STUDIES ON COTTON STENOSIS--I. AUXIN METABOLISM OF THE DISEASED PLANT

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1 STUDIES ON COTTON STENOSIS--I. AUXIN METABOLISM OF THE DISEASED PLANT BY P. NARAYANASAMY, T. JAGANATHAN, K. RANGANATHAN AND A. PALANISWAMI (Department of Plant Pathology, Agricultural College and Research Institute, Tamil Nadu Agricultural University, Coimbatore) Received March 16, 1972 (Communicated by Dr. G. Rangaswami, F.A.Sc), ABSTRACT Cotton plants infected by stenosis disease show growth abnormalities which may possibly be due to an abnormal reduction in the IAA-contents of the diseased plants. The accelerated activity ofperoxidase might be responsible for the oxidation of IAA. The activity of IAA-oxidase was reduced in infected plants. This may be due to the higher concentrations of pheaolicsinduced by higher activity of polyphenolase. INTRODUCTION VIRUS-INDUCED disturbances of auxin metabolism of diseased plants are considered to be the possible causes of the various growth abnormalities like retardation of growth and stimulation of axillary buds (Diener, 1963). Cotton stenosis caused by a graft-transmissible virus, is characterised by stunting, smalling of leaves, stimulation of axillary buds resulting in bushy growth of the plants and sterility. As the symptoms of the disease indicate the possibility of auxin metabolism being deranged, the present study to throw some light on the pathophysiology of the infected plants was :taken up. MATERIAL AND METHODS Healthy and stenosis-affected cotton (variety K.7) plants were used, When they were about three months old. Leaf samples of same age were collected in the mornings from healthy and diseased plants and used for different estimations. The averages of two estimations of samples are presented in different tables. Auxins were extracted by the method of Ito et al (1969). One hundred grams of leaves were collected and extlacted with ether at 0 C for 24 hours. B4 79

2 i0 P. NARAYANASAMY AND OTHERS The ether was changed and decanted three times and filtered. The filtrate was shaken with 8 per cent sodium bicarbonate solution and after adjusting the ph to with hydrochloric acid, the aqueous solution was shaken with ether. The ether extracts were concentrated at 40 C and chromatographed on Whatman No. 1 paper. One set of chromatograms was sprayed with a mixture of isopropanol-ammonia-water (10:1:1) to develop the colour. The auxins were identified based on the Ry values. Sheets of another set of chromatograms were cut in the corresponding areas and the auxins and growth inhibitors present in the chromatograms were extracted with 2 ml of 3 per cent sucrose solution. The rice coleoptile straight growth test (Das et al., 1966) was employed for the bioassay of auxins using paddy (variety ASD 5) seeds. The activity of peroxidase in healthy and diseased leaves of cotton was estimated by the method of Mudd et al. (1959) and expressed as changes in optical density at 420 mtz. The amounts of indoleacetic acid (IAA) oxidised by the activity of IAA-oxidase, two hours after incubation were determined by the method of Goldacre et al. (1953). The polyphenolase activity was measured by the change in the optical density at 410 m tz (Haskins, 1955). The total phenolics contents of healthy and diseased leaves were estimated and expressed as mg of catechol equivalents per g of leaf tissue (Bray and Thorpe, 1954). RESULTS 1. Auxin contents of healthy and diseased leaves Chromatographic analyses revealed that healthy as well as diseased cotton leaves contained only indoleacetic acid (IA_A), while the other auxins were not observed in detectable amounts. Further, no growth inhibiting substance was observed in the samples analysed. The levels of IAA present in the healthy and diseased leaves estimated by the rice coleoptile bioassay method are presented in Table I. The IAA contents in healthy leaves ranged from 32.6 to 33.8tzg/g, while the diseased leaves contained only 4"3 I~g/g, showing a reduction of per cent over comparable healthy leaves. 2. Oxidases Since the IAA contents of the diseased leaves were greatly reduced due to infection, the activity of peroxidase and IAA.-oxidase which are known

3 Studies on Cotton Stenosis--I 81 TABLE I IAA contents of healthy and diseased cotton leaves fresh leaf) (expressed as #g/g of No. Healthy Diseased ~ of decrease Mean decrease over healthy (~) "3 86" "4 87"57 87" " to play a significant role in the oxidation of IAA was studied, Peroxidase activity was found to be significantly higher in diseased leaves than in healthy ones, the mean percentage of increase being (Table ]I). TABLE II Peroxidase activity in healthy and diseased cotton leaves No. Optical density at 420 mtz Healthy Diseased of increase Mean incl ease over healthy (%) The activity of IAA-oxidase was determined by estimating the amounts of IAA oxidised by the enzyme present in 2 g of leaves in two hours. The data presented in Table III indicate that the activity of IAA-oxidase was lowered by virus infection, the reduction being per cent. 3. Total phenolies Total phenolics and the activity of polyphenolase were estimated to find out whether they are responsible for the decreased IAA-oxidase activity

4 82 P. NARAYANASAMY AND OTHERS TABLE III IAA-oxidase activity in healthy and diseased leaves of cotton /~g of IAA-oxidised No. ~ of decrease Mean decrease Healthy Diseased over healthy (~) in the diseased plants. The total phenolics contents of the diseased plants were higher, when compared to the healthy plants (Table IV). TABLE IV Total phenolics contents of healthy and disease,t cotton leaves mg of catechol equiva~nts per g ofleaves No. ~ of iacrease Mean increase Healthy Diseased over healthy (~) The activity of the enzyme polyphenolase in the healthy and diseased cotton leaves was assayed, since this enzyme has definite influence on the synthesis of phenolics. The activity of polyphenolase was greatly enhanced in the diseased leaves (Table V). TABLE V Polyphenolase activity in healthy and diseased leaves of cotton Optical density at 410 m/z No. ~ of increase Mean increase Healthy Diseased over healthy (~o)

5 Studies on Cotton Stenosis--I 83 DISCUSSION Virus infection is known to interfere with the auxin levels of the host plant resulting in various types of growth abnormalities. Pavillard (1952) reported the depressing effect of virus infection on auxin contents of leaves. The decreased auxin content of diseased tissues coupled with stunting was observed for a number of virus-host combinations by Hirata (1954). In the present study also retardation of growth of cotton affected by stenosis might be due to a reduction in IAA content rather than due to arty growth inhibiting substance. The activity of polyphenolase remained at a higher level in diseased leaves than in the healthy leaves. This trend is similar to that observed in the case of plants affected by different viruses (Farkas et al, 1960; Kikuchi and Yamaguchi, 1960; and van Kammen and Brouwer, 1964) and myco. plasma (Parthasarathy et al., 1970). Higher activity of this enzyme naturally results in the formation of greater amounts of phenolics in the diseased tissues of cotton. Similar increase in the phenolics contents of diseased plants has been reported by Parthasarathy and Ramaswamy (1961). Pheno!ics are known to adversely affect the activity of IAA-oxidase (Andre.e, 1952; Tomaszewski and Thimann, 1966). It is likely that the increased level of phenolics of the diseased leaves may be a contributing factor for the decreased activity of IAA-oxidase in the diseased cotton plants. Even though the activity of IAA-oxidase activity was less, the IA.A content of diseased plants was abnormally low, when compared with healthy ones. The reduced levels of IAA in diseased plants may be either due to reduced synthesis of auxins or due to accelerated activity of oxidases. As oxidases are known to destroy auxins (Diener, 1963), the significant increase in the activity of peroxidase indicates that this enzyme might be responsible for the destruction of IA~_ in diseased cotton plants rather than IAA-oxidase. Moreover, peroxidase and other oxidases may play some role in the conversion of tryptophane to IAA. resulting in the reduced synthesis of IAA. However, further investigations are sine qua non to quantity the role of another IAA-destroying system involving tyrosinase and phenols (Briggs and Ray, 1956). ACKNOWLEDGEMENT The authors wish to express their gratitude and thanks to Dr. G. Rangaswami, Vice-Chancellor, Tamil Nadu Agricultural University, Coimbator% for the valuable suggestions.

6 84 P. NARAYANASAMY AND OTHERS Androa, W. A. Bray, G. G. and Thorpe, W. V. Briggs, W. R. and Ray, P. M. Das, V. S. R., Raju, P. V. and Rao, M. P. Diener, T. O. Farkas, G. L., Kiraly, Z. and Solymosy, F. Goldacre, P. L., Galstoa, A. W. and Weintraub, R. L. Haskins, F.A... Hirata, S. Itc, H., Motomura, Y., Konno, Y. and Hatayama, T. Kikuchi, M. and Yamaguchi, A. Mudd, J. B., Johnson, B. J., Burris, R. H. and Buchholtz, K. P. Parthasarathy, K., Babu, D. R. C. and Rao, P. S. REFERENCES.. "Effect of scopoletin on indoleacetic acid metabolism." Nature, 1952, 170, 83. "Analysis of phenolic compounds of interest in metabolism," Meth. Biochem. AnaL, 1954, 1~ 27. " An auxin inactivating system involving tyrosinase," Plant Physiol., 1956, 31, 165. "The rice coleoptile straight growth test for auxin bioassay," Curr. Sci., 1966, "Physiology of virus-infected plants," Ann. Rev. Phytopath., 1963, 1, 197. "Role of oxidative rnatabolism in the localization of plant viruses," Virology, 1960, 12, 408. "The effect of substituted phenols on the activity of IAA-oxidase of Peas" Arch. Biochlm. Biophys., 1953, 43, 358. "Changes in the activities of several enzymes during the germination and seedling development in corn (Zea mays L.)," Plant Physiol., 1953, 30, "Studies on the phytohormone in the malformed portion of the diseased plants. 1. The relation between growth rate and amount of free auxin in the fungus galls and virusinfected plants," Ann. phytopath. Soc. Japan, 1954, 19, 33. "Exogenous gibberellin as responsible for the seedless berry development of grapes. I. Physiological studies on the development of Seedless I~lware grapes," Tohoku J. agric. Res., 1969, 20, 1. "Polyphenoloxidase activity of Nicotiana glutinosa leaves infected with Tobacco mosaic virus," Nature, 1960, 187, "Oxidation of indoleacetic acid by quack Plant Physiol., 1959, 34, 144. grass rhizomes," "Studies on sandal spike. VIII. Polyphenolase activity and metabolism of sandal (Santalum album L.) in health and disease," Prec. Ind. Acad. Sci., 1970, 72B, ~ and Ramaswamy, M.N. "The role of phenolic bodies in the metabolism of sandal (Santalum album L.) in health and disease," J. Sci. Industr. Res., 1061, 20C, 273. Pavillarc, J... "Recherces sur la Croissance des plantes virosees: virus et auxines," C.r. Acad. Sci., 1952, 235, 87. Tomaszewski, M. and "Interactions cf phenolic acids, metallic ions and chelating Thimann, K.V. agents on auxin-induced growth," Plant PhysioL, 1966, 41, van Kammen, A. and ~couwer, D. "Increase of polyphenoloxidase activit3, by a local virus infection in urinocutated parts of leaves," Virology, 1964, 22, 9 r