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Permeability and membrane lipid metabolism of Phaseolus vulgaris infected with Uromyces phaseoli III. Changes in relative concentration of lipid bound fatty acids and phospholipase activity
Physiological
Plant Pathology
(1974)
4, 25-35
Permeability and membrane lipid metabolism of Phaseolus vulgaris infected with Uromyces phaseoli? Ill. Changes in relative concentration phospholipase activity H. H.
HOPPE
and R.
HEITEFUSS
Institut f& Pjlan~enpathologie und PJlanzemchutz, 34 Giittingen, Federal Republic of Germany (Accepted for publication
of lipid bound fatty acids and
Georg August
Uniuersitiit,
June 1973)
Fatty acids from membrane lipids and phospholipase activity of rust-infected half leaves and uninfected halves of the same leaves were determined and compared with healthy primary leaves. The following lipids were analysed for their fatty acid composition: monogalactosyl diglyceride (MGG), digalactosyl diglyceride (DGG), phosphatidyl-choline (PCH), phosphatidyl-ethanolamine (PE) and phosphatidyl-glycerol (PG). The major fatty acids detected in these lipids of both bean leaves and fungal uredospores were pa&tic, stearic, oleic, linoleic, linolenic and in PG, trans-three-hexadecanoic acid. The PE and PCH isolated from the infected half leaves contained a higher amount of unsaturated fatty acids than the corresponding lipids from the two other tissues. The PE and PCH from fungal uredospores showed a higher degree of unsaturation than the lipids from bean leaves. The changes in fatty acid composition of PG, MGG and DGG were small and possibly not significant. The differences in fatty acid composition were localized mainly in the region of the pustule. The adjacent fungus free host tissue was affected only to a small extent. The infected tissue showed an increase in phospholipase activity as compared with the controls and the uninfected leaf halves. The results were discussed mainly in relation to permeability changes of rustinfected bean tissue.
INTRODUCTION
In an earlier publication [14] we reported changes in the concentration of certain phospholipids and glycolipids and in the incorporation of 32P into phospholipids in bean leaves infected with Uromyces phaseoli. These results were discussed with regard to previously observed changes in membrane permeability of rust infected bean tissue [13]. Investigations concerning lipid metabolism during pathogenesis were extended to the fatty acid composition of membrane lipids and to the phospholipase activity. The results of this investigations are reported here. MATERIALS
AND
METHODS
The materials used in this study, the preparation of leaf disks and rings, the extraction and isolation of phospholipids and glycolipids from bean leaves and uredospores 7 The following abbreviations will be used in this paper: days p.i. = days after inoculation; I infected leaf half; H = uninfected half of the same leaf; C = control leaf; PCH, PE, PG phosphatidyl-choline, -ethanolamine and -glycerol, respectively; PX = unidentified phospholipid; MGG = monogalactosyl diglyceride; DGG = digalactosyl diglyceride; 16:0 = palmitic acid; 16 :l trans-n3-hexadecenoic acid; 18:0 = stearic acid; 18: 1 = oleic acid; 18:2 = linoleic acid;l8: 3 linolenic acid.
= = = =
26
H. H. Hoppe
of the parasite by thin-layer previous papers [13, 141. Determination
of fat&
chromatography
acid content of phospholipidr
and
R. Heitefuss
have already been described in two
and glycolipids
To prevent oxidative damage of fatty acids during thin-layer chromatography butylated hydroxy toluene was added to the solvents (50 mg/lOO ml in the first dimension, 5 mg/ 100 ml in the second dimension). The developed plates were dried in air for 20 min and sprayed with 0.04% 2’,7’-dichlorofluorescein in methanol, previously purified following the method of Parker & Peterson [25]. Using this method only the higher concentrated lipids (PG, PE, PCH, MGG, DGG) were visible under U.V. light and were marked. The spots from four replicate plates were pooled for fatty acid analysis. Fatty acid methyl esters were prepared by direct transmethylation with Na-methylate in the presence of silica gel following the method described by Husek [15]. The methyl esters were dissolved in hexane and injected into a Tricarb-Packard gas chromatograph equipped with a flame ionization detector, which had the following set features: column: 10% diethyleneglycolsuccinate on chromosorb W, temperature 180 “C isothermal, injector and detector temperature: 200 “C, carrier gas: N,, flow rates: N, 30 ml/mm, H, 40 ml/mm, air 400 ml/min. For identification of the peaks fatty acid methyl ester standards were run under the same conditions. Quantitative analysis of fatty acids was based on peak area measurement, multiplying the height of the peak by the width of the peak at half height. Concentration was determined using calibration factors previously calculated by the measurement of peak area produced by known quantities of fatty acid methyl esters. The amount of each fatty acid component was expressed as a percentage of the total area. Phospholipase
assay procedure
extraction. Plant material (3 to 5 g) was ground in a mortar with the same amount of buffer (O-1 M-succinate-NaOH buffer, pH 5.0) and squeezed through four layers of cheesecloth. When the effect of pH on phosphatidase activity was determined the incubation buffer was used for extraction and the pH of the extract was readjusted. The sap was centrifLged at 10 000 g for 20 min and the supernatant fluid assayed for phosphatidase activity. Substrate. Soybean lecithin (Sigma, U.S.A.) was purified by chromatography on a silica acid column [2]. The MeOH fraction was taken to dryness and the residue suspended in buffer (0.05 M-succinate-NaOH, pH 5.0) by blending in an ice-bath using a Virtis homogenizer operated at full speed for 30 min. In the experiments with different pH during incubation the substrate was suspended in the incubation buffer using a succinate-NaOH buffer in the range of pH 3-O to 6-O and a Tris-HCl buffer in the range pH 7.0 to 9.0. Enzyme assay. Phosphatidase was assayed by measuring the release of trichloroacetic acid soluble phosphate [21, 2.21. Standard enzyme reaction mixtures contained 0.5 ml enzyme extract and 0.5 ml of a 1o/o substrate suspension. After Enzyme
Permeability
and
lipid
metabolism
in infected
bean
leaves
27
incubation for 2 h at 30 “C the reaction was stopped by the addition of 0.1 ml 5% serum albumin to aid the precipitation of phosphatides and 0.9 ml 20% trichloroacetic acid [Zl]. After centrifugation the supernatant was assayed for soluble phosphate using the method of Allen [I]. Blanks were analysed in which the serum albumin and trichloroacetic acid were added to the substrate before the addition of the enzyme preparation. Enzyme activity was normally expressed as pg P released per mg protein in 2 h. Protein was determined according to the method of Lowry et al. [ZO] after precipitation with trichloroacetic acid. RESULTS
Efect
of infection on the fatty acid composition of membrane lipids
The distribution of the major fatty acids in isolated lipids from infected and uninfected leaf halves and from control leaves of bean plants is shown in Table 1. 1
TABLE
Fatty
acid composition of bean lipids from rust-infected bean leaf halves (I), non-infected halves (H) and control leaves (C). Values are a percentage of total fatty acids Days
p.i. I
PCH
PG
PE
Days PCH
PG
PE
16:0 16:2 18:0 18:l 18:2 18:3 16:0 16:l 18:0 18:l 18:2 18:3 16:0 18:0 18:l 18:2 18:3
19.3 7.2 2.4 29.3 41.8 36.1 33.5 3-5 1.3 3.4 22.2 25.9 4.0 1.5 32.6 36.0
16:0 16:2 18:0 18:l 18:2 18:3 16:0 16:l 18:0 18:l 18:2 18:3 16:0 18:0 18:l 18:2 18:3
19.7 4.8 1.0 15.5 59.0 33.5 35.0 4.0 0.5 2.0 25.0 27*0 3.6 1.0 21.4 47.0
p.i.
4 H 21.7 7.4 2:.; 44.2 37.1 34.1 4.6 0.9 2.3 21.0 31.9 4.8 1.2 30.3 31.8 10 19.8 2-9 4.6 o-4 10.5 61.8 35.7 34.8 3.9 0.8 l-6 23.2 28.2 3.9 o-7 17.6 49.6
C
I
21.8 7.2 2-4 25.6 43.0 38.6 34.8 4.4 1.1 2.0 19-l 32.5 4.9 1.7 31.4 29.5
18.7 6.6 3.0 26.5 45.2 33.0 31.2 4.1 1.0 2.4 28.3 18.2 2.9 2.2 31.8 44.9
22.0 5.3 o-7 13.6 58.4 38.2 36.2 3.9 0.4 l-3 19.9 30.5 2.5
16.3 0.6 3.8 1.3 15.0 63.0 33.1 33.1 4.1 0.6 2.5 26.6 24.6 2.7 1.6 21.5 49.6
2Z.E 45.7
6 H 22.0 2.6 1.5 23.9 45.0 35.8 34.0 3.9 0.6 1.7 24.0 29.6 5.4 32 32.3 12 16.5 6.9 2.8 1.1 9.6 63.1 39.3 32.6 3.8 0.8 2.0 20.5 28.5 2-4 1.1 16.5 51.5
C 21.4 7.9 2:.: 44.2 37.9 34.5 4.4 0.7 1.9 20.6 30% 4.8 1.2 30.3 33.1 17.7 3.5 3.2 0.9 9.0 65.7 37.3 35.8 4.0 0.6 l-5 20.8 26.5 3.2 0.7 16.1 53.5
leaf
28
H. H. Hoppe
and
R. Heitefuss
The fatty acid composition is expressed as weight per cent of total fatty acids. The values are averages of two (4, 12 days p.i.) or three (6 days p.i.) separate experiments. The data of each experiment were obtained by duplicate determinations (two different samples used for transesterification). The deviation from the average for duplicate determinations was less than 5%, for the separate experiments it was about 10%. The variations do not apply to the low concentration (less than 10%) fatty acids in which percentage variation sometimes was greater as a consequence of the smaller amounts of these fatty acids. The principal fatty acids detected in all lipids of both diseased and healthy bean leaf tissue were palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic acid (18 : 3). In PG a fatty acid appeared (16 : 1) with a retention time similar to palmitoleic acid and was tentatively identified by us as trans3-hexadecanoic acid. This reached up to 35% of the total fatty acids in the PG of our material. It occurs in many plants, particularly chlorophyllous tissues [18, 231. At the later stages of our experimental period a fatty acid in PCH was detected whose retention time lay between 16 : 1 and 18: 0. This acid was not identified by us. According to its retention time it could be a 16:2. The results indicate that the appearance of this acid was related to the progress of senescence in the bean tissue. The fatty acid was first detected 10 days after inoculation in the PCH of the non-infected half leaves and was present 12 days after inoculation in all three treatments. It was highest in the non-infected leaf halves, which were already yellow, and lowest in the infected ones, which were still green. Other fatty acids, not shown in Table 1, were present only in trace amounts (< 1o/o of the fatty acids in each lipid). The fatty acid composition of plant lipids reported here is in good agreement with the results of other authors [16]. Comparing single lipids it becomes evident that PCH and PE have a similar fatty acid composition, PG is relatively rich in palmitic acid and poor in linoleic acid whereas MGG and DGG contained a high portion of linolenic acid, about 94 and 87%, respectively. The fatty acid composition of the two galactolipids was the same in both diseased and healthy tissue and did not change during the experimental period. The data of these lipids are therefore not presented in Table 1. The phospholipids (PE, PCH, PG) of the infected leaf halves contained a higher amount of unsaturated fatty acids between 4 and 8 days after inoculation than the uninfected leaf halves and the controls. This difference was more pronounced in the PE than in the two other phospholipids (compare also Fig. 1). No difference was observed between the lipids of the uninfected leaf halves and the controls. To make the difference in fatty acid composition clearer the ratio Z unsaturated fatty acids C saturated fatty acids was calculated for each of the three phospholipids and is presented in Fig. 1. Increased amounts of unsaturated fatty acids occurred in the diseased tissue at 4 days after inoculation when chlorotic spots appeared. In PE the content of unsaturated fatty acids reached a maximum 8 days after inoculation and decreased later ( 10 and 12 days after inoculation) under the low light conditions of our experiments. In PCH the increase in unsaturated fatty acids was lower than in PE;
Permeability
and lipid
metabolism
in infected
bean I
I
29
leaves
I
’
I
I’
l- C PE
lC
PG I 2
I 4
I 6
I 8
I IO
I 12
Days after inoculation
FIG.
1. Bean
leaves
infected
with
U. phaseoli:
C unsaturated 2 saturated in PE, PCH
and
PG.
I, infected
leaf halves;
The
fatty
acid
ratio
fatty acids fatty acids H, uninfected
leaf halves;
C, control
leaves.
however, it began earlier and was already observed 3 days after inoculation. In contrast to PE and PG an increase in unsaturated fatty acids occurred in PCH at the later stages of the infection (10 and 12 days after inoculation) in all three treatments. This increase was mainly due to the unidentified acid which was possibly 16: 2 and therefore added to the unsaturated acids. The variations in acid composition of PG were small and can possibly be attributed to experimental error. However, between 4 and 12 days after inoculation the mean values of the fatty acid ratio were always higher in the infected leaf halves than in the two other treatments.
H. H. Hoppe
30
and
R. Heitefuss
As mentioned in a previous paper [la] it is not possible to decide whether the changes in fatty acid composition are due to changes in plant lipid metabolism or to a direct contribution of the fungal lipids or to both of these causes. We tried again to clarify this problem by two experiments. In the first experiment an attempt was made to find out whether the non-infected tissue surrounding the pustule was influenced by the infection. Changes in this tissue region can clearly be attributed to host plant metabolism. In the second experiment the germinated and ungerminated uredospores of the parasite were analysed to get information on the fatty acid composition of the fungal lipids. The fatty acid composition of the spore lipids (PCH and PE) is shown in Table 2. In addition the results of both experiments are expressed in the fatty acid ratio mentioned earlier and summarized in Table 3. TABLE
Fatty
acid
Uredospores: Ungerminated Germinated
composition of PCH U. phaseoli.
2
PE from ungerminated and gemtinated Values are a percentage of total fat& acids
uredospores
and
16:0
18:0
PCH I8:l
18:2
18:3
16:0
18:0
PE 18:l
18:2
18:3
24.9 22.4
4.5 5.1
2.4 1.5
10.7 7.6
57.5 63.4
7.4 4.5
1.7 0.7
2.9 1.7
152 10.7
72.8 82.4
The PCH and PE of the uredospores contained the same major fatty acids as the corresponding lipids of the bean leaves. In PCH the per cent distribution of the acids was similar to bean leaves; however, the PE of the uredospores had a higher content of linolenic acid (18 : 3). During germination the linolenic acid increased in both lipids leading tb a higher degree of unsaturation in the PE and PCH of the germinated spores (Table 3). The experiments with leaf disks confirmed the previously demonstrated differences in the fatty acid composition of membrane lipids between the infected and healthy TABLE
3
‘T7zefatty acid ratio (Z: unsaturated fatty acids)/@ saturated fatty acids) of several membrane lipids in leaf disks and leaf rings from bean leaves and in the uredosfiores of U. phaseoli Sample
Id
Cd
Ir
Cr
SPg*
Spung.
2.1 8.3 57.8
1.7 8.2 51.6
l-9 8.2 54.6
1.8 8.3 51.7
-
-
PE PG
3.3 1.5
1.5 I.4
1.9 1.5
1.5 1.4
18.2 -
10.0
PCH
2.4
2-O
2.1
1.9
2.6
2-1
PX DGG MGG
Leaf disks from infected (Id) and control leaves (Cd) respectively. Disks from infected leaves contained a pustule in the centre. Leaf rings from infected (Ir) and control leaves (Cr) respectively. Rings from infected leaves were prepared by removal of the central pustule from disks (for further details see Materials and Methods in reference [14J). Spg, germinated uredospores; Spung, ungerminated uredospores.
Permeability
and
lipid
metabolism
in infected
bean
leaves
31
tissue. The PCH and PE from the disks with a pustule in the centre (Id) had a higher degree of unsaturation than the compounds from corresponding disks of the controls (Cd). After removing the pustules only small differences between rings from infected leaves (Ir) and from controls (Cr) were observed (Table 3). The results indicated that the shift in fatty acid composition occurred mainly in the region of the pustule, whereas the surrounding fungus free tissue was affected only to a small extent by the parasite. E$ect of infection on the @hospholipase activity of bean leaves
Five types of phospholipases, e.g. phospholipases A, B, C, D and lysophospholipase have been described [12], each of which is specific for one of the ester linkages in the phospholipid molecule. Using the acid-soluble phosphorus procedure it is not possible to detect phospholipase D (phosphatidyl choline phosphatidohydrolase, EC 3.1.4.4), which catalyses the removal of choline, ethanolamine, etc. from the phospholipids with the consequent formation of PA, and phospholipase A (phosphatide acyl hydrolase, EC 3.1.1.4) which specifically removes one of the fatty acids. With this method only the activities of the following enzymes can be detected : phospholipase B, which removes both fatty acids, phospholipase C, which removes the polar head group and the combined activity of phospholipase A+lysophospholipase (lysolecithin acyl hydrolase, EC 3.1.1.5). A mixture of these different phospholipases could also be responsible for the release of acid-soluble phosphate in our reaction mixtures. Properties of phospholipase reaction. Maximum phospholipase activity of all extracts was between pH 4.0 and 5.0. The activity dropped off sharply at pH 3.5 and decreased between pH 6.0 and 7.0. Very low activities were detected above pH 7.0. All subsequent tests were performed at pH 5.0. CaCl, had no effect on phospholipase activity at pH 5-O when concentrations of CaCl, between 0 and 6 mM were present in the reaction mixtures. The optimum pH for activity is consistent with previous reports [22, 291. The lack of a response to CaCl, can be explained by the crude enzyme extract containing possibly enough Ca2+ for optimal enzyme activity. Phospholi.ase activity in diseased and healthy tissue. The enzyme activities in the controls and the uninfected half leaves were relatively low and remained rather constant over the experimental period. The activities were measured at 30 “C, which may not give a good estimation of the fungal enzymes since Knoche & Horner [19] found a marked maximum at around 18 “C for a lipase isolated from wheat rust uredospores. However, at 20 “C we found in our experiments almost no phospholipase activity in the control leaves and we therefore incubated the enzyme assays at 30 “C. The infected leaf halves showed an increase in enzyme activity beginning between 4 (experiment I) and 6 (experiment II) days after inoculation. Maximum activity was reached 6 to 8 days p.i. The differences between the two experiments are possibly due to variations in infection density. In experiment I the infection density was higher and the parasite developed faster than in experiment II. In the first experiment chlorotic spots appeared 4 days after inoculation whereas they were detected in the second experiment 1 day later (Fig. 2).
32
H. H. Hoppe
and
R. Heitefuss
x Experiment1
_
Days after inoculation
FIG. 2. I, infected
Phospholipase activities leaf halves; H, uninfected
in extracts leaf halves;
of bean leaves infected C, control leaves.
with
U. phmeoli:
DISCUSSION
The barrier properties of biological membranes depend to a considerable extent on the nature of the hydrocarbon chains of the membrane lipids. Force-area characteristics of monomolecular films of phospholipids with different fatty acid constituents show that the mean molecular area occupied per phospholipid molecule increases when the hydrocarbon tails become shorter or when unsaturated fatty acids are introduced into the phospholipid molecule. Extrapolation from such systems to biological membranes is difficult although they give some information on lipid-lipid interaction [3]. A more relevant approach is possibly the study of the permeability properties of liposomes, which are prepared by spontaneous swelling of membrane lipids in salt solutions and which are composed of multibilayers behaving as osmometers ES]. Liposomes prepared in salt solutions were exposed to an isotonic solution of glycerol or erythritol. The permeability of lecithin liposomes towards these two non-electrolytes increased markedly with an increasing degree of unsaturation or decreasing chain length of the lipid acyl groups. These results and studies performed with natural systems preferably with
Permeability
and lipid metabolism
in infected bean leaves
33
Mycooplasmalaidlawii B [7,9, 261 and Eschrichia coli [lo] indicate that permeability of membranes towards water-soluble non-electrolytes may depend at least partly on the unsaturation and on the length of fatty acids in the membrane lipids. From this viewpoint it seems possible that in our experiments the higher degree of unsaturation in the phospholipids, especially in the PE, of the infected tissue is responsible for the higher leakage of non-electrolytes like sugars [13] from this tissue. This explanation depends on the supposition that the observed changes in fatty acid constituents are at least partly due to changes in host lipids and are not restricted to a direct contribution by the fungus. The high degree of unsaturation in the PE from the uredospores indicates that the differences can possibly be attributed to the fungal PE although the results of the experiments with leaf disks and leaf rings show that the fungus-free tissue surrounding the pustule is also affected to a small extent by the infection. From these results it is concluded that both the host and the parasite are involved in the changes of the lipid acyl groups and that these changes might be important for the alterations of membrane permeability observed in the infected tissue. The results described in the second part of this paper show a higher phospholipid hydrolase activity in the infected half leaves than in the uninfected ones and the controls. Production of phospholipases by phytopathogens or higher phospholipase activities in infected plant tissues were reported by several authors [17, 21, 28, 291 and discussed in relation to membrane permeability. In a previous paper we mentioned that the changes in the amount of phospholipid observed in our experiments may reflect an enhanced degradation of membrane lipids in the infected leaf halves and we suggested that the enhanced degradation could be responsible for the higher leakage from this tissue [13]. However, the onset of the changes does not support this theory. The leakage showed a sharp increase 4 days after inoculation whereas the decrease in lipid concentration (MGG, DGG, PG) and the increase in phospholipase activity was hardly detectable 4 days after inoculation and reached a maximum 6 to 8 days after inoculation. Because of the late beginning of the changes in lipid amount and phospholipase activity it seems to be improbable that these changes induce the leakage stimulation. Increased phospholipase activity results in an increased release of free fatty acids. The addition of free fatty acids to isolated mitochondria causes a swelling of the organelles and uncouples oxidative phosphorylation [31]. Wojtczak & Lehninger [30] showed that the spontaneous swelling of rat liver mitochondria can be ascribed to the enzymatic release of free fatty acids from the membrane lipids. Similar results have been reported for mitochondria from Phaseolw vulgaris [4-61. From rust-infected wheat plants it is known that a partial uncoupling occurs in the later stages of development of infections on a susceptible variety [27]. Olah et al. [24] observed, however, in contrast to other authors [II], swollen plant mitochondria in rust-infected cells. The similarity between free fatty acid and fungus-induced swelling of mitochondria supports the idea that the increased phospholipase activity causes a partial damage to mitochondria in the infected tissue. However, the maximum phospholipase activity in our experiments was reached between 6 and 8 days p.i. whereas the damage to mitochondria and uncoupling of respiration occurs at later stages of infection. 3
34
H. H. Hoppe and R. Heitefuss
The assistance of Dr Manocha and Dr Williams with the translation scripts of this series is gratefully acknowledged.
of the manu-
REFERENCES 1. ALLEN, R. T. L. (1940). The estimation of phosphorus. Biochemical Journal 34, 858-865. 2. BEISS, U. (1963). Phosphatide und Glykolipide. In Moderne Methoden akr Pjancenanabse VI, Ed. by H. F. Linskens & M. V. Tracey, pp. 52-80. Springer-Verlag, Berlin, Gijttingen, Heidelberg. 3. DEENEN, L. L. M. VAN (1969). Membrane lipids and lipophilic proteins. In The Molecular Basis of Membrane Function, Ed. by D. C. Testeson, pp. 47-78. Prentice-Hall, Inc., Englewood Cliffs, New Jersey. 4. EARNSHAW, M. J. & TRUELOVE, B. (1968). Swelling and contraction of Phaseolus hypocotyl mitochondria. Plant Physiology 43, 121-129. 5. EARNSHAW, M. J. & TRUELOVE, B. (1970). Swelling of Phaseolus mitochondria in relation to free fatty acid level. Plant Physiology 45, 318-321. 6. EARNSHAW, M. J. & TRUELOVE, B. (1970). Swelling of Phaseolus mitochondria induced by the action of phospholipase A. Plant Physiology 45, 322-326. 7. MCELHANEY, R. N., DE GIER, J. & VAN DEENEN, L. L. M. (1970). The effect of alterations in fatty acid composition and cholesterol content on the permeability of Mycoplasma laidlawii B cells and derived liposomes. Biochimica et Biophysics 219, 245-247. 8. DE GIER, J., MANDERSLOOT, J. G. & VAN DEENEN, L. L. M. (1968). Lipid composition and permeability of liposomes. Biochimica et Biophysics Acta 150, 666-675. 9. HAEST, C. W. M., DE GIER, J. & VAN DEENEN, L. L. M. (1969). Changes in the chemical and the barrier properties of the membrane lipids of E. coli by variation of the temperature of growth. Chemistry and Physics of Lipids 3,413-417. 10. HAEST, C. W. M., DE GIER, J., VAN Es, G. A., VERKLEIJ, A. J. & VAN DEENEN, L. L. M. (1972). Fragility of the permeability barrier of Escherichia coli. Biochimica et Biophysics Acta 288, 43-53. 11. %RDU?CK, N. V., GREENWOOD, A. D. & WOOD, R. K. S (1971). The line structure of the haustorium of Uromyces appendiculatus in Phoseolus vulgaris. CanadianJournal of Botany 49,383-390. 12. HITCHCOCK, C. & NICHOLS, B. W. (1971). Plant Lipid Biochemistry. Academic Press, London, New York. 13. HOPPE, H. H. & HEITEFUSS, R. (1974). Permeability and membrane lipid metabolism of Phaseolus vulgaris infected with Uromyces phaseoli. I. Changes in the efflux of cell constituents. Physiologkal Plant Pathology 4, 5-9. 14. HOPPE, H. H. & HEITEIWS, R. (1974). Permeability and membrane lipid metabolism of Phaseolus vulgaris infected with Uromyces phaseoli. II. Changes in lipid concentration and 3sP incorporation into phospholipids. Physiological Plant Pathology 4, 1 l-23. 15. HUSEK, P. (1969). Methode zur direkten Methylierung diinnschichtchromatographisch aulgetrennter Lipide ohne deren Elution aus Kieselgel.
Permeability
26. ROMIJN,
27. 28.
29. 30. 31.
and
lipid
metabolism
in infected
bean
leaves
35
J. C., VAN GOLDE, L. M. G., MCELHANEY, R. N. & VAN DEENEN, L. L. M. (1972). Some studies on the fatty acid composition of total lipids and phosphatidyl glycerol from Acholeplanna laidlawii B and their relation to the permeability of intact cells of this organism. Biochimica et Biophyka Acta 280, 22-32. SHAW, M. (1963). The physiology and host-parasite relations of the rusts. Annual Review oj Phytopatholou 1, 259-294. TSUNG-CHE TSENG & BATEMAN, D. F. (1968). Production of phosphatidases by phytopathogens. Phytopathology 58, 1437-1438. TSUNG-CHE TSENG & BATEMAN, D. F. (1969). A phosphatidase produced by Sclerotium rolfsii. PhytopatholoQ 59, 359-363. WOJTCZAK, L. & LEHNINGER, A. L. (1961). Formation and disappearance of an endogenous uncoupling factor during swelling and contraction of mitochondria. Biochimica et Biophy.rica Acta 51, 442-456. WOJTCZAK, L. & ZALLJSICA, H. (1969). Inhibition by oleate of the binding and exchange of ATP by mitochondrial membranes. Biochimica et Biophysics Acta 189, 455-456.
Plant Pathology
(1974)
4, 25-35
Permeability and membrane lipid metabolism of Phaseolus vulgaris infected with Uromyces phaseoli? Ill. Changes in relative concentration phospholipase activity H. H.
HOPPE
and R.
HEITEFUSS
Institut f& Pjlan~enpathologie und PJlanzemchutz, 34 Giittingen, Federal Republic of Germany (Accepted for publication
of lipid bound fatty acids and
Georg August
Uniuersitiit,
June 1973)
Fatty acids from membrane lipids and phospholipase activity of rust-infected half leaves and uninfected halves of the same leaves were determined and compared with healthy primary leaves. The following lipids were analysed for their fatty acid composition: monogalactosyl diglyceride (MGG), digalactosyl diglyceride (DGG), phosphatidyl-choline (PCH), phosphatidyl-ethanolamine (PE) and phosphatidyl-glycerol (PG). The major fatty acids detected in these lipids of both bean leaves and fungal uredospores were pa&tic, stearic, oleic, linoleic, linolenic and in PG, trans-three-hexadecanoic acid. The PE and PCH isolated from the infected half leaves contained a higher amount of unsaturated fatty acids than the corresponding lipids from the two other tissues. The PE and PCH from fungal uredospores showed a higher degree of unsaturation than the lipids from bean leaves. The changes in fatty acid composition of PG, MGG and DGG were small and possibly not significant. The differences in fatty acid composition were localized mainly in the region of the pustule. The adjacent fungus free host tissue was affected only to a small extent. The infected tissue showed an increase in phospholipase activity as compared with the controls and the uninfected leaf halves. The results were discussed mainly in relation to permeability changes of rustinfected bean tissue.
INTRODUCTION
In an earlier publication [14] we reported changes in the concentration of certain phospholipids and glycolipids and in the incorporation of 32P into phospholipids in bean leaves infected with Uromyces phaseoli. These results were discussed with regard to previously observed changes in membrane permeability of rust infected bean tissue [13]. Investigations concerning lipid metabolism during pathogenesis were extended to the fatty acid composition of membrane lipids and to the phospholipase activity. The results of this investigations are reported here. MATERIALS
AND
METHODS
The materials used in this study, the preparation of leaf disks and rings, the extraction and isolation of phospholipids and glycolipids from bean leaves and uredospores 7 The following abbreviations will be used in this paper: days p.i. = days after inoculation; I infected leaf half; H = uninfected half of the same leaf; C = control leaf; PCH, PE, PG phosphatidyl-choline, -ethanolamine and -glycerol, respectively; PX = unidentified phospholipid; MGG = monogalactosyl diglyceride; DGG = digalactosyl diglyceride; 16:0 = palmitic acid; 16 :l trans-n3-hexadecenoic acid; 18:0 = stearic acid; 18: 1 = oleic acid; 18:2 = linoleic acid;l8: 3 linolenic acid.
= = = =
26
H. H. Hoppe
of the parasite by thin-layer previous papers [13, 141. Determination
of fat&
chromatography
acid content of phospholipidr
and
R. Heitefuss
have already been described in two
and glycolipids
To prevent oxidative damage of fatty acids during thin-layer chromatography butylated hydroxy toluene was added to the solvents (50 mg/lOO ml in the first dimension, 5 mg/ 100 ml in the second dimension). The developed plates were dried in air for 20 min and sprayed with 0.04% 2’,7’-dichlorofluorescein in methanol, previously purified following the method of Parker & Peterson [25]. Using this method only the higher concentrated lipids (PG, PE, PCH, MGG, DGG) were visible under U.V. light and were marked. The spots from four replicate plates were pooled for fatty acid analysis. Fatty acid methyl esters were prepared by direct transmethylation with Na-methylate in the presence of silica gel following the method described by Husek [15]. The methyl esters were dissolved in hexane and injected into a Tricarb-Packard gas chromatograph equipped with a flame ionization detector, which had the following set features: column: 10% diethyleneglycolsuccinate on chromosorb W, temperature 180 “C isothermal, injector and detector temperature: 200 “C, carrier gas: N,, flow rates: N, 30 ml/mm, H, 40 ml/mm, air 400 ml/min. For identification of the peaks fatty acid methyl ester standards were run under the same conditions. Quantitative analysis of fatty acids was based on peak area measurement, multiplying the height of the peak by the width of the peak at half height. Concentration was determined using calibration factors previously calculated by the measurement of peak area produced by known quantities of fatty acid methyl esters. The amount of each fatty acid component was expressed as a percentage of the total area. Phospholipase
assay procedure
extraction. Plant material (3 to 5 g) was ground in a mortar with the same amount of buffer (O-1 M-succinate-NaOH buffer, pH 5.0) and squeezed through four layers of cheesecloth. When the effect of pH on phosphatidase activity was determined the incubation buffer was used for extraction and the pH of the extract was readjusted. The sap was centrifLged at 10 000 g for 20 min and the supernatant fluid assayed for phosphatidase activity. Substrate. Soybean lecithin (Sigma, U.S.A.) was purified by chromatography on a silica acid column [2]. The MeOH fraction was taken to dryness and the residue suspended in buffer (0.05 M-succinate-NaOH, pH 5.0) by blending in an ice-bath using a Virtis homogenizer operated at full speed for 30 min. In the experiments with different pH during incubation the substrate was suspended in the incubation buffer using a succinate-NaOH buffer in the range of pH 3-O to 6-O and a Tris-HCl buffer in the range pH 7.0 to 9.0. Enzyme assay. Phosphatidase was assayed by measuring the release of trichloroacetic acid soluble phosphate [21, 2.21. Standard enzyme reaction mixtures contained 0.5 ml enzyme extract and 0.5 ml of a 1o/o substrate suspension. After Enzyme
Permeability
and
lipid
metabolism
in infected
bean
leaves
27
incubation for 2 h at 30 “C the reaction was stopped by the addition of 0.1 ml 5% serum albumin to aid the precipitation of phosphatides and 0.9 ml 20% trichloroacetic acid [Zl]. After centrifugation the supernatant was assayed for soluble phosphate using the method of Allen [I]. Blanks were analysed in which the serum albumin and trichloroacetic acid were added to the substrate before the addition of the enzyme preparation. Enzyme activity was normally expressed as pg P released per mg protein in 2 h. Protein was determined according to the method of Lowry et al. [ZO] after precipitation with trichloroacetic acid. RESULTS
Efect
of infection on the fatty acid composition of membrane lipids
The distribution of the major fatty acids in isolated lipids from infected and uninfected leaf halves and from control leaves of bean plants is shown in Table 1. 1
TABLE
Fatty
acid composition of bean lipids from rust-infected bean leaf halves (I), non-infected halves (H) and control leaves (C). Values are a percentage of total fatty acids Days
p.i. I
PCH
PG
PE
Days PCH
PG
PE
16:0 16:2 18:0 18:l 18:2 18:3 16:0 16:l 18:0 18:l 18:2 18:3 16:0 18:0 18:l 18:2 18:3
19.3 7.2 2.4 29.3 41.8 36.1 33.5 3-5 1.3 3.4 22.2 25.9 4.0 1.5 32.6 36.0
16:0 16:2 18:0 18:l 18:2 18:3 16:0 16:l 18:0 18:l 18:2 18:3 16:0 18:0 18:l 18:2 18:3
19.7 4.8 1.0 15.5 59.0 33.5 35.0 4.0 0.5 2.0 25.0 27*0 3.6 1.0 21.4 47.0
p.i.
4 H 21.7 7.4 2:.; 44.2 37.1 34.1 4.6 0.9 2.3 21.0 31.9 4.8 1.2 30.3 31.8 10 19.8 2-9 4.6 o-4 10.5 61.8 35.7 34.8 3.9 0.8 l-6 23.2 28.2 3.9 o-7 17.6 49.6
C
I
21.8 7.2 2-4 25.6 43.0 38.6 34.8 4.4 1.1 2.0 19-l 32.5 4.9 1.7 31.4 29.5
18.7 6.6 3.0 26.5 45.2 33.0 31.2 4.1 1.0 2.4 28.3 18.2 2.9 2.2 31.8 44.9
22.0 5.3 o-7 13.6 58.4 38.2 36.2 3.9 0.4 l-3 19.9 30.5 2.5
16.3 0.6 3.8 1.3 15.0 63.0 33.1 33.1 4.1 0.6 2.5 26.6 24.6 2.7 1.6 21.5 49.6
2Z.E 45.7
6 H 22.0 2.6 1.5 23.9 45.0 35.8 34.0 3.9 0.6 1.7 24.0 29.6 5.4 32 32.3 12 16.5 6.9 2.8 1.1 9.6 63.1 39.3 32.6 3.8 0.8 2.0 20.5 28.5 2-4 1.1 16.5 51.5
C 21.4 7.9 2:.: 44.2 37.9 34.5 4.4 0.7 1.9 20.6 30% 4.8 1.2 30.3 33.1 17.7 3.5 3.2 0.9 9.0 65.7 37.3 35.8 4.0 0.6 l-5 20.8 26.5 3.2 0.7 16.1 53.5
leaf
28
H. H. Hoppe
and
R. Heitefuss
The fatty acid composition is expressed as weight per cent of total fatty acids. The values are averages of two (4, 12 days p.i.) or three (6 days p.i.) separate experiments. The data of each experiment were obtained by duplicate determinations (two different samples used for transesterification). The deviation from the average for duplicate determinations was less than 5%, for the separate experiments it was about 10%. The variations do not apply to the low concentration (less than 10%) fatty acids in which percentage variation sometimes was greater as a consequence of the smaller amounts of these fatty acids. The principal fatty acids detected in all lipids of both diseased and healthy bean leaf tissue were palmitic (16:0), stearic (18:0), oleic (18:1), linoleic (18:2) and linolenic acid (18 : 3). In PG a fatty acid appeared (16 : 1) with a retention time similar to palmitoleic acid and was tentatively identified by us as trans3-hexadecanoic acid. This reached up to 35% of the total fatty acids in the PG of our material. It occurs in many plants, particularly chlorophyllous tissues [18, 231. At the later stages of our experimental period a fatty acid in PCH was detected whose retention time lay between 16 : 1 and 18: 0. This acid was not identified by us. According to its retention time it could be a 16:2. The results indicate that the appearance of this acid was related to the progress of senescence in the bean tissue. The fatty acid was first detected 10 days after inoculation in the PCH of the non-infected half leaves and was present 12 days after inoculation in all three treatments. It was highest in the non-infected leaf halves, which were already yellow, and lowest in the infected ones, which were still green. Other fatty acids, not shown in Table 1, were present only in trace amounts (< 1o/o of the fatty acids in each lipid). The fatty acid composition of plant lipids reported here is in good agreement with the results of other authors [16]. Comparing single lipids it becomes evident that PCH and PE have a similar fatty acid composition, PG is relatively rich in palmitic acid and poor in linoleic acid whereas MGG and DGG contained a high portion of linolenic acid, about 94 and 87%, respectively. The fatty acid composition of the two galactolipids was the same in both diseased and healthy tissue and did not change during the experimental period. The data of these lipids are therefore not presented in Table 1. The phospholipids (PE, PCH, PG) of the infected leaf halves contained a higher amount of unsaturated fatty acids between 4 and 8 days after inoculation than the uninfected leaf halves and the controls. This difference was more pronounced in the PE than in the two other phospholipids (compare also Fig. 1). No difference was observed between the lipids of the uninfected leaf halves and the controls. To make the difference in fatty acid composition clearer the ratio Z unsaturated fatty acids C saturated fatty acids was calculated for each of the three phospholipids and is presented in Fig. 1. Increased amounts of unsaturated fatty acids occurred in the diseased tissue at 4 days after inoculation when chlorotic spots appeared. In PE the content of unsaturated fatty acids reached a maximum 8 days after inoculation and decreased later ( 10 and 12 days after inoculation) under the low light conditions of our experiments. In PCH the increase in unsaturated fatty acids was lower than in PE;
Permeability
and lipid
metabolism
in infected
bean I
I
29
leaves
I
’
I
I’
l- C PE
lC
PG I 2
I 4
I 6
I 8
I IO
I 12
Days after inoculation
FIG.
1. Bean
leaves
infected
with
U. phaseoli:
C unsaturated 2 saturated in PE, PCH
and
PG.
I, infected
leaf halves;
The
fatty
acid
ratio
fatty acids fatty acids H, uninfected
leaf halves;
C, control
leaves.
however, it began earlier and was already observed 3 days after inoculation. In contrast to PE and PG an increase in unsaturated fatty acids occurred in PCH at the later stages of the infection (10 and 12 days after inoculation) in all three treatments. This increase was mainly due to the unidentified acid which was possibly 16: 2 and therefore added to the unsaturated acids. The variations in acid composition of PG were small and can possibly be attributed to experimental error. However, between 4 and 12 days after inoculation the mean values of the fatty acid ratio were always higher in the infected leaf halves than in the two other treatments.
H. H. Hoppe
30
and
R. Heitefuss
As mentioned in a previous paper [la] it is not possible to decide whether the changes in fatty acid composition are due to changes in plant lipid metabolism or to a direct contribution of the fungal lipids or to both of these causes. We tried again to clarify this problem by two experiments. In the first experiment an attempt was made to find out whether the non-infected tissue surrounding the pustule was influenced by the infection. Changes in this tissue region can clearly be attributed to host plant metabolism. In the second experiment the germinated and ungerminated uredospores of the parasite were analysed to get information on the fatty acid composition of the fungal lipids. The fatty acid composition of the spore lipids (PCH and PE) is shown in Table 2. In addition the results of both experiments are expressed in the fatty acid ratio mentioned earlier and summarized in Table 3. TABLE
Fatty
acid
Uredospores: Ungerminated Germinated
composition of PCH U. phaseoli.
2
PE from ungerminated and gemtinated Values are a percentage of total fat& acids
uredospores
and
16:0
18:0
PCH I8:l
18:2
18:3
16:0
18:0
PE 18:l
18:2
18:3
24.9 22.4
4.5 5.1
2.4 1.5
10.7 7.6
57.5 63.4
7.4 4.5
1.7 0.7
2.9 1.7
152 10.7
72.8 82.4
The PCH and PE of the uredospores contained the same major fatty acids as the corresponding lipids of the bean leaves. In PCH the per cent distribution of the acids was similar to bean leaves; however, the PE of the uredospores had a higher content of linolenic acid (18 : 3). During germination the linolenic acid increased in both lipids leading tb a higher degree of unsaturation in the PE and PCH of the germinated spores (Table 3). The experiments with leaf disks confirmed the previously demonstrated differences in the fatty acid composition of membrane lipids between the infected and healthy TABLE
3
‘T7zefatty acid ratio (Z: unsaturated fatty acids)/@ saturated fatty acids) of several membrane lipids in leaf disks and leaf rings from bean leaves and in the uredosfiores of U. phaseoli Sample
Id
Cd
Ir
Cr
SPg*
Spung.
2.1 8.3 57.8
1.7 8.2 51.6
l-9 8.2 54.6
1.8 8.3 51.7
-
-
PE PG
3.3 1.5
1.5 I.4
1.9 1.5
1.5 1.4
18.2 -
10.0
PCH
2.4
2-O
2.1
1.9
2.6
2-1
PX DGG MGG
Leaf disks from infected (Id) and control leaves (Cd) respectively. Disks from infected leaves contained a pustule in the centre. Leaf rings from infected (Ir) and control leaves (Cr) respectively. Rings from infected leaves were prepared by removal of the central pustule from disks (for further details see Materials and Methods in reference [14J). Spg, germinated uredospores; Spung, ungerminated uredospores.
Permeability
and
lipid
metabolism
in infected
bean
leaves
31
tissue. The PCH and PE from the disks with a pustule in the centre (Id) had a higher degree of unsaturation than the compounds from corresponding disks of the controls (Cd). After removing the pustules only small differences between rings from infected leaves (Ir) and from controls (Cr) were observed (Table 3). The results indicated that the shift in fatty acid composition occurred mainly in the region of the pustule, whereas the surrounding fungus free tissue was affected only to a small extent by the parasite. E$ect of infection on the @hospholipase activity of bean leaves
Five types of phospholipases, e.g. phospholipases A, B, C, D and lysophospholipase have been described [12], each of which is specific for one of the ester linkages in the phospholipid molecule. Using the acid-soluble phosphorus procedure it is not possible to detect phospholipase D (phosphatidyl choline phosphatidohydrolase, EC 3.1.4.4), which catalyses the removal of choline, ethanolamine, etc. from the phospholipids with the consequent formation of PA, and phospholipase A (phosphatide acyl hydrolase, EC 3.1.1.4) which specifically removes one of the fatty acids. With this method only the activities of the following enzymes can be detected : phospholipase B, which removes both fatty acids, phospholipase C, which removes the polar head group and the combined activity of phospholipase A+lysophospholipase (lysolecithin acyl hydrolase, EC 3.1.1.5). A mixture of these different phospholipases could also be responsible for the release of acid-soluble phosphate in our reaction mixtures. Properties of phospholipase reaction. Maximum phospholipase activity of all extracts was between pH 4.0 and 5.0. The activity dropped off sharply at pH 3.5 and decreased between pH 6.0 and 7.0. Very low activities were detected above pH 7.0. All subsequent tests were performed at pH 5.0. CaCl, had no effect on phospholipase activity at pH 5-O when concentrations of CaCl, between 0 and 6 mM were present in the reaction mixtures. The optimum pH for activity is consistent with previous reports [22, 291. The lack of a response to CaCl, can be explained by the crude enzyme extract containing possibly enough Ca2+ for optimal enzyme activity. Phospholi.ase activity in diseased and healthy tissue. The enzyme activities in the controls and the uninfected half leaves were relatively low and remained rather constant over the experimental period. The activities were measured at 30 “C, which may not give a good estimation of the fungal enzymes since Knoche & Horner [19] found a marked maximum at around 18 “C for a lipase isolated from wheat rust uredospores. However, at 20 “C we found in our experiments almost no phospholipase activity in the control leaves and we therefore incubated the enzyme assays at 30 “C. The infected leaf halves showed an increase in enzyme activity beginning between 4 (experiment I) and 6 (experiment II) days after inoculation. Maximum activity was reached 6 to 8 days p.i. The differences between the two experiments are possibly due to variations in infection density. In experiment I the infection density was higher and the parasite developed faster than in experiment II. In the first experiment chlorotic spots appeared 4 days after inoculation whereas they were detected in the second experiment 1 day later (Fig. 2).
32
H. H. Hoppe
and
R. Heitefuss
x Experiment1
_
Days after inoculation
FIG. 2. I, infected
Phospholipase activities leaf halves; H, uninfected
in extracts leaf halves;
of bean leaves infected C, control leaves.
with
U. phmeoli:
DISCUSSION
The barrier properties of biological membranes depend to a considerable extent on the nature of the hydrocarbon chains of the membrane lipids. Force-area characteristics of monomolecular films of phospholipids with different fatty acid constituents show that the mean molecular area occupied per phospholipid molecule increases when the hydrocarbon tails become shorter or when unsaturated fatty acids are introduced into the phospholipid molecule. Extrapolation from such systems to biological membranes is difficult although they give some information on lipid-lipid interaction [3]. A more relevant approach is possibly the study of the permeability properties of liposomes, which are prepared by spontaneous swelling of membrane lipids in salt solutions and which are composed of multibilayers behaving as osmometers ES]. Liposomes prepared in salt solutions were exposed to an isotonic solution of glycerol or erythritol. The permeability of lecithin liposomes towards these two non-electrolytes increased markedly with an increasing degree of unsaturation or decreasing chain length of the lipid acyl groups. These results and studies performed with natural systems preferably with
Permeability
and lipid metabolism
in infected bean leaves
33
Mycooplasmalaidlawii B [7,9, 261 and Eschrichia coli [lo] indicate that permeability of membranes towards water-soluble non-electrolytes may depend at least partly on the unsaturation and on the length of fatty acids in the membrane lipids. From this viewpoint it seems possible that in our experiments the higher degree of unsaturation in the phospholipids, especially in the PE, of the infected tissue is responsible for the higher leakage of non-electrolytes like sugars [13] from this tissue. This explanation depends on the supposition that the observed changes in fatty acid constituents are at least partly due to changes in host lipids and are not restricted to a direct contribution by the fungus. The high degree of unsaturation in the PE from the uredospores indicates that the differences can possibly be attributed to the fungal PE although the results of the experiments with leaf disks and leaf rings show that the fungus-free tissue surrounding the pustule is also affected to a small extent by the infection. From these results it is concluded that both the host and the parasite are involved in the changes of the lipid acyl groups and that these changes might be important for the alterations of membrane permeability observed in the infected tissue. The results described in the second part of this paper show a higher phospholipid hydrolase activity in the infected half leaves than in the uninfected ones and the controls. Production of phospholipases by phytopathogens or higher phospholipase activities in infected plant tissues were reported by several authors [17, 21, 28, 291 and discussed in relation to membrane permeability. In a previous paper we mentioned that the changes in the amount of phospholipid observed in our experiments may reflect an enhanced degradation of membrane lipids in the infected leaf halves and we suggested that the enhanced degradation could be responsible for the higher leakage from this tissue [13]. However, the onset of the changes does not support this theory. The leakage showed a sharp increase 4 days after inoculation whereas the decrease in lipid concentration (MGG, DGG, PG) and the increase in phospholipase activity was hardly detectable 4 days after inoculation and reached a maximum 6 to 8 days after inoculation. Because of the late beginning of the changes in lipid amount and phospholipase activity it seems to be improbable that these changes induce the leakage stimulation. Increased phospholipase activity results in an increased release of free fatty acids. The addition of free fatty acids to isolated mitochondria causes a swelling of the organelles and uncouples oxidative phosphorylation [31]. Wojtczak & Lehninger [30] showed that the spontaneous swelling of rat liver mitochondria can be ascribed to the enzymatic release of free fatty acids from the membrane lipids. Similar results have been reported for mitochondria from Phaseolw vulgaris [4-61. From rust-infected wheat plants it is known that a partial uncoupling occurs in the later stages of development of infections on a susceptible variety [27]. Olah et al. [24] observed, however, in contrast to other authors [II], swollen plant mitochondria in rust-infected cells. The similarity between free fatty acid and fungus-induced swelling of mitochondria supports the idea that the increased phospholipase activity causes a partial damage to mitochondria in the infected tissue. However, the maximum phospholipase activity in our experiments was reached between 6 and 8 days p.i. whereas the damage to mitochondria and uncoupling of respiration occurs at later stages of infection. 3
34
H. H. Hoppe and R. Heitefuss
The assistance of Dr Manocha and Dr Williams with the translation scripts of this series is gratefully acknowledged.
of the manu-
REFERENCES 1. ALLEN, R. T. L. (1940). The estimation of phosphorus. Biochemical Journal 34, 858-865. 2. BEISS, U. (1963). Phosphatide und Glykolipide. In Moderne Methoden akr Pjancenanabse VI, Ed. by H. F. Linskens & M. V. Tracey, pp. 52-80. Springer-Verlag, Berlin, Gijttingen, Heidelberg. 3. DEENEN, L. L. M. VAN (1969). Membrane lipids and lipophilic proteins. In The Molecular Basis of Membrane Function, Ed. by D. C. Testeson, pp. 47-78. Prentice-Hall, Inc., Englewood Cliffs, New Jersey. 4. EARNSHAW, M. J. & TRUELOVE, B. (1968). Swelling and contraction of Phaseolus hypocotyl mitochondria. Plant Physiology 43, 121-129. 5. EARNSHAW, M. J. & TRUELOVE, B. (1970). Swelling of Phaseolus mitochondria in relation to free fatty acid level. Plant Physiology 45, 318-321. 6. EARNSHAW, M. J. & TRUELOVE, B. (1970). Swelling of Phaseolus mitochondria induced by the action of phospholipase A. Plant Physiology 45, 322-326. 7. MCELHANEY, R. N., DE GIER, J. & VAN DEENEN, L. L. M. (1970). The effect of alterations in fatty acid composition and cholesterol content on the permeability of Mycoplasma laidlawii B cells and derived liposomes. Biochimica et Biophysics 219, 245-247. 8. DE GIER, J., MANDERSLOOT, J. G. & VAN DEENEN, L. L. M. (1968). Lipid composition and permeability of liposomes. Biochimica et Biophysics Acta 150, 666-675. 9. HAEST, C. W. M., DE GIER, J. & VAN DEENEN, L. L. M. (1969). Changes in the chemical and the barrier properties of the membrane lipids of E. coli by variation of the temperature of growth. Chemistry and Physics of Lipids 3,413-417. 10. HAEST, C. W. M., DE GIER, J., VAN Es, G. A., VERKLEIJ, A. J. & VAN DEENEN, L. L. M. (1972). Fragility of the permeability barrier of Escherichia coli. Biochimica et Biophysics Acta 288, 43-53. 11. %RDU?CK, N. V., GREENWOOD, A. D. & WOOD, R. K. S (1971). The line structure of the haustorium of Uromyces appendiculatus in Phoseolus vulgaris. CanadianJournal of Botany 49,383-390. 12. HITCHCOCK, C. & NICHOLS, B. W. (1971). Plant Lipid Biochemistry. Academic Press, London, New York. 13. HOPPE, H. H. & HEITEFUSS, R. (1974). Permeability and membrane lipid metabolism of Phaseolus vulgaris infected with Uromyces phaseoli. I. Changes in the efflux of cell constituents. Physiologkal Plant Pathology 4, 5-9. 14. HOPPE, H. H. & HEITEIWS, R. (1974). Permeability and membrane lipid metabolism of Phaseolus vulgaris infected with Uromyces phaseoli. II. Changes in lipid concentration and 3sP incorporation into phospholipids. Physiological Plant Pathology 4, 1 l-23. 15. HUSEK, P. (1969). Methode zur direkten Methylierung diinnschichtchromatographisch aulgetrennter Lipide ohne deren Elution aus Kieselgel.
Permeability
26. ROMIJN,
27. 28.
29. 30. 31.
and
lipid
metabolism
in infected
bean
leaves
35
J. C., VAN GOLDE, L. M. G., MCELHANEY, R. N. & VAN DEENEN, L. L. M. (1972). Some studies on the fatty acid composition of total lipids and phosphatidyl glycerol from Acholeplanna laidlawii B and their relation to the permeability of intact cells of this organism. Biochimica et Biophyka Acta 280, 22-32. SHAW, M. (1963). The physiology and host-parasite relations of the rusts. Annual Review oj Phytopatholou 1, 259-294. TSUNG-CHE TSENG & BATEMAN, D. F. (1968). Production of phosphatidases by phytopathogens. Phytopathology 58, 1437-1438. TSUNG-CHE TSENG & BATEMAN, D. F. (1969). A phosphatidase produced by Sclerotium rolfsii. PhytopatholoQ 59, 359-363. WOJTCZAK, L. & LEHNINGER, A. L. (1961). Formation and disappearance of an endogenous uncoupling factor during swelling and contraction of mitochondria. Biochimica et Biophy.rica Acta 51, 442-456. WOJTCZAK, L. & ZALLJSICA, H. (1969). Inhibition by oleate of the binding and exchange of ATP by mitochondrial membranes. Biochimica et Biophysics Acta 189, 455-456.