QUANTITATIVE DETERMINATION OF STARCH AND GLYCOGEN AND THEIR METABOLISM IN LEAVES OF TUSSILAGO FARFARA DURING INFECTION BY PUCCINIA POARUM

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1 New Phytol. (1974) 73, QUANTITATIVE DETERMINATION OF STARCH AND GLYCOGEN AND THEIR METABOLISM IN LEAVES OF TUSSILAGO FARFARA DURING INFECTION BY PUCCINIA POARUM BY P. M. HOLLIGAN*, E. E. M. MCGEE AND D. H. LEWIS Department of Botany, The University, Sheffield Sio 2TN (Received 8 February 1974) SUMMARY Methods for the differential extraction of starch and glycogen from rust-infected leaf tissue and their assay using amyloglucosidasc and glucose oxidase are described. In rusted leaves of Tussilago, the low starch levels show a typical diurnal variation whereas there is continued synthesis of glycogen by the fungus in the dark. INTRODUCTION The most widely used methods for determining a-glucans are based on yields of glucose following their acid or enzymic hydrolysis. For extracts from plants, a specific degradation technique must be applied to prevent interference from other polysaccharides that contain glucose. The enzyme, amyloglucosidase (E.C ), which hydrolyses i->4 and 1-^6 bonds in a-glucans, is suitable for this purpose and crude preparations of fungal origin that contain small amounts of a-amylase give a complete conversion of starch and glycogen to glucose (Marshall and Whelan, 1970). For quantitative analysis of a-glucans, care must be taken to eliminate any j?-glucanase activity of the amyloglucosidase (Mac- Rae, 1971) or to show that j5-glucans are absent in the samples. Yields of glucose can be readily determined by the glucose oxidase method (Lloyd and Whelan, 1969) but, again, the possibility of hydrolytic activity towards water-soluble glucans by this enzyme preparation must be considered. To our knowledge, there is no technique that permits the independent hydrolysis of starch and glycogen and, to distinguish the two a-glucans when they occur together as in green plants infected by fungi, a preliminary fractionation must be made. In this paper, investigations of these problems in relation to studies on glucan metabolism in rust-infected leaves of Tussilago farfara L. are described. MATERIALS AND METHODS Assay of a-glucans Chemicals. Source of chemicals were as follows: glucose oxidase (Type II), peroxidase (Type II), o-dianisidine dihydrochloride, amyloglucosidase (Type III from Aspergillus oryzae) and glycogen (Type III from rabbit liver) from Sigma Chemical * Present address: The Laboratory, Marine Biological Association of the United Kingdom, Citadel Hill, Plymouth, PLi 2PB. 873

2 874 P- M. HoLLiGAN, E. E. M. MCGEE AND D. H. LEWIS Co; starch, amylose and cellobiose from B.D.H.; and laminaran and lichenan from Koch-Light. Enzymic hydrolysis of polymers. Solutions of amyloglucosidase were prepared by shaking a suspension of the crude enzyme in 0.05 M citrate buffer, ph 4.5, for 10 min. After centrifugation, the supernatant was diluted as necessary with the same buffer. One unit of enzyme is defined as i ^g soluble protein (as determined by the method of Lowry et al (1951) using bovine albumin as a standard) and is equivalent to approximately 7.7 //g of the enzyme as received. Samples of aqueous solutions of glucans, aqueous extracts of leaf tissue or aqueous suspensions of homogenized leaf tissues were mixed with equal volumes of enzyme solution and incubated at 45 C for 2 hr. The samples were then heated at 100 C for 5 min and centrifuged. The supernatant and washings from any pellet were combined and diluted with distilled water to give a final glucose concentration of 5-75 /^g/ml. Acid hydrolysis of polymers. Samples were mixed with equal volumes of 3 N H2SO4 and heated at 100 C for 2 h. After neutralization with NaOH,the hydrolysates were diluted as above. Corrections for loss of glucose (3-4%) were determined for each batch of samples by including control samples of free glucose. Assay of glucose. Glucose was determined by the glucose oxidase method (Lloyd and Whelan, 1969). In a preliminary experiment, the hydrolytic activity of glucose oxidase in different buffers was compared (for details, see 'Results'). Plant material and its exposure to Healthy plants of Tussilago farfara L. and plants infected by the rust, Puccinia poarum Niels., were raised as previously described (McGee et al.., 1973). Discs (1.4 cm diameter) were excised from leaves with a no. 10 cork borer. Those from diseased leaves each included a central mature pustule of the rust which occupied one-quarter to one-third of the surface area. Discs were exposed to ^*C02 in the light for 3 h at 20 C by placing them on moist filter paper within Petri dishes (14 cm diameter). The source of ^*C02 was 5.0 ml of diethanolamine-hcl-[^'^c]bicarbonate buffer that was poured into a small glass container just before sealing each dish. The composition of this buffer was adjusted to give 0.2% carbon dioxide in the gas phase (Macfadyen, 1970). Samples from the discs were analysed either immediately after the ^''^CO2 feeding period or after a further 20 h in the dark. The sampling procedure, using cork borers of appropriate sizes, was as follows: Diseased discs: Sample D, centre of each disc (0.20 cm^) within limits of pustule. Sample Am, annulus surrounding sample D (0.75 cm^) of fungal and host tissue. Sample An2, annulus surrounding sample Am (0.48 cm^) of host tissue only. Healthy discs: Sample H, centre of each disc (0.20 cm^); remainder of disc discarded. Extraction of tissue Samples were killed by immersion in boiling ethanol and then extracted as shown in Fig. I.

3 TISSUE: Extracted in 80% ethanol under reflux RESIDUE: Homogenized and extracted in cold and hot water RESIDUE: Treated with amyloglucosidase RESIDUE: Treated with 1.5 N H2SO4 RESIDUE: Discarded, but would include chitin, cellulose and other polysaccharides Starch and glycogen in infected leaves 875 SUPERNATANT: LOW molecular weight sugars (including fructans) {fraction i) SUPERNATANT: Water soluble polysaccharides {fraction 2) Concentrated and mixed with 2 vols ethanol. Stored at 4 C overnight and centrifuged RESIDUE: Redissolved in water and treated with amyloglucosidase (325 units/ml). Mixed with 2 vols ethanol, stored at 4 C overnight and centrifuged RESIDUE: Treated with 1.5 N H2SO4 > SUPERNATANT: glucose derived from waterinsoluble a-glucans {fraction 3) > SUPERNATANT: Sugars (mainly glucose, galactose, mannose and xylose) derived from other water-insoluble polysaccharides {fraction 4) SUPERNATANT: Some fructans hut no other detectable carbohydrate {fraction 2A) -+ SUPERNATANT: Glucose derived from watersoluble a-glucans {fraction zb) - SUPERNATANT: sugars (mainly glucose and arabinose) derived from; other watersoluble polysaccharides {fraction 2C) Fig. I. Procedure for the extraction and hydrolysis of polysaccharides from leaf tissue of Tussilago. Assay of radioactivity Radioactivity in each fraction of the extracts was assayed by liquid scintillation counting using a toluene-triton X-ioo scintillant (Turner, 1968). RESULTS Hydrolytic activity of glucose oxidase Impurities in commercial glucose oxidase cause considerable degradation of both a- and js-glucans in phosphate and dilute tris (o.i M) buffers (Table i). However, in the tris (0.3 M)-phosphate-glycerol buffer devised by Lloyd and Whelan (1969), this effect

4 876 p. M. HoLLiGAN, E. E. M. MCGEE AND D. H. LEWIS Table i. Hydrolytic activity of glucose oxidase in different buffers at ph 7.0 {values as percentage degradation of glucan during glucose assay compared with control treatments in which glucose yields from equivalent samples treated with 1.5 N H2SO4. were determined) Glucose polymers (50 //g/ml) a-glucans Starch Glycogen ^-Glucans Cellobiose Laminaran Tris-phosphate-glycerol (0.3 M tris) 0.3 O.I O.I 2.1* Buffers * Mainly contaminant glucose. Tris-maleate (0.1 M tris) Phosphate (0.1 M phosphate) is very much reduced. This buffer was used for subsequent assays and the residual hydrolytic activity from the glucose oxidase was considered insignificant. Hydrolytic activity of amyloglucosidase Treatment of a- and j8-glucans with varying quantities of amyloglucosidase showed that the activity of ^-glucanase was reduced significantly in relation to that of a-glucanase at low enzyme concentrations (Table 2). After samples of both healthy and diseased leaves of Tussilago had been incubated with amyloglucosidase at 65 units/ml, no further glucose was released by a second treatment with the enzyme at 325 units/ml. It follows that hydrolysable ^-glucan was either absent or present in only very small amounts in both Tussilago and Puccinia. Therefore, for the complete hydrolysis of a-glucans of these plants, all samples were treated with high concentrations of amyloglucosidase ( units/ml depending on the amounts of tissue). Metabolism of cc-glucan in healthy and diseased leaf tissue of Tussilago Tables 3 and 4 present data on the distribution of ^'*'C and of glucose polymers in healthy and diseased leaf discs after they had been exposed to ^^COj. During the 3 h in ^^^002 in the light, ^*C is assimilated by host tissue mainly into sucrose and a-glucan (Holligan et al., 1974). Values here were 60% and 25% respectively. In the subsequent dark period, there is a rapid decline in absolute levels of, and ^"^C in, a-glucan in host Table 2. Hydrolysis of glucans by amyloglucosidase {values as percentage degradation of glucan compared with control treatments in which glucose yields were determined from equivalent samples treated with 1.5 N H2SO4) Glucose polymers (50 ngjmx) a-glucans Starch Glycogen Amylose )?-Glucans Cellobiose Laminaran Lichenan Concentration of amyloglucosidase (units/ml) IOI , Not determined I 15 I

5 Starch and glycogen in infected leaves 877 Table 3. Incorporation of radioactivity into healthy and diseased discs 0/Tussilago Sample 3 h light D Am An2 H 3 h light + 20 h dark D Am An2 H Total in fractions I, 2, 3 and 4 (ct/min/cm^) 8, ,700 18,400 20,750 7,200 5,400 10,800 Soluble glucan* t (fraction 2B) (ct/min/cm^) Insoluble glucan* (fraction 3) (ct/min/cm^) Total glucan (ct/min/cm^) ^*Cin glucans (7o) * Paper chromatography showed that after the dark period some radioactivity is present in substances other than glucan. This was probably in protein, small amounts of which were detected in these fractions. t Up to 30% of the radioactivity in fraction 2 was lost during the preparation of fractions 2A, 2B and 2C. The cause of this is not known. tissue (H and An2 samples). Radioactivity in sucrose also declines (Holligan et al., 1974). Much of the radioactivity is either lost as respiratory CO2 or redistributed into structural polymers (discarded in the final residue, see Fig. i) and, in the case of diseased discs, into fungal metabolites. The a-glucan from host tissue gives a black colour with iodine and is considered to be a starch which is partly soluble in water and which is a primary product of photosynthesis. Although fructans accumulate in leaf tissue in much larger amounts (>2ooo figlcm^ within pustules of P. poarum (Holligan et al., 1973)), they incorporate little or no label during ^^COj experiments of this type (Holligan et al.., 1974) and are apparently a secondary product of carbon dioxide fixation. Incorporation of radioactivity into the D samples follows an opposite pattern. As described by Holligan et al. (1974), sucrose is translocated rapidly to within the pustule and is assimilated by the fungus. This process continues in the dark so that, by 23 h, more than 50% of the ^'^C fixed by the diseased discs is in fungal metabolites. The major portion of the a-glucan in the D samples is water soluble and the absolute level does not decline significantly in the dark (Table 4). The level of radioactivity in this fraction is low at the end of the light period but increases in the dark. The intact water-soluble polymer from D samples gives a brown colour with iodine and is considered to be a glycogen synthesized by the fungus. The small amount of insoluble glucan in the D Table 4. Glucose released by degradation of polysaccharides from various fractions from various regions of healthy and diseased leaves of Tussilago (see ^Materials and Methods^ and Fig. i) (values as ^ Sample 3 h light D Am An2 H 3 h light+ 20 h dark D Am An2 H a-glucans Soluble Insoluble (fraction 2B) (fraction 3) II 9 < < <5 Total <I0 Other Soluble (fraction 2C) 13a polysaccharides Insoluble (fraction 4) Total

6 878 p. M. HoLLiGAN, E. E. M. M C G E E AND D. H. L E W I S samples, relatively heavily labelled in the light, disappears almost completely in the dark. The solubility of a-glucans in the Am samples which contain some fungal material is intermediate between those of the D and An2 samples, probably due to the presence of some glycogen in this zone. The levels of glucose derived from water-soluble and insoluble polysaccharides other than a-glucans show little or no change during the dark period. Incorporation of ^*C into these fractions is low (25% of the total recovered) except for fraction 4 of the D samples after the dark period where levels reach approximately 8%. This fraction contains newly synthesized 'glucomannan' (see Holligan, Chen and Lewis, 1973; Holligan et al.., 1974)DISCUSSION By using appropriate sampling, extraction and assay techniques, it is possible, in leaves of Tussilago infected by Puccinia poarum, to distinguish a-glucans from host and fungal tissue that probably correspond to starch and glycogen respectively. By refinements of the extraction procedure, e.g. by extraction of homogenized tissue in cold water only, it may be possible to improve the separation between these two polymers. Although there is a considerable literature on the gross changes and metabolism of starch in infected tissues (reviewed by Scott, 1972), there had been little attempt previously to distinguish glycogen from starch. The Tussilago I Puccinia poarum symbiosis is especially suitable for these studies since healthy leaves are low in starch and pustules are large in size. Also, unlike leaves of cereals, bean and clover, fructans and not starch accumulate in host cells in response to infection (Holligan et al., 1973). Our analyses do not invalidate previous observations on a-glucan levels in other rust-infected plants since it is probable that, quantitatively, there is a much greater synthesis of starch around pre-sporulating pustules than glycogen accumulation within. This, however, needs to be verified. The nature of the insoluble glucan which is present in small quantities in pustules on Tussilago leaves remains to be investigated. It becomes relatively heavily labelled in the light and is metabolized in the dark. These are features of host starch but, within pustules of Puccinia poarum, chloroplasts disintegrate (Orcival, 1968) and chlorophyll is barely detectable (unpublished results). Starch in Tussilago, about a third of which is extractable by hot water, shows a typical diurnal variation such that radioactivity, photosynthetically incorporated into it from ^'*C02, disappears as it is re-utilized in the dark. Fungal glycogen, by contrast, is almost entirely soluble in hot water and continues to be synthesized in the dark. The specific activities of both soluble and insoluble starch in H and An2 samples after 3 h photosynthesis are very similar (38 and 27, and 34 and 23 ct/min/^g respectively) whereas the value for soluble glycogen is 2 ct/min/fig. Observed values for the specific activities of host glucans after the dark period are of little significance since residual radioactivity is low and absolute levels approach the lower limits of detectability. However, soluble glycogen levels remain high and, after 20 h, its specific activity rises to 13 d/min/fig. As the percentage radioactivity in host glucans declines, that in glycogen does not rise significantly (Table 3) since there is concomitant incorporation of ^"^C into the soluble fraction as mannitol, arabitol and trehalose are synthesized (Holligan et al., 1974). In addition to glycogen, the only other insoluble glucan which incorporates substantial amounts of radioactivity is a glucomannan, probably a structural component of fungal cell walls (Holligan et al., 1973).

7 Starch and glycogen in infected leaves 879 ACKNOW^LEDGMENTS We are grateful to both the Science Research Council and Agricultural Research Council for financial support and wish to thank Mrs Jean Bacon for technical assistance. REFERENCES HOLLIGAN, P. M., CHEN, C. & LEWIS, D. H. (1973). Changes in the carbohydrate composition of leaves of Tussilago farfara during infection by Puccinia poarum. New Phytol., 72, 947. HOLLIGAN, P. M., CHEN, C, MCGEE, E. E. M. & LEWIS, D. H. (1974). Carbohydrate metabolism in healthy and rusted leaves of coltsfoot. New Phytol., 73, 881. LLOYD, J. B. & WHELAN, W. J. (1969). An improved method for enzymic determination of glucose in the presence of maltose. Anal. Biochem., 30, 467. LowRY, O. H., RosENBROUGH, N. J., EARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Eolin phenol reagent. J. biol. Chem., 193, 265. MACFADYEN, A. (1970). Simple methods for measuring and maintaining the proportion of carbon dioxide in air, for use in ecological studies of soil respiration. Soil Biol. Biochem., 2, 9. MACRAE, J. C. (1971). Quantitative measurement of starch in very small amounts of leaf tissue. Planta, 96, IOI. MARSHALL, J. J. & WHELAN, W. J. (1970). Incomplete conversion of glycogen and starch by crystalline amyloglucosidase and its importance in the determination of amylaceous polymers. FEBS Letters, 9,85. MCGEE, E. E. M., HOLLIGAN, P. M., EUNG, A. K. & LEWIS, D. H. (1973). Maintenance of the rust, Puccinia poarum, on its alternate hosts under controlled conditions. New Phytol., 72, 937. ORCIVAL, J. (1968). Sur des modifications particulieres provoquees par une Uredinale sur la structure des chloroplasts de Tussilago farfara L. C. r. hebd. Seanc. Acad. Sci. Paris, D, 266, SCOTT, K. J. (1972). Obligate parasitism by phytopathogenic fungi. Biol. Rev., 47, 537. TURNER, J. C. (1968). Triton X-ioo scintillant for carbon-14 labelled materials. Int. J. appl. Rad. Isotopes

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