*8), filtered and reextracted with 70 ml MeOH in a mortar. The residue was washed with 100 ml CHCI,. After combining all extracts in a separation funnel 90 ml Ha0 was added, the relation of CHCI,MeOH-H,O was now 2 : 2 : 1.8 and the extract separated into two distinct layers. The lipid layer was removed and the water layer was extracted three times with 50 ml CHCl,. The combined MeOH-CHCl, layers were evaporated to dryness at 40 “C, the lipid residue was dissolved in CHCl,, filtered through a sintered glass funnel (pore size G 3) and again dried under vacuum by rotary evaporation. The residue was dissolved in 2 ml benzene-amyl-alcohol-CHCls (1 : 1 : 1) and stored in tightly closed bottles at -20 “C. Thin-layer chromatography. Two-dimensional thin-layer chromatography was performed on 500-km layers of silica gel G (Merck, Darmstadt) following activation of the air dried layers at 110 “C for 30 min. Samples (O-04 ml) were applied on a 1 cm long streak. Solvent systems used were [21] : first dimension: CHCls-MeOH-NH,OH-Hz0 (163 : 75 : 6 : 4), second dimension: CHCla-MeOH-acetic acid-H,0 (170 : 25 : 25 : 6). The phospho- and glycolipids were detected by different colour reactions [2] (i) iodine vapour, (ii) ninhydrin reagent, (iii) phosphoric acid ester reagent and (iv) choline-phospholipid reagent, using pure PS and PCH as standards for reference. The coloured spots were compared for their RF-values with those of other authors

Extraction.

[S, 7, 21-23, 301. Quantitative lipid determination.

The quantitative analysis of phospholipids and glycolipids was performed by phosphate and sugar determination respectively after staining the spots with iodine vapour. To obtain enough lipid for the analysis of phosphorus and sugar we developed eight plates of each extract, scraped the spots off the plates and combined two of them from the following lipids: MGG, DGG, SL, PI, PS, DPG, PA. From these lipids we analysed four replications. From the higher concentrated lipids (PCH, PG, PE) we analysed only one spot and made

Permeability

and lipid

metabolism

in infected

bean

leaves

13

six replications. Appropriate blank areas were scraped off the plate and analysed for phosphate and sugar. The phosphate determination was carried out in the presence of the adsorbent by a method described by Beiss [3] which was slightly modified by us. After the digestion of the lipids with O-5 ml of H,S04HCI0, (1 : I)-mixture, 4 ml of 0.75% ammonium molybdate in water and 0.25 ml of a reducing reagent containing 1.2.4aminonaphtholsulfonic acid was added. After heating at 100 “C for 30 min the optical densities were read at 820 nm. The reducing reagent was prepared following the method of Stanton [31]. Sugars were determined after glycolipid hydrolysis by the phenol-HsSO,-method according to Roughan & Batt [26, 271. Lipid concentration was expressed in pm01 per g dry weight. Spore germination

and lipid extraction

Uredospores (2 g) were germinated on the surface of 10 1 distilled water for 12 h at room temperature in the dark. The spores were collected, rinsed thoroughly with water and extracted as described for plant material. The ungerminated uredospores were extracted by refluxing 2 g of spores for 12 h in a CHCls-MeOH (2 : 1) mixture. Incorporation

of 32P into phospholipids

Bean plants were excised at the soil level and incubated for 7 h in 25 ml of l/l0 Knop’s nutrient solution containing 5 @i 32P/ml [II], The procedures for extraction and thin-layer chromatography were the same as described before. To determine the specific activity of each phospholipid the extract was run on six chromatograms. The spots of the same phospholipid were combined and digested with 3 ml acid mixture. Of the digested material O-5 ml was used for determining the radioactivity and 0.5 ml (in three replicates) for the phosphate content. To determine the specific activity in the total lipid extract its volume was brought to 50 ml and 1.0 ml of this was used for measuring the radioactivity and 0.5 ml (four replicates) for phosphate content. The amount of 32P in the entire plant was measured by the specific activity of total phosphates in the dried material of which 30 mg were digested with 1 ml of the acid mixture, the volume was then brought to 5 ml with water. Phosphate determinations were done with 0.25 ml (four replicates), the radioactivity assays with O-5 ml using a gas flow counter model FHZ 448 (Frieseke und Hoepfner, Erlangen). The radioactivity was calculated as specific activity (cts/min per pg of P). Autoradiograms were prepared from some of the chromatograms using X-ray film. Preparation

of leaf-disks and leaf-rings

Leaf disks (0.5 cm diameter) were cut with a cork borer from sparsely infected bean leaves. Each disk was centred on a pustule. Leaf rings were prepared from the infected disks by removal of the central pustule with a steel cylinder (O-15 cm diameter). These rings were free of fungal contamination when observed under the microscope [3.5]. Similar disks and rings were also taken from control plants. AI1 samples were analysed for their lipid composition.

14 RESULTS Phospholipids the parasite Identijcation

H. H. Hoppe and R. Heitefuss and glycolipids

in infected and uninfected

bean leaves and in the uredospores of

of 1ipioJ.s. Two-dimensional thin-layer chromatograms of lipid extracts from bean leaves and from the ungerminated uredospores of the parasite are shown in Fig. 1. MGG, DGG and SL were identified as glycolipids. There were at least two other spots which contained no phosphate and which were tentatively identified by us from their R, value as cerebroside (spot 10) and steryl glucoside (spot 11) [S, 71. PCH, PE, PG and PI were the main phospholipids while PS, PA, DPG and PX appeared only in low concentrations. PX was not identified with certainty; however, it appeared to have an RF value very similar to Nacetyl-PE [30]. This lipid appeared in larger amounts only in experiments with leaf disks and leaf rings. In other experiments it was in too low concentration for quantitative determination. The contents of DPG and SL were in all experiments too low to get exact quantitative data, hence we could not observe any changes in these two lipids as the result of infection. The exact identification of PS was difficult and problematic. Separations were performed with the solvent system described in the “Materials and Methods Section” and in addition with the system of Beiss [3] on silicic acid impregnated paper. The compound showed a positive ninhydrin reaction in all cases and cochromatographed with synthetic PS which was added to the lipid extract. The same compound appeared in the germinated and ungerminated uredospores of the parasite. This is not in agreement with the results of Langenbach & Knoche [IS, 171 who detected no PS in the spores of U. phaseoli but a ninhydrin positive, inositol containing lipid. The compound in our material was unfortunately in too low concentration to get enough lipid for identification of hydrolysis products. However, from the chromatographic behaviour we conclude that it is PS, bearing in mind the limits of identification by cochromatography and ninhydrin reaction. On the chromatograms of extracts from uredospores some further spots appeared after staining with iodine vapour. The spots were very faint and were not identified by us (spot 16 to 21). Spot 19 was detected only in the extracts of ungerminated, but not in germinated uredospores. Spot 2 1 appeared only in traces in ungerminated but in larger amounts in the germinated uredospores of the parasite. The mobility of this compound (spot 21) in solvent system I indicates that it could possibly be phosphatidylmonomethylethanolamine or phosphatidyldimethylethanolamine which were found by Langenbach & Knoche [IS, 171 in germinating spores after labelling with ssP or 2-[W]methioninemethyl. Except for these two spots (spots 19 and 21) we observed no change in lipid composition after germination. E$ect of infection on lipid content. The lipid concentrations found in our material are in agreement with the data published by other authors [9, 27, 381. The effect of infection on the phospholipid and glycolipid concentration of bean leaves is shown in Figs 2-4. The type of changes occurring can be divided into three groups. The first group includes those membrane lipids (PCH, PE, PI) whose concentration in the infected leaf halves showed no significant change compared with the control; on the other hand, these lipids showed a more or less pronounced

Permeability

and lipid metabolism

FIG. 1. Two-dimensional the ungerminated uredospores NH,OH-HsO (163 : 75 : 6 (17 : 25 : 25 : 6). The numbers 5, DPG; 6, PE; 7, DGG; 8, 15, origin; 10, 11, 16, 17, 18,

in infected bean leaves

15

thin-layer chromatograms of bean leaf lipids (A) and lipids in of U. phaseoli (B). Solvent system I, CHCls-MeOH-l4n : 4). Solvent system II, CHCls-MeOH -acetic acid-H,0 represent the following lipids: 1, PA; 2, PS; 3, PI; 4, PCH; SL; 9, PG; 12, PX; 13, MGG; 14, neutral lipids and pigments; unidentified spots.

decrease in the uninfected leaf halves. This decrease was more pronounced with PI from 6 to 12 days after inoculation (Fig. 2). The second group is comprised of those lipids (PG, MGG, DGG) whose concentration in the infected leaf halves showed a decrease compared to the controls (Fig. 3). This decrease was first noticed 4 days (MGG) and 6 days after inoculation

16

H. H. Hoppe

I

I

I

I

I

I

and

R. Heitefuss

--I

Days after inoculation

FIG. 2. The effect of infection with U. phaseoli on the concentration of PCH, PE and PI (values are means of two experiments) in bean leaves. I, infected leaf halves; H, non-infected leaf halves; C, control leaves.

(PG and DGG). Th e v al ues for non-infected leaf halves were always between the infected ones and the controls. The curves for these lipids are similar to the chlorophyll concentration curve (Fig. 4) which confirms the previous reports that these lipids are constituents of chloroplast membranes [9, 1.21. The lipids of the third group showed an increase in the infected leaf halves, PS after 4 days and PA after 8 days, compared to the control (Fig. 4). The increase in PA during later stages of infection could possibly be due to the more severe damage by the parasite and to the onset of senescence. The concentration of PS reached a maximum 8 days after inoculation and then showed a continuous decline.

Permeability

and lipid metabolism

17

in infected bean leaves

i60q5i$ 1 s ‘Z e 5

40-

\ x .

20-

MGG

s u

I

I

I

I

I

I

2

4

6

8

IO

12

Days after inoculation FIG.

(values halves;

3. The effect of infection with U. phadi on the concentration of PG, MGG and DGG are means of two experiments) in bean leaves. I, infected leaf halves; H, non-infected leaf C, control leaves.

In these experiments it was not possible to attribute the changes in lipid concentration to either changes in plant lipid metabolism or to a direct contribution by the parasite. We tried to clarify this problem by two experiments: (i) In the first experiment an attempt was made to find out whether the noninfected tissue surrounding the pustule showed an alteration in lipid composition and concentration, Any observed changes in this tissue region cannot be the result of fungus lipids. Lipids extracted from leaf disks, collected from sparsely infected tissue and from controls, were compared with those prepared from leaf rings (see Materials and Methods). (ii) In the second experiment we analysed the germinated and ungerminated uredospores of the parasite to obtain information on the lipids of the fungus and the direct contribution of the parasite.

H. H. Hoppe

18

I

and

R. Heitefuss

Chlorophyll

PS

I.O-

1 2

FIG. 4. The effect of infection and PS (values are means of two non-infected leaf halves; C, control

I 4

I I I 6 8 IO Days after inoculation

I 12’

with U. phaseok on the concentration of chlorophyll, experiments) in bean leaves. I, infected leaf halves.; leaves.

PA H,

The results of these two experiments are presented in Table 1. The most striking difference between the results of these experiments and that with whole leaves (Figs 2 to 4) was the high content of PX in the disks and rings. This phospholipid (PX) was detectable in all the experiments; however, its conIn the experiments with contration was too low to allow quantitative determination. leaf disks and leaf rings this lipid (PX) reached a concentration up to 24% of total phospholipids (Table 1). The main differences in phospholipid concentration of the infected bean tissue and the non-infected one were confirmed by the experiments with leaf disks. The disks with pustules in the centres had a higher PS and a lower PG concentration

Permeability

and

lipid

metabolism

in infected

bean TABLE

The distribution

Sample PX DPG PE PG PCH PS PI PA Leaf disks from Leaf rings from Spg, germinated

19

1

in leaf disks and uredospores of U. phaseoli

leaf rings of bean leaves and in the

Id

Cd

Ir

Cr

Spg

23.8 0.8 13.5 19.7 24.7 4.6 8.7 4.2

20.6 0.9 11.6 28.7 25.3 1.2 6.8 4.9

22.3 1.5 10.0 26.5 22.1 3.3 8.4 5.9

16.1 2.5 10.5 29.3 24.5 2.9 7-5 6.7

0.6 32.4 525 8.0 4.6 1.9

(per cent)

of phospholipid

leaves

control leaves control leaves uredospores.

Spung 2.8 27.2 54.4 7.9 6.1 1.6

(Cd) and infected leaves (Id). (Cr) and infected leaves (Ir) , Spung, ungerminated uredospores

than the disks from the control leaves. After removing the pustules only small differences between leaf rings from infected leaves and controls were observed (Table 1). This implies that all differences in phospholipid concentration observed after infection were due mainly to changes in the region of the pustule and not to changes in the surrounding fungus-free tissue. The lipid composition of the uredospores differed significantly from that of bean leaves (Table 1). The spores contained no MGG, DGG, SL and PG. They had a high content of PCH and PE and contained more PS than the bean leaves. Incorporation

of 32P into phos~holipids

of infected and non-infected

bean leaves

Eight days after infection all phospholipids of the infected half leaves were more highly labelled than those of the control leaves. The PS showed the most striking difference. It was scarcely detectable on the autoradiograms of the controls but appeared as a dark spot on those of the infected half leaves (Plate 1). In Table 2 the specific activities (cts/min per pg P) of the phospholipids, of the total lipid extract and of the total phosphate of the dry material are compared. In all samples PA showed the highest specific activity followed by PI and the remaining phospholipids. These results are in agreement with the data on soybean phosphatides published by Singh & Privett [30]. The high labelling of the PA emphasized the central position of this phospholipid in lipid metabolism [9]. Four days after inoculation the specific activity of all phospholipids in the infected half leaves increased as compared with the control leaves. The increase in activity of the PS was very distinct at 3 days after inoculation and this level of higher activity over the control was maintained throughout the experimental period. The differences between the phospholipids of the uninfected half leaves and the controls were not well marked. Four and 6 days after inoculation the specific activity of all phospholipids in uninfected half leaves was higher than in the controls, but 8 days after inoculation there was no difference between the two samples. The specific activity of the total lipid extract showed a clear trend. Four, 6 and 8 days after inoculation the infected half leaves were found to have the highest

H. H. Hoppe The specific activity

Days p.i.

(ctslmin

TABLET per pg P) in phosfiholipids, weights)

total lipids

and

R. Heitefuss

and in total ghosphate

PA

PI

PS

PCH

PG

PE

Total lipid extract

200 195 275

420 381 520

568 490 636

4810 5250 5810

325 166 136

775 371 340

999 530 438

4440 3700 1800 5230 3280 2440 3100 1780 2110

3

I H c

4780 4960 6160

850 660 950

393 30 50

4

I H C

8780 7260 4320

1660 950 780

830 86 63

179 184 223 410 187 149

6

I H c

9190 6720 4810

1410 684 456

799 66 24

450 161 114

213 211 86

568 271 185

933 450 330

8

I H C

9120 7790 7280

1710 1080 1100

550 74 44

610 278 283

226 123 164

686 340 346

475 400 303

I, bean leaves control leaves.

infected

with

U. phareoli

leaf

halves;

H,

non-infected

leaf

(dry

Dry material

halves;

C,

incorporation into total phospholipids followed by the uninfected half leaves and the controls. The specific activity of the total phosphate in the dried plant material should represent the 32P uptake. The dry material from the infected half leaves showed a higher specific activity than the controls throughout the whole experimental period. The activity of the uninfected half leaves was higher only 4 and 6 days after inoculation compared to the controls.

DISCUSSION

Biological membranes are diffusion barriers. They govern all permeability processes from simple diffusion to active transport against a concentration gradient. In a previous paper it was reported that the barrier properties of the cell membranes of P. vulgaris are reduced as a result of the infection with U. phaseoli. Since membrane lipids play an important role in the structure and function of biological membranes we would first like to discuss whether the permeability changes are related to the alterations in lipid metabolism presented in this paper. The idea that lipids may be involved in membrane permeability has been frequently propounded in a variety of ways. It has been shown that phosphate fluxes of mammalian red blood cells increase with the content in phosphatidyl cholines [4]. Kuiper CD-151 observed that glycolipids strongly increased Cltransport into all parts of bean, cotton and grape plants. The plants showed a greater diversity in response to other lipids. By other experiments it was demonstrated that negatively charged lipids were involved in the transport of cations across membranes. Lipoprotein complexes from biological membranes bound cations in a specific manner. Using synthetic bilayers it was shown that negatively

PLATE

1. Autoradiogram

of aP-labelied

phospholipids and

from bepn leaves infected control leaves (right).

with

U. phaseoK:

leaf halves

(left;

8 days

p.i.)

Permeability

and lipid

metabolism

in infected

bean

leaves

21

charged lipids in particular were responsible for the selective binding of cations, whereas membranes from uncharged glycolipids exhibited only slight cation selectivity [9]. Consistent with these results are studies on the permeability of spontaneously formed liquid crystals of membrane lipids in salt solutions (liposomes). Bangham et al. [I] observed that the permeability of liposomes towards cations was facilitated by a negative charge of the membranes. Membranes without a negative charge showed only a very low cation permeation. Since predominantly PE and PS are the carriers of the negative charge of a phospholipid membrane [24, 371 these studies are of interest for our results. The higher amount of PS in the infected half leaves could possibly be involved in the enhanced leakage of electrolytes from this tissue. However, this view is only correct if the increase in PS concentration at least partly reflects a change in plant metabolism and not a direct contribution of the fungal lipids. The relative high content of PS in the germinated and ungerminated uredospores (Table 1) supports the idea that the changes in PS concentration reflect the presence of fungal lipids. On the other hand, the parasite would have to contribute about 40% of the dry matter of the host-parasite complex if the PS increase is to be explained only with the fungal lipids, assuming an equal PS content in parasitic hyphae and uredospores. As it seems to be improbable that the infected leaves contain so much fungal material it is suggested that the higher PS content of the infected tissue at least partly reflects a higher PS synthesis in the bean leaves. Consistent with this view are the results obtained by the 32P incorporation into the phospholipids. The increase in specific activity was most striking with the PS of the infected half leaves. The increase was already measurable 3 days after inoculation and surpassed the activity increase of the other phospholipids, indicating a stimulated biosynthesis of this lipid as a result of the infection. The decline in glycolipid and PG concentration, which was similar to the decrease in chlorophyll content of the infected half leaves, indicated a specific damage more probably to the chloroplast than of other plasma membranes. The chloroplast lipids consist nearly exclusively of MGG, DGG, SL and PG. However, these lipids are present at lower concentrations also in other cytoplasmic membranes [I,?]. Damage to chloroplasts in rust-infected tissue has been reported by several authors [5, 291 and can possibly lead to changes in cell compartmentation. Ryrie 82 Scott observed changes in the intracellular distribution of NADP+ in barley leaves infected with powdery mildew. In healthy leaves almost all of the NADP+ was localized in the chloroplasts. In infected leaves, however, about 60% of the NADP+ was detected in the non-chloroplast part of the cells. The increased availability of NADP+ or other metabolites in the cytoplasm&z parts of the cells may be an important factor in regulating the pentose-phosphate cycle or other metabolic pathways [28]. The results presented in this paper may be discussed from another viewpoint. In the infected tissue we observed no changes in the concentration of the main phospholipids PCH, PE and PI, lipids which were also detected in the parasite. We observed a decrease in those lipids, which were not present in the fungus (MGG, DGG, PG). These findings might be explained by an enhanced degradation of all plant membrane lipids in the infected tissue. If that were the case, then the decrease in plant lipid concentration would be detectable only with those lipids which are not present in the parasite (MGG, DGG, PG). H owever, it would not be detectable

22

H. H. Hoppe

and

I?. Heitefuss

with the lipids which are common to both host and parasite (PCH, PE, PS), because in this case the decrease in plant lipids would be compensated or possibly overcompensated for by the fungal lipids. An increased membrane lipid degradation in the infected tissue might be of great importance for the enhanced leakage of cell constituents from this tissue. However, it has to be taken into consideration that the leakage stimulation occurred earlier during pathogenesis than the main changes in lipid content. This view has been discussed by other authors in connection with stimulation of phosphatidase activity in infected plant tissues [19, 20, 361. From other experiments it was concluded that the permeability of membranes may depend on the fatty acid composition of membrane lipids [9]. In the next paper of the present series changes in phosphatidase activity and in lipid bound fatty acids of our host-parasite model will be described. REFERENCES 1. BANGHAM, A. D., STANDISH, M. M. & WATKINS, J. C. (1965). Diffusion of univalent ions across the lamellae of swollen phospholipids. Journal of Molecular Bioloa 13, 238-252. 2. BEISS, U. (1963). Phosphatide und Glykolipide. In Moderne Methoden der Pjanzenanalyse VI, Ed. by H. F. Linskens & N. V. Tracey, pp. 52-80. Springer Verlag, Berlin, Gottingen, Heidelberg. 3. BEISS, U. (1969). Beitrag zur quantitativen Bestimmung von Phosphatiden. Fette Se@ Anstrichmittel 71, 363-365. 4. DEUTICKE, B. & GRUBER, W. (1970). Anion permeability of mammalian red blood cells: possible relation to membrane phospholipid patterns. Biochimka et Biophysics Acta 211, 369-372. 5. FUCHS, W. H. & TSCHEN, J. (1969). Syntheseaktivitat und Grosse der Zellkerne von Phaseolus vulgaris nach Infektion mit Uromyces phaseoli typica. Netherlands Journal of Plant PatholoQ 75, 86-95. 6. GALLIARD, T. (1968). Aspects of lipid metabolism in higher plants. I. Identification and quantitative determination of the lipids in potato tubers. Phytochemistry 7, 1907-1914. 7. GALLIARD, T. (1968). Aspects of lipid metabolism in higher plants. II. The identification and quantitative analysis of lipids from the pulp of pre- and post-climacteric apples. Phytochemistry 7, 1915-1922. 8. HEITEFUSS, R. & FUCHS, W. H. (1963). Phosphatstoffwechsel und Sauerstoffaufnahme in Weizenkeimpflanzen nach Infektion mit Puccinia graminis tritici. Phytopathologischs es phaseoi var. typica. Dissertation, Gottingen. 12. KATES. M. (1970). Plant vhosuholiwids and elvcoliwids. Advances in L&id Research 8, 225-265. 13. KUIPE& P. J. C.‘( 1968). Lipids in grape ro&‘in rklation to chloride transport. Piant PhysioloD 43, 1367-1371. 14. KUIPER, P. J. C. (1968). Ion transport characteristics of grape root lipids in relation to chloride transport. Plant Physioloa 43, 1372-1374. 15. KUIPER, P. J. C. (1969). Effect of lipids on chloride and sodium transport in bean and cotton plants. Plant Physioloa 44, 968-972. 16. LANGENBACH, R. .I. & KNOCHE, H. W. (1971). Phospholipids in the uredospores of Uromyces phaseoli. I. Identification and localization. Plant Phjsiolo& 48, 728-734. 17. LANGENBACH, R. J. & KNOCHE, H. W. (1971). Phospholipids in the uredospores of Uromyces phaseoli. II. Metabolism during germination. Plant Physiology 48, 735-739. 18. L~~DECKE, H. & BEISS, U. (1966). Untersuchungen tiber Zuckerrtibenlipide.
Permeability 21. 22. 23. 24. 25. 26.

27. 28.

29. 30. 31. 32. 33. 34. 35.

36. 37. 38. 39.

and

lipid

metabolism

in infected

bean

leaves

23

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