School of Biological Sciences, University of Bradford. {Received 22 October 1968) SUMMARY

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1 New Phytol. (1969) 68, ADENOSINE TRIPHOSPHATE CONCENTRATION IN RELATION TO RESPIRATION AND RESISTANCE TO INFECTION IN TISSUES INFECTED BY VIRUS BY JILL BAYLEY AND M. J. MERRETT School of Biological Sciences, University of Bradford {Received 22 October 1968) SUMMARY When leaves of Nicotiana tabacum L. var. Xanthi were infected with tobacco etch virus, local lesion formation was accompanied by an increased rate of respiration which coincided with an increase in their adenosine triphosphate concentration. Stimulation of respiration by 2.4 dinitrophenol decreased to a low value with increasing lesion density. A combination of increased adenosine triphosphate concentration and greater adenosine triphosphate turnover may contribute towards the increased respiration of the local lesion host. Adenine greatly increased the adenosine triphosphate concentration of healthy leaves but not the respiration rate. With the local lesion leaf, where adenosine triphosphate concentration was increased by virus infection, adenosine triphosphate concentration was unaffected by adenine. Treatment of leaves with adenine increases resistance to virus infection, while healthy tissue surrounding virus-induced local lesions is also resistant to reinfection; both give increased adenosine triphosphate concentration suggesting that this is an important factor in resistance to virus infection. Increased adenosine triphosphate concentration in the leaf may result in the production of additional host ribonucleic acid which then occupies receptor sites usually available to virus ribonucleic acid. INTRODUCTION When plant tissues are inoculated with virus there can either be a systemic spread of virus through the tissue or localization of virus in a small group of cells. There is a slow spread of virus from this small group of cells into adjoining cells and this is accompanied by the death of the first cells giving a necrotic local lesion. The tissue surrounding virusinduced local lesions is highly resistant to infection by virus (Ross, 1961). The early events and consequent changes that lead to cell death in these local lesions and the biochemical changes in the surrounding resistant area are little understood. A significant feature of local lesion formation is an increased rate of respiration of the host-tissue (Yamaguchi and Hirai, 1959; Weintraub, Kemp and Ragetli, i960; Sunderland and Merrett, 1965). The increase in respiration of leaves of Nicotiana tabacum L. var. Xanthi infected with tobacco mosaic virus (TMV) was proportional to lesion number and was related to the necrotic process rather than virus increase (Sunderland and Merrett, 1965). Local lesion formation results in the uncoupling of respiration (Sunderland and Merrett, 1965) and an increase in adenosine triphosphate (ATP) concentration in the host-plant (Sunderland and Merrett, 1967). The present work was to determine whether other host-virus combinations, resulting in necrotic local lesions, also give increased ATP concentration in the host and whether this ATP increase would provide an explanation for enhanced respiration or virus-induced acquired resistance to infection. 257

2 258 JILL BAYLEY AND M. J. MERRETT MATERIALS AND METHODS Plant material Plants of Nicotiana tabacum L. var. Xanthi or L. var. Samsun were grown in a glasshouse at a mean temperature of 19 C. Fully expanded leaves were used in all experiments. Leaves of the same age on different plants were selected for controls and for inoculation with virus. Healthy conditioned callus tissues of N. tabacum type White Burley variety Judy's Pride were grown as described previously (Merrett and Handoll, 1967). Infection of plant tissue Healthy and TEV-infected tissue cultures derived from the same clone were obtained by cutting a tissue culture in two, sub-culturing one-half and inoculating the other with TEV. The inoculum was the expressed sap of leaves of A'^. tabacum L. var. White Burley showing characteristic symptoms of TEV infection. The sap was passed through a Seitz-filter, then poured over the surface of the callus and the callus pricked with a needle under sterile conditions. The callus was allowed to grow for 3 weeks before being surface sterilized and sub-cultured. Attempts to infect tissue cultures with tobacco ringspot virus and cucumber mosaic virus by this method proved unsuccessful. Leaves of N. tabacum L. var. Xanthi were inoculated with TEV by rubbing with the expressed sap of leaves of N. tabacum L. var. White Burley infected with TEV. The sap was serially diluted with o.oi M phosphate buffer, ph 7.0 when different lesion densities on the leaves were required. Control leaves were rubbed with o.oi M phosphate buffer, ph 7.0. Measurements Respiration rates of tissue cultures were determined as described previously (Merrett and Handoll, 1967). Respiration rates of leaf tissues were determined by removing discs, 11 mm in diameter, from the leaves by means of a leaf punch and recording oxygen uptake by conventional Warburg manometry. All measurements were made at 25 C in the dark. The same experimental conditions in Warburg flasks were used as for tissue cultures (Merrett and Handoll, 1967). ATP was extracted from plant tissues and determined by the luciferin-luciferase method of Strehler and McElroy (1957) as described previously (Merrett and Handoll, 1967). RESULTS Respiration of etch-infected tissue cultures There were no apparent symptoms resulting from infection of tobacco callus tissue cultures by TEV, the growth rate of the tissue was unaffected (Table i). In several experiments no significant difference was recorded between the respiration rates of healthy and TEV-infected tissue cultures. Respiration rate and lesion development and number The respiration rate of infected leaf tissue was followed during lesion development by using an inoculum known to give good lesion cover. At intervals leaf discs were removed from healthy and infected plants and their respiration rate determined (Fig. i). The rapid increase in respiration rate of the leaf corresponded with the appearance of

3 A TP and resistance to virus 259 lesions. A maximum rate of respiration, reached 50 hours after inoculation, was maintained for approximately 20 hours. Then the respiration rate decreased as the proportion of necrotic tissue in the leaf was increased by lesion growth. Different lesion densities were produced on leaves by serially diluting the sap from TEV-infeeted leaves before inoculation. Shortly after the appearance of lesions leaf discs were removed and their respiration determined. Increasing lesion density on the leaf was accompanied by greater rates of respiration (Fig. 2). In previous experiments with tobacco mosaic virus the increase in respiration rate was proportional to lesion density Table i. The effect of infection with tobacco etch virus on growth and respiration of tobacco callus tissue cultures Respiration rate (Q02) Final dry weight (mg) Healthy tissue i.26±o.i4 24* ±1-5 Tobacco etch infected tissue I.2O±O.I5 23 ±0.9 * Each value represents mean from ten cultures ± standard error Hours after inoculation Fig I The respiration of healthy and TEV-infected leaf-discs of Nicotiana tabacum h. var Xanthi in relation to lesion development. O, Healthy leaf discs;, infected leat discs. Plants were inoculated at o hours and kept in glasshouse. Leaf discs were removed at intervals and respiration rate determined. Following appearance of lesions, twenty-five lesions were present on six infected discs (11 mm diameter) used in each Warburg flask. at all except the highest lesion densities (Sunderland and Merrett, 1965), whereas in the present experiments with TEV at lesion densities greater than five per leaf disc there was a much smaller increment in respiration rate per lesion (Fig. 2). The lesions produced by TEV on leaves of N. tabacum L. var. Xanthi are more diffuse than those produced by tobacco mosaic virus and it is possible that interference between lesions takes place at lower lesion densities with TEV than occurs with tobacco mosaic virus. In the healthy leaf respiration rate was increased by lo"^ M dinitrophenol showing that respiration rate is probably limited by the availability of phosphate acceptors for respiratory chain phosphorylation. With the maximum lesion density employed in these experiments the stimulation of respiration achieved by dinitrophenol was less than 10% (Fig. 2). High 100

4 26o JILL BAYLEY AND M. J. MERRETT lesion density or dinitrophenol will increase the respiration rate of leaf discs but the two effects are not additive. Lesion development and ATP concentration Leaves were infected with a virus inoculum known to give a dense lesion cover. At intervals infected leaves and comparable healthy control leaves were removed from the plants and ATP concentrations determined. A rapid increase in ATP concentration of the leaf was accompanied by the appearance of lesions (Fig. 3). The maximum ATP concentration of the leaf was reached sometime after the appearance of lesions, then as the proportion of necrotic tissue in the leaf increased the ATP concentration fell away Total lesion number on six leaf discs Fig. 2. The effect of lesion density on the respiration rate of leaf discs., Respiration rate in the presence of phosphate buffer; O, respiration rate in the presence of io~^m dinitrophenol. Plants were inoculated with TEV and 24 hours after the appearance of lesions leaf discs were removed from healthy and infected plants and respiration rate determined. As previously shown with necrotic lesion development using tobacco mosaic virus (Sunderland and Merrett, 1967), the increase in ATP concentration was greater at higher lesion densities. Adenine and ATP concentration Plants were inoculated with TEV and at intervals leaf discs were removed from infected and healthy plants and floated on a solution of adenine (200 mg/1) for 3 hours before determining ATP concentration. This resulted in a higher ATP concentration than comparable leaf-discs floated on distilled water (Fig. 4) (see also Merrett and Handoll, 1967). Following the appearance of lesions on leaf discs on distilled water the ATP concentration increased to that of healthy and infected discs treated with adenine. It can be concluded that adenine and local lesion formation both have the same effect in increasing the ATP concentration in the leaf. The effect of adenine on respiration rate was determined by floating leaf discs on buffer in Warburg flasks and recording respiration before and after addition of adenine from the side-arm. It was found that respiration was unaffected by adenine.

5 ATP and resistance to virus 261 ioo S 80 o I o Q- 40 c o i 20 oli Hours after inoculation Fig. 3. The effect of local lesion development on the ATP concentration of leaves oinicotiana tabacum L. var Xanthi. Leaves inoculated with TEV and healthy and infected leaves sampled at intervals. ATP expressed as percentage increase in infected leaf compared with healthy control. Lesion density g.o cm^ ^ 50 "o e e 2: 40 Appearance of lesions Hours after inoculation 80 Fig. 4. The effect of adenine on the ATP concentration of healthy and TEV-infected leaves oinicotiana tabacum L. var Xanthi. A, TEV-infected leaf discs floated on adenine; A, healthy leaf discs floated on adenine;, TEV-infected leaf discs floated on distilled water; O, healthy leaf discs floated on distilled water.

6 262 JILL BAYLEY AND M. J. MERRETT DISCUSSION In several experiments with leaves of Nicotiana tabacum L. var. Xanthi infected with tobacco etch virus the respiration rate increased over the same period of time as the concentration of adenosine triphosphate in the leaf increased. However, increased adenosine triphosphate concentration in a tissue does not necessarily result in an increase in respiration rate. When tobacco callus tissue cultures were supplied with adenine the ATP concentration in the tissue was increased but respiration rate remained the same, showing that respiratory control was unaltered (Merrett and Handoll, 1967). Enhanced respiration requires an increase in ATP turnover, experiments with dinitrophenol suggest this occurs in the local lesion leaf. Dinitrophenol uncouples respiration from phosphorylation resulting in an increased rate of respiration when the availability of phosphate acceptors is limiting respiration (Loomis and Lipmann, 1948). When the healthy leaf is treated with 10"* M 2:4 dinitrophenol there is an increase in oxygen uptake showing that respiration is probably limited by the availability of phosphate acceptors but once local lesions have appeared the stimulatory effect on respiration of dinitrophenol decreases suggesting an increased availability of phosphate acceptors. In addition to the observed net synthesis of ATP, it is also necessary to postulate enhanced ATP turnover to explain the respiratory increase and the effect of dinitrophenol when local lesions appear. These results, when considered with the results from the experiments with adenine, add to our understanding of another feature of virus diseases characterized by local lesion formation, that of 'localized acquired resistance' (Ross, 1961). Ross originally demonstrated this phenomenon by first inculating leaves with a dilute suspension of tobacco mosaic virus followed by subsequently challenge inoculating with a concentrated suspension of tobacco mosaic virus. The challenge inoculum produced fewer lesions of much smaller size in the zone around each old lesion when compared with the result of inoculation with a previously uninoculated control leaf (Ross, 1961). Treatment of leaves with adenine resembles localized resistance. It was shown by Kiraly and Szirmai (1964) that floating leaf discs of N. glutinosa on adenine solution immediately after inoculation increased resistance to infection. Increased oxygen uptake by tissues surrounding local lesions (Weintraub etal., i960; Sunderland and Merrett, 1965) has been demonstrated, and increased activity of a number of respiratory enzymes in this area has also been demonstrated (Solymosy and Farkas, 1963). However, enhanced respiration or activation of respiratory enzymes are not the primary causes of resistance to infection in local lesion hosts, as lesion number is decreased by adenine but respiration is unaffected. Local lesion development and treatment with adenine have one effect in common on the metabolism of leaves, both result in increased ATP concentration of the tissue. The development of necrotic local lesions induced by tobacco mosaic virus (Sunderland and Merrett, 1963, 1967) or by TEV (Fig. 3) is accompanied by a rapid increase in ATP concentration of the tissues. Thus in conclusion it would appear that enhanced ATP concentration and turnover in local lesion hosts is probably the reason for the increase in respiration rate. In addition enhanced ATP concentration of the tissue may be a determining factor in virus induced localized acquired resistance, though this may not be the primary cause of the phenomenon. One possibility is that overproduction of ATP in a tissue results in increased ribonucleic acid production and this additional host ribonucleic acid occupies receptor sites in the cell usually available to virus ribonucleic acid thereby increasing resistance to virus.

7 ATP and resistance to virus 263; ACKNOWLEDGMENTS The authors are grateful to Dr A. A. Brunt, Glasshouse Research Institute for providing tobacco etch virus. This work was supported by a grant from the Agricultural Research Council. REFERENCES KiRALY, Z. & SziRMAi, J. (1964). The influence of kinetin on Tobacco Mosaic Virus production in Nicotiana glutinosa leaf discs. Virology, 23, 286. LooMis, W. F. & LiPMANN, F. (1948). Reversible inhibition of the coupling between phosphor5'lation and oxidation. J. biol. Chem., 173, 807. MERBETT, M. J. & HANDOLL, C. (1967). Some effects of adenine on growth, respiration, adenosine diphosphate and triphosphate concentrations in plant tissues. New PhytoL, 66, 569. Ross, A. F. (1961). Localized acquired resistance to plant virus infection in hypersensitive hosts. Virology, 14, 329. SoLYMOSY, F. & FARKAS, G. L. (1963). Metabolic characteristics at the enzymatic level of tobacco tissues exhibiting localized acquired resistance to viral infection. Virology, 21, 210. STREHLER, B. L. & MCELROY, W. D. (1957). Assay of adenosine triphosphate. In: Methods in Enzymology^ Vol. 3 (Ed. by S. P. Colowick & N. O. Kaplan), pp Academic Press, London and New York. SuNDERLAND, D. W. & MERRETT, M. J. (1963). Adenosine diphosphate and adenosine triphosphate concentrations in leaves showing necrotic local virus lesions. Virology, 23, 274. SuNDERLAND, D. W. & MERRETT, M. J. (1965). The respiration of leaves showing necrotic local lesions following infection by tobacco mosaic virus. Ann. appl. Biol., 56, 477. SuNDERLAND, D. W. & MERRETT, M. J. (1967). Adenosine diphosphate, adenosine triphosphate concentrations and respiration rate of leaves showing necrotic lesions following infection by tobacco-mosaic virus. Physiologia PL, 20, 368. WEINTRAUB, M., KEMP, W. G. & RAGETLI, H. W. J. (i960). Studies on the metabolism of leaves with localized virus infections. L Oxygen uptake. Can. J. Microbiol., 6, 407. YAMAGUCHI, A. & HiRAi, H. (1959). The effect of local infection with tobacco mosaic virus on respiration; in leaves of Nicotiana glutinosa L. Phytopathology, 49, 447. C N.P.

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