LIPID METABOLISM IN LEAVES OF TUSSILAGO FARFARA DURING INFECTION BY PUCCINIA POARUM

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1 New PhytoL (197) 7, LPD METABOLSM N LEAVES OF TUSSLAGO FARFARA DURNG NFECTON BY PUCCNA POARUM BY D. M. LOSEL AND D. H. LEWS {Received 1 June 197) SUMMARY Following infection by Puccinia poarum, there is an accumulation of lipid in leaf tissue of Tussilago farfara. Most of this increase is in the fungus, which accounts for nearly half of the dry weight of mature aecial pustules. While the lipid content of infected tissue rises to more than three times that of healthy leaves, that of the uninfected parts of diseased leaves is reduced by one-third. Aecial pustules accumulate a high proportion of neutral lipid, particularly triglycerides and fatty acids. As infection progresses, a loss of chloroplast glycolipids and phosphatidyl glycerol is accompanied hy increases in phosphatidyl ethanolamine and phosphatidyl choline and the formation of phosphatidyl serine, which is not readily detectable in healthy leaf tissue. The lipid changes following rust infection have been compared with those of senescing Tussilago leaves. Both neutral lipids and phospholipids incorporated more photosynthetically assimilated '*C in rust-infected tissue than in healthy leaves. Greatest differences were in free fatty acids, triglycerides, phosphatidyl ethanolamine and phosphatidyl choline with significant increases also in sterols, phosphatidyl inositol and phosphatidyl serine. Whereas three-quarters of the activity incorporated from ''*CO2 into lipid appears in the neutral lipids of diseased tissue, in healthy tissue these contain only one-quarter. NTRODUCTON Progress towards an understanding of the physiology of biotrophic associations has been much greater in the fields of carbohydrate and amino-acid metabolism (Shaw, 196; Smith, Muscatine and Lewis, 1969; Scott, 1972) than in that of lipid metabolism, where relatively few studies have so far been made. The comprehensive investigation by von Sydow (1966b) of the distribution of label in healthy and rust-infected tissues of susceptible and resistant varieties of wheat after photosynthesis in ^'^COj makes little mention of lipid components, although analysing in some detail the activities of individual sugars and amino and organic acids. Much of the host assimilate in rust-infected leaves must be converted to the conspicuous oil droplets long recognized in aeciospores, urediospores and teliospores where lipid levels of 6-2% of dry weight have been reported (Tulloch and Ledingham, i96). n their extensive survey of the fatty acid composition of oils from rust spores, Tulloch and Ledingham (i96, 1962, 196) and Tulloch (196) found that, independent of host, there were generally similarities between different spore forms of individual species of Puccinia. There were, however, exceptions where oil composition differed in rusts of the same genus on host plants belonging to different families. Thus, the extent to which host and rust lipid metabolism are related is not clear. Studies on spores, while relevant to the physiology of germination and the initiation of infection, provide information mainly on the end product of the association rather on the "57

2 1158 D. M. LosEL AND D. H. LEWS active phases of the host-parasite interaction. Among the few observations on the lipid metabolism of host and parasite as a physiological unit are the finding of increased lipid levels in cabbage hypocotyls infected with Plasmodiophora brassicae (Williams et al., 1968), a report of changes in total fatty acid composition of Phaseolus vulgaris at different stages of infection with Uromyces phaseoli (Schipper and Mirocha, 197) and the investigation of the fate of absorbed propionate in rust-infected tissue by Reisener and Jager (1969). Earlier, Schmidt (192) provided microscopic evidence of lipid accumulation in cells of sugar-beet leaves adjacent to infection hyphae. Detailed information on the membrane lipid classes of rust-infected tissue and their fatty acid composition has recently been provided by Hoppe and Heitefuss (197b, c) in an attempt to explain the increased permeability demonstrated by them in Phaseolus leaves following infection with Uromyces phaseoli (Hoppe and Heitefuss, 197a). The present paper describes changes in lipid content and metabolism in tissues of Tussilago following infection with Puccinia poarum. Aspects of the carbohydrate metabolism of this association have already been reported (Holligan, Chen and Lewis, 197; HoUigan e< a/., 197a; Holligan, McGee and Lewis, 197b). MATERALS AND METHODS Plant material Leaves were obtained from healthy and rust-infected plants of Tussilago, maintained in growth-room conditions as described by McGee et al. (197). The nomenclature used to describe the leaf tissues sampled corresponds to that of Holligan et al. (197) and is as follows: H, healthy tissue from uninfected leaves; HD, healthy tissue from diseased leaves; Di, pycnial pustules with no aecia visible; D2, aecial pustules with aecia visible but still closed; D, aecial pustules with less than 5 % of aecia dehisced; D, aecial pustules with more than 5% of aecia dehisced. Staining of lipid in leaf tissue Hand-cut sections and partially macerated tissue of healthy and rust-infected leaves of Tussilago were cleared in chloral hydrate and stained with Sudan V by a method similar to that of Schmidt (192). Lipid extraction Batches of discs, 5-1 mm diam. according to size of pustules, were cut with a corkborer. Fresh weight and area were quickly recorded and the discs transferred to isopropanol, in which they were left overnight at 15 C before being ground in 2 volumes of this solvent and extracted. Kates (1957) recommends initial extraction with isopropanol to inactivate lipolytic enzymes present in leaf tissue. After further extraction with chloroform : methanol (2:1, followed by 1:2), the combined extracts were reduced to near dryness with a rotary evaporator, taken up in a small volume of Folch lower phase solvent (Folch, Lees and Sloane-Stanley, 1957) and washed twice with upper phase solvent. The lipid extracts in the lower phase were evaporated to a small volume, dried with sodium sulphate and centrifuged. The supernatant was transferred to weighed vials, dried to constant weight (first with a stream of nitrogen, then under vacuum in a desiccator) and made to a convenient volume corresponding to a known area of leaf or to a concentration of the order of 1 mg/ml.

3 Lipid metabolism in rust-infected leaves To avoid oxidation,.5% butylated hydroxytoluene (BHT) was added routinely to solvents in which lipids were stored. Separation of lipids on silicic acid columns Aliquots (5 mg lipid) of extracts were loaded on i g columns of silicic acid. Neutral lipids were eluted with 5 ml chloroform and polar lipids with 1 ml chloroform: methanol (1:1 v/v), followed by 5 ml methanol. The eluted fractions were then reduced to a standard volume and further separated on thin-layer plates. Thin-layer chromatography Eor one-dimensional separations, extracts containing 5-2 /<g lipid were spotted on 2 cm X 2 cm plates of 2^o-{.im thick silica gel on aluminium foil (Merck), Neutral lipids were separated in the double solvent system of Skipski et al. (1965): (i) wo-propyl ether:acetic acid (2:1 v/v), run to 1 cm from the base-line; (2) petroleum ether:diethyl etherracetic acid (9:1: i) run to i cm from the top of the plate. Polar lipids were generally separated in the solvent system of Nichols, Harris and James (1965), i.e, chloroform:methanol:acetic acid:water (17::2:7). Eor two-way separations of polar lipids, volumes of extracts containing.5- "^g ^ip'^ were applied along an origin i cm long. The successive solvents used (Nichols, 196) were: (i) Chloroform:methanol:7N ammonium hydroxide (65::); (2) chloroform: methanol: acetic acid: water (17:25:25:6), dentification of lipids on thin-layer plates Lipids were visualized in iodine vapour or by spraying with 5% sulphuric acid and heating at 9O C, dentification was (a) by comparison with available standard lipids and with published diagrams for these solvent systems and (b) by reactions of specific classes of lipids to various reagents. Sterols and sterol esters gave a reddish purple colour reaction after spraying with sulphuric acid and heating, other lipids giving shades of brown and appearing in characteristic order. Phospholipids were recognized by their blue reaction with Molybdenum Blue reagent, phosphatidyl ethanolamine and phosphatidyl serine by their pink colour with ninhydrin (.2% in moist butanol, heated in steam at 1 ) and chloroplast glycolipids by their reddish-violet reaction with.2% orcinol in 75% sulphuric acid. Quantitative estimation of lipids Total lipids were determined by the sulphuric acid charring method of Marsh and Weinstein (1966), Neutral lipid spots separated by thin-layer chromatography were estimated in the presence of silica gel by the method of Amenta (196) and phospholipids by the procedure of Rouser, Siakotos and Eleischer (1966). n order to allow for the leaf pigments present in extracts used for total lipid determinations, chlorophylls were estimated by the method of Holden (1965). Chitosan estimations The method of Ride and Drysdale (1972) was used to give an estimate of the amount of fungus present in infected host tissue, using aeciospores as pure fungal material for comparison. Since, however, it is likely that the wall composition of these differs greatly from that of the vegetative, intercellular hyphae, this can only be an approximate estimation. n repeated assays of Tussilago tissue infected with Puccinia

4 ii6o D, M. LosEL AND D, H. LEWS poarum, the results obtained by this technique corresponded well to the stage of infection studied and the amount of fungus observed microscopically. ncorporation of For photosynthetic assimilation of '"^COj, detached leaves with their cut ends immersed in a beaker of water were enclosed in a clear Perspex box (22 cm x 12 cm x8 cm), sealed with 'Vaseline'. ''^COz was released by injecting 1% lactic acid through a needle-hole in the lid into an appropriate volume (usually equivalent to 1 ;ici) of NaH'^COj, contained in a small dish within the chamber. After exposure to light for 15 min, 1% KOH was injected through another hole in the lid into a second dish, in order to absorb any residual '*CO2. After a further 5 min, the leaves were removed from the chamber and left in darkness for 18-2 ^ ^ allow further metabolism of the labelled assimilate before lipid extraction. Autoradiographs were prepared by exposing thin-layer plates covered with 'Melinex' to X-ray film for periods of between 2 and weeks. Counts of activity in aliquots of extracts pipetted on to planchets were made with a Nuclear-Chicago gas-flow counter. The distribution of activity in thin-layer chromatograms was estimated by cutting the aluminium foil thin-layer plates into strips and counting these on a Nuclear-Chicago Actigraph, fitted with a thin-layer plate carrier. The activity of polar lipids separated by two-way thin-layer chromatography was determined by eluting the lipid from silica gel, scraped from spots previously revealed by exposure to iodine vapour, evaporating the solvent from these extracts in scintillation vials and counting on a Nuclear-Chicago Unilux liquid scintillation counter in 5 ml Triton-toluene solution. RESULTS Light microscope observations Following infection of Tussilago leaves with Puccinia poarum, the distribution of lipid was as follows. n young pustules with pycnia but no aecia (stage Di), abundant oil drops were present in cells of the upper epidermis and mesophyll. A similar situation but with fewer oil drops recurred in the lower epidermis (Plate i, Nos. 1 and 2). n the fungus, lipid was prominent in the pycnial tissue although sparse in the intercellular hyphae. Young aecial pustules in which only the outermost ring of aecia had opened (D2) had much less oil in host tissue. Lipid was abundant in all cells of the intercellular hyphae and the aecial peridium (Plate i, Nos. -5 and Plate 2, No. 5). n mature aecial pustules (D), the oil had decreased further in epidermal cells and had disappeared from the mesophyll tissue but lipid was still present throughout the fungus as well as in the aeciospores. The oldest stage studied with all aecia sporulating freely (D) was characterized by a lack of oil droplets in intercellular hyphae. Visible accumulation of lipid was confined to the aecium and the immediately adjacent hyphae. At all these phases of development, the leaf cells outside the pustule appeared to be free of lipid drops, as were those of entirely healthy leaves. Analysis of lipid extracts n order to assess the qualitative and quantitative changes in lipid during disease development, lipid extracts were prepared from healthy and diseased leaves of Tussilago of comparable age. Discs from healthy leaves were compared with those from young pustules not yet showing aecia; from mature aecial pustules and from uninfected parts of

5 Lipid metabolism in rust-infected leaves 1161 diseased leaves. n each case, five replicate batches of each type of tissue were sampled and extracted. Table i shows lipid content, fresh and dry weights of leaf tissue, together with an estimate of the amount of fungus present, based on the chitosan assay of Ride and Drysdale (1972). Total lipid increased as infection progressed, reaching, in mature aecial pustules, more than three times the lipid content of entirely healthy leaves and more than five times that of the healthy parts of diseased leaves. At the same time, the estimated dry weight of fungal structures present rose to approximately 11 % of the dry weight of young aecial pustules and 5% of the dry weight of mature pustule tissue. Table i. Composition of healthy and rust-infected leaves of Tussilago Fresh weight (mg/cm^) Dry weight (mg/cm^) Estimated mg fungus/cm^ Lipid (/ig/cm^) Lipid as % dry weight Healtliy leaves H* Leaves with young aecial pustules HD2* D2* Leaves with mature aecial pustules HD* D* * For notation in this and subsequent Tables, Figs, and Plates, see Methods. Numbers following HD indicate healthy tissues from leaves bearing pustules of corresponding stage. Table 2. Neutral and polar lipids of healthy and rust-infected leaves of Tussilago Sterol esters Triglyceride Free fatty acids Sterols Total polar lipids //g lipid H a 18 per cm* D Thin-layer chromatography of lipids from fifteen series of extracts from healthy and rust-infected tissues revealed a consistent pattern of changes in lipid composition during the development of the fungus in host tissue. All classes of neutral lipids increased in rust pustules, particularly free fatty acids, triglycerides and sterols (Fig. i. Table 2). All these neutral lipids occurred also in aeciospore extracts (Fig. i). As infection progressed, there was a loss of the chloroplast components MGDG,* DGDG, SL and PG, while PC increased and PS, which was present only in trace amounts in healthy tissue, became readily detectable. Since PS tended to run with SL in the single solvent system most frequently used, its presence in low concentration was confirmed by two-dimensional separation of extracts containing 2 to mg of lipid (Fig. 2). t was identified by comparison with the chromatographic behaviour of an authentic standard and its colour reactions with ninhydrin and Molybdenum Blue. Phosphate estimations on silica gel scraped from appropriate areas of chromatograms allowed the relative amounts of individual phospholipids in healthy and infected tissue to be compared (Table ). Abbreviations used: MGDG = monogalacto.syl diglyceride; DGDG = digalactosyl diglyceridc- SL = sulphoquinovosyl diglyceride; PG = phosphatidyl glycerol; DPG = diphosphatidyl glycerol' PE = phcsphatidyl cthanolamine; PC = phosphatidyl choline; P = phosphatidyl inositol; PS = phosphatidyl serine; PA = phosphatidic acid.

6 l62 D. M. LosEL AND D. H. LEWS ^ o ^ Srerol esters O Triglycerides Free fatty adds ^ > C 5 diglycerides 1:2 diglycerides Sterols? o H DA 1 1 Standards Monoglycerides Polar lipids Fig.. Thin-layer chromatogram of neutral lipids from healthy (H) and rust-infected (D) tissue from leaves of Tussilago and from aeciospores (A), using the double-solvent system of Skipski et al. (1965) (see 'Methods'). (a) Healthy leaf (b) Aecial pustule Fig. 2. Two dimensional thin-layer chromatograms of polar lipids from (a) healthy and (b) rust-infected tissue from leaves of Tussilago. Sequential development in directions and with solvent systems of Nichols (196) (see 'Methods').

7 -''«- - - * Lipid metabolism in rust-infected leaves 116 The possibility that changes in lipid content of rust-infected leaves might be similar to those of senescence had to be considered. Diseased Tussilago leaves frequently showed symptoms of senescence, such as yellowing or increased amounts of red, anthocyanin pigment in the uninfected areas. Lipid extracts from leaves with mature aecial pustules were therefore compared with those of healthy leaves of comparable age and from uninfected, senescent leaves which had, in one case, developed anthocyanin coloration and, in another, become mainly yellow in colour. For comparison of the effects of senescence and infection, extracts of the following were analysed, (a) Young (D2) aecial pustules; (b) discs cut from uninfeeted tissue, 1 cm from margins of D2 pustules; (c) mature (D) aecial pustules from other leaves; (d) tissue adjacent to D pustules, forming a 7-mm wide ring around the infected region. Table, Phospholipid composition of healthy rust-infected leaves of Tussilago and m P per cm* % of total phospholipid H D2 H D2 PE PC PG PS o O. o. P O. C PA Table, Comparison of lipid composition of uninfected and senescent leaf tissue of Tussilago with that of rust pustules {relative lipid levels {scale -) assessed from TLC plates) Neutral lipids Sterol esters Triglycerides free fatty acids diglyceride8 sterols Polar lipids MGDG DGDG PA PE PC Healthy leaves H -. Leaves with aecial pustules \ jnopened HD2 D2 a 2 a X a Leaves with mature aecial pustules HD D 2 a 2 a t Senescent leaves Yellow Ke a 2 2 Although amounts of fatty acid and triglyceride in senescent tissue increased compared with healthy leaves, this was less than the increase in these lipid classes in young and, especially, in mature aecial pustules (Table ), Senescent leaves had a higher level of sterol esters than had healthy leaves but less than in pustules. Little increase in diglyceride was observed in senescence, while diglycerides were more prominent in infected leaves, particularly in uninfeeted tissue adjacent to pustules. Separation of the neutral lipids of these extracts in a different solvent system (Freeman and West, 1966) confirmed these results. While senescent Tussilago leaves resembled rust-infected tissue in their loss of chloroplast components, they did not show the increased PE and PC characteristic of

8 116 D, M, LosEL AND D, H, LEWS rust pustules (Table ). The lipids of yellow senescent leaves and of those with anthocyanin pigment showed no clear differences. rkorporation of label in lipids after photosynthesis in The transfer of host assimilate to the lipid fractions of healthy and diseased leaves was investigated in a series of experiments as detailed in 'Methods' above. AU neutral lipids, particularly triglycerides, fatty acids, diglycerides and sterols, became strongly labelled in diseased tissue, whereas healthy tissue showed relatively little ^"^C in neutral lipids (Plate 2, No. 6). When polar lipids were separated on silica gel plates, much less label was found in MGDG, DGDG, SL and PG in diseased tissue than in healthy Table 5. ncorporation of ^^COj by lipids of healthy and rust-infected leaves of Tussilago Activity of total lipid (cpm/cm^) Polar lipids (%) Neutral lipids (%) H HD D Table 6. Distribution of activity among lipid classes after photosynthesis of ^*CO2 and a translocation period of 2 h {see 'Methods') (A) Neutral lipids Carotenoids Sterol esters Fatty acid methyl esters Triglycerides Free fatty acids Diglycerides Sterols Monoglycerides Total lipid (B) Polar lipids PG PE PC P PS PA DPG Total DGDG Total polar lipid % of total lipid label dpm/cm^ H cpm/cm^* Yo of total phospholipid S. 1.9 D Ya of total lipid label.5 J ' 5 dpm/cm^ " *9 cpm/cm^* D % of total phospholipid * Estimated from Actigraph scans (see Methods). (MGMG is not included since it failed to separate clearly from neutral lipids). tissue (Plate 2, No. 7). At the same time, pustule extracts showed greatly increased '^C incorporation in PE and PC, a smaller increase in activity of P and marked labelling of PS which was barely detectable in uninfected tissue. Table 5 shows the relative activities of total lipid extracts from healthy and infected

9 Lipid metabolism in rust-infected leaves 1165 leaves together with the percentage distribution of labelling between the neutral and polar lipid fractions. Compared with healthy leaves, pustules showed a six-fold increase in incorporation of ^*C into lipids, while lipids of uninfected tissue away from pustules incorporated less than half. Whereas three-quarters of *'*C in lipids of healthy leaf tissue appeared in the membrane lipids and chloroplast glycolipids with only one-quarter in the neutral lipid fraction, these proportions were reversed in rust-infected leaf tissue. n uninfected parts of infected leaves, polar and neutral lipids were approximately equally labelled. The distribution of ''*C among individual lipid classes evaluated by scanning of chromatograph strips for neutral lipids (Table 6A) and by scintillation counting for phospholipids and glycolipids eluted from silica gel spots on two-way chromatograms (Table 6B) confirmed that the most striking consequence of infection was the high level of incorporation into neutral lipids. Also, there was a marked reduction of activity in chloroplast lipids, PG and DGDG, in infected tissues accompanied by greatly increased activity in PC, PS, PA and DPG. Although autoradiography repeatedly recorded increases of '*C in P and PE in infected over healthy tissue (Plate 2, No. 7), owing to problems of recovery, this was not always evident following elution from TLC plates and radioactive assay (Table 6). DSCUSSON Direct microscopical observation demonstrates that infection by Puccinia poarum induces an initial formation of lipid droplets in Tussilago leaf cells, comparable to that observed by Schmidt (192) in sugar beet tissues infected by Uromyces betae. This is followed by loss of lipid from host cells and extensive accumulation of oil droplets in intercellular hyphae and all fungal structures. The abundant lipid detected in peridial cells suggests that these have more than a protective function for the development of aeciospores and may be of nutritive importance. The biochemical studies reported confirm this accumulation of lipid in rust-infected tissues and provide preliminary indications of the metabolic changes occurring. The high levels of neutral lipid characteristic of maturing aecial pustules must correspond to the dense accumulation of oil droplets seen throughout the fungus at this stage but, in earlier phases of infection while pycnia are developing, much of the neutral lipid extracted may be contributed by host cells. Orcival's (1968) examination of ultrastructure of chloroplasts of Tussilago during development of aecial pustules of Puccinia poarum revealed altered membrane organization accompanied by the occurrence of lipid globules between membranes and dense, thickened regions of the plastid envelope which suggested degradation of the membrane complex with release of lipids. t has not, so far, been possible to distinguish host and parasite changes in polar lipid, apart from the obvious loss of chloroplast lipids. t is likely that changes in the host cells of pustules resemble those occurring in senescent uninfected leaves. Triglycerides and free fatty acids of both tissues increase, although the levels present during senescence were lower than in diseased tissues and there appeared to be no accumulation of sterols in the senescent leaves. Orcival (1968) commented on the resemblance of chloroplast changes in aecial pustules on Tussilago to those observed by Barton (1966) in mesophyll cells of senescent Phaseolus leaves. The high sterol levels of mature pustules may be largely in the aeciospores since, in other Basidiomycetes, e.g. Agaricus bisporus (O'Sullivan and Losel, 1971; Holtz and Schisler, 1971), sterols are low or absent in mycelium

10 ii66 D. M. LosEL AND D. H. LEWS and more abundant in spores. Sterols were present in the aeciospore extracts examined in the present study. The greater prominence of diglycerides in and around rust-infected tissue would appear to indicate more active lipid metabolism in these than in senescent tissue, as also would the higher levels of phosphatidic acid and free fatty acids. Ferguson and Simon (197) found losses of membrane phospholipids from senescing cucumber cotyledons to be accompanied by leakage of electrolytes. That such changes, characteristic of senescence, may also be occurring in host cells of rust-infected Tussilago leaves is indicated by the increased permeability detected by Hoppe and Heitefuss (197a) in Phaseolus leaves infected with Uromyces phaseoli. Hoppe and Heitefuss (197b, c) investigated the membrane lipids of rust-infected hean leaves with a view to interpreting these changes in permeability in infected tissue. n their elegant comparison of the polar lipids of infected and uninfected halves of rusted hean leaves, they found no significant change in the levels of PC, PE and P in rustinfected tissue compared with controls, while these, especially P, decreased in uninfected halves of rusted leaves. MGDG, DGDG and PG decreased while PS and PA increased in infected tissue. ncorporation of ^^P by all phospholipids, particularly PS, increased in infected tissue, the highest specific activity being in PA, followed by PL As in the Uromyces-'ini&ctcA bean leaves studied by Hoppe and Heitefuss, the phosphatidyl serine detected in aecial pustules of Tussilago is likely to be a fungal component, although traces occur in healthy Tussilago leaves. PS tends to he absent or barely detectable in leaves (Hitchcock and Nichols, 1972) but was identified by Hoppe and Heitefuss (197b) in both resting and germinating uredospores of Uromyces phaseoli. Similarly, the increases in other phospholipids in Tussilago leaves following infection probably reflect the increased synthesis of fungal membranes required in the development of the remarkably dense fungal tissue which permeates the entire intercellular space system of the pustule. Rice (192) already commented on the 'intercellular hyphal masses' of Puccinia sorghi forming a 'pseudoparenchyma'. ndeed, in much of the pustule on Tussilago, the mesophyll cells appear to have relatively little direct contact with each other and are surrounded by closely adpressed fungal hyphae, a situation structurally analogous to that of the algal layer in a lichen. Hoppe and Heitefuss were inclined to regard the increased PS as an aheration in host membranes rather than new fungal phospholipid, although PS was present in uredospores and sporelings, arguing that 'the parasite would have to contribute about % of the dry matter of the host-parasite complex if the PS increase is to be explained only with fungal lipids'. The presence of this amount of fungal material, which they considered improbable, is indicated in our present results by the chitosan estimation of aecial pustules on Tussilago. Direct microscopic observation suggests the proportion of fungal material in the infected tissue may be even higher than this. Thus, the dominant synthesis of fungal membranes must mask any lipid changes in the host cells, except of such characteristic components as the chloroplast membrane lipids. n the work reported here, as in previous studies with this host-parasite system (Holligan et al., 197; 197a, b), it has been found more satisfactory to express the amounts of substances present per unit area of leaf, rather than on a dry-weight basis as was chosen hy Hoppe and Heitefuss. Priestley (197) recommends extracted dry weight of plant tissues as a reference basis for biochemical studies but both dry weight and extracted dry weight present difficulties in tissues such as infected leaves, which accumulate unusually large amounts of insoluble as well as soluble storage materials. Von Sydow (1966a) working with rust-infected wheat leaves, similarly found the dry weight data of

11 Lipid metabolism in rust-infected leaves 1167 greatest interest were those related to leaf surface. From a comparison of the amounts of lipid recorded in the present work (Table i) with the values for carbohydrates given by Table 2 of Holligan et al. (197), it can be seen (Table 7) that mature aecial pustules accumulate lipid to a level comparable to that of free sugars and fructans. Table 7. Carbohydrates and lipids from rust-infected leaves of Tussilago [values as ^igjcm^) Stage of Tt infection lipid sugars* fructan* H Da»ix 9 7 D 6 ago 78 Converted from data for 12-mm discs given in Holligan et al. (197)- These discs would include host tissue around pustule. Holligan et al. [ 197a) noted that, following their exposure to ^ ^COj and a 2-h period of translocation, leaves of Tussilago infected by Puccinia poarum incorporated more radioactivity into lipids than healthy leaves (16% of total ^"^C incorporated compared with 6%). This effect is more pronounced than in the rusted wheat leaves studied by von Sydow (1966). The results of lipid analysis here show that this enhanced incorporation into lipids is not only into neutral lipids but also into phospholipids (Table 5). Clearly, as noted above concerning absolute amounts of lipid, any decreased conversion of '*Cphotosynthate into non-chloroplastic membrane components of host cells is outweighed by the synthesis of fungal membranes. The intensity of labelling of diglyceride is much higher than would be expected from the relatively low amounts of diglycerides detected on thin layer plates. This most likely indicates the rapid turn-over of diglycerides involved in breakdown and synthesis of both neutral and polar lipids. Similarly PA and PG, which were often difficult to detect on thin layer plates were readily seen on autoradiograms, suggesting high metabolic activity in quantitatively minor components. The visible changes in lipid content in host cells and fungus at different stages of infection and the differing destinations of fixed *'*C have stimulated a more detailed biochemical comparison of all phases of the life cycle of this fungus-host association, which will be reported in subsequent papers. ACKNOWLEDGMENTS We are grateful to the Agricultural Research Council for financial support. We wish to thank Drs P. M. Holligan and E. M. Carey for helpful discussion, Mrs Jean Bacon for technical assistance and Mr G. Woods for photography. REFERENCES AMENTA, J. S. (196). A rapid method for quantification of lipids separated by thin-layer chromatorraphv y. Lipid Res., 5, 27.» i'. BARTON, R. (1966). Fine structure of mesophyll ct-lls in scnescing leaves of Phaseolus. Planta {Bed) ' FERGUSON, C. H. R. & SMON, E. W. (197). Membrane lipids in senescing green tissues. J. c\p. Bot ZA 7- ' ' FoLCH, J., LEKS, M. & SLOANE-STANLEY, G. H. (1957). solation and purification of total lipids. J. bioi. Chem., 226, 97. FREEMAN, C. P. & WEST, D. (1966). Complete separation of lipid classes on a single thin-layer plate. Jf. Lipid Res., J, 2.

12 ii68 D. M. LosEL AND D. H. LEWS HTCHCOCK, C. & NCHOLS, B. W. (1971). Plant Lipid Biochemistry. Academic Press, London and New York. HoLDEN, M. (1965). Analytical methods for chlorophylls. n: Chemistry and Biochemistry of Plant Pigments (Ed. by T. W. Goodwin), p. 61. Academic Press, London and New York. HOLLGAN, P. M., CHEN, C. & LEWS, D. H. (197). Changes in the carbobydrate composition of leaves of Tussilago farfara during infection by Puccinia poarum. New PhytoL, 72, 97. HOLLGAN, P. M., CHEN, C, MCGEE, E. E. M. & LEWS, D. H. (197a). Carbohydrate metabolism in healthy and rusted leaves of Coltsfoot. Nevi PhytoL, 7, 881. HOLLGAN, P. M., MCGEE, E. E. M. & LEWS, D. H. (197b). Quantitative determination of starch and glycogen and their metabolism in leaves of Tussilago farfara during infection by Puccinia poarum. New PhytoL, 7, 87. HoLTZ, R. B. & ScHisLER, L. C. (1971). Lipid metabolism of Agaricus bisporus (Lange) Sing. i. Analysis of sporopbore and mycelial lipids. Lipids, 6, 176. 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(192). The haustoria of certain rusts and the relation between host and pathogen. Bull. Torrey Bot. Club, 5, 6. RDE, J. P. & DRYSDALE, R. B. (1972). A rapid method for the chemical estimation of filamentous fungi in plant tissue. PhysioL Plant PathoL, 2, 7. RouSER, G., SiAKOTOS, A. N. & FLESCHER, S. (1966). Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids, i, 85. SCHPPER, A. L. & MiRocHA, C. J. (i97o)- Change in fatty acid content of fugus-host tissue during pathogenesis of Uromyces phaseoli typica in Phaseolus vulgaris. Phytopathology, 6, 77. SCHMDT, E. W. (192). Uber eine pathologische Fettbildung im Zuckerrubenblatt. Ber. deutsch. bot. Ges., SO, 72. SCOTT, K. J. (1972). Obligate parasitism by phytopathogenic fungi. Biol. Rev., 7, 57. SHAW, M. (196). The physiology and host-parasite relations of the rusts. A. Rev. Phytopathology, i, 259. SKPSK, V. P., SMOLOWE, A. F., SULLVAN, R. C. & BAHKLEY, M. (1965). 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Microbiol., 8, 79. TULLOCH, A. P. & LEJNGHAM, Ci. A. (196). The component fatty acids of oils found in spores of plant rusts and other fungi. Part. Can. J. Microbiol., 1, 51. WLLAMS, P. H., KEEN, N. T., STRANDBERG, J. D. & MCNABOLA, S. S. (1968). Metabolite synthesis and degradation during clubroot development in cabbage hypocotyls. Phytopathology, 58, 921.

13 TK NKW 'lytologls'l', 7, 6 PLATE ). \. [,OSi:[, AND ).,.KVVS /.//'/ ) METABOLSM N RUST-NFECTED LEAVES (fociirg p. 1168)

14 THE NEW PHYTOLOGST, 7, 6 PLATE 2 sterol esters - t MGDG Trigiyeerides Fatty acids Diglycerides Sterols PG DGDG PC SL PS Monoglycerides P Polar lipids 6 H HD D 7H HD Origin D. M. LOSEL AND D. H. LEWS LPD METABOLSM N RUST-NFECTED LEAVES

15 Lipid metabolism in rust-infected leaves 1169 EXPLANATON OF PLATES PLATE Oil drops in pustules of Puccinia poarum on leaves of Tussilago No.. Upper epidermis, stage Di. (Oil drops arrowed.) No. 2. Lower epidermis, stage Di. (Oil drops arrowed.) Nos. and. ntercellular hyphae. Stage D2. PLATE 2 No. 5. Lipid in cells of aecial peridium. No. 6. Autoradiograph of thin-layer chromatogram of neutral lipids from total lipid extracts of healthy and rust-infected leaves of Tussila.^o after exposure to ""COj in liglit followed by 2 h in darkness. (For notation, see 'Methods', and footnote, p ) No. 7. Autoradiograph of thin-layer chromatogram of polar lipid extracts from healthy and rust-infected leaves of Tussilago after exposure to '"COj in light followed by a 2-h period of incorporation in darkness. (For notation, see 'Methods' and footnote, p )

16