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Endogenous levels of indoleacetic acid in leaves of tobacco reacting hypersensitively to tobacco mosaic virus
Kurze Mitteilung' Short Communication Department of Plant Physiology, Agricultural University, Wageningen, The Netherlands
Endogenous Levels of Indoleacetic Acid in Leaves of Tobacco Reacting Hypersensitively to Tobacco Mosaic Virus
1. C. VAN LOON and A. TH. BERBEE Received 10 April 1978 . Accepted 24 May 1978
Summary The formation of necrotic local lesions on Samsun NN tobacco leaves after infection with tobacco mosaic virus was accompanied by an increase in the IAA content of the inoculated leaves. After lesion growth had stopped, IAA levels in both inoculated and in non-inoculated, symptomless, virus free leaves did not significantly differ from those in leaves of water-inoculated plants. No connection could be established between IAA level and extent of virus localization. Key words: T MV injection, tobacco leaves, hypersensitive reaction, indoleacetic acid.
Tobacco varieties carrying the NN genes react to infection with tobacco mosaic virus (TMV) with the formation of necrotic local lesions on the inoculated leaves. At 20°, lesions on young expanding leaves attain sizes up to 1 mm in diameter, whereas on leaves ranging from mature to senescent they remain progressively smaller (VAN LOON, 1976). However, when after lesion development such leaves are inoculated for a second time with TMV, no lesions develop in the immediate vicinity of the already existing lesions and lesions that are formed further away remain considerably smaller (Ross, 1961 a). Such acquired resistance is not confined to the originally inoculated leaves, but is likewise manifested in non-inoculated young leaves that neither show symptoms, nor contain demonstrable virus (Ross, 1961 b). In tobacco leaves endogenous levels of auxins are high during rapid expansion and fall gradually to low levels during leaf senescence (WIGHTMAN, 1977). In varieties that develop systemic mosaic symptoms as a result of TMV infection a reduction in IAA content is well established (PAVILLARD, 1955; BEAUCHAMP, 1958; RAJAGOPAL, 1977). Therefore, it became of interest to investigate whether in a tobacco variety reacting with local lesions, this hypersensitive reaction is also accompanied by a decrease in IAA level and whether the slower rate of lesion enlargement resulting in small local lesions in older leaves and in leaves with acquired resistance might be connected with lower levels of endogenous auxin. Z. Pjlanzenphysiol. Bd. 89. S. 373-375. 1978.
374
L. C. VAN LOON and A. TH. BERBEE
Tobacco plants (Nicotiana tabacum L. cv. Samsun NN) were grown from seed in a growth chamber under controlled conditions of 18 to 20 DC temperature, 16 h daylength, 25,000 erg sec-1 cm-2 fluorescent light at plant height and 65 to 80 Ofo relative humidity. After 9 to 11 weeks the leaves were dusted with carborundum and inoculated with purified TMV W VI (100 ,ug/ml) using a gauze pad (VAN LOON and VAN KAMMEN, 1970), followed by a water rinse. Control leaves were similarly inoculated with water only. For IAA determination, three consecutive, almost to just full-grown leaves per plant were harvested, deribbed and frozen in liquid nitrogen. Immediately after liquid evaporation, 5 g was weighed and extracted with methanol, as described by KNEGT and BRUINSMA (1973). Following their procedure, IAA, after purification, was converted to indolo-a-pyrone and determined spectrophotofluorimetrically at 490 nm. Each determination was performed at least in duplicate, and each experiment was repeated at least twice.
When IAA was determined daily after inoculation, the amount in water-inoculated leaves tended to decline, in accordance with the increase in mean leaf age. Except at day 1 after inoculation, the values for TMV-infected leaves were always higher than those .:::Jf water-inoculated leaves (Table 1). Since local lesions became visible about 2 days after inoculation, the appearance of lesions and their subsequent enlargement up to day 7 are accompanied by an increase in IAA content of the leaves. Whereas in leaves developing few lesiQns, IAA content Table 1: Changes in IAA content (ng/g fro wt.) of tobacco leaves after inoculation with water or TMV. Lesions appeared on the leaves 2 days after inoculation with TMV and enlarged up to day 7. Mean values from three experiments. Water-inoculated leaves
Days after inoculation
o
10.2 13.7 9.5 5.7 6.1 8.1 2.8 3.2
1 2
3 4 5 6 7
TMV- inoculated leaves 10.7 12.0 12.4 8.0 20.4 14.8 4.7
Table 2: IAA content (ng/g fro wt.) of leaves from water-inoculated and TMV-inoculated plants. Leaf condition
Water-inoculated plants IAA Number of content experiments
TMV-inoculated plants IAA Number of content experiments
Inoculated leaves 7 days after inoculation
6.3
± 1.9
7
7.0± 1.0
4
Non-inoculated upper leaves 14 days after inoculation
5.3 ± 2.4
6
4.7 ± 2.4
5
Z. Pjlanzenphysiol. Ed. 89. S. 373-375.1978.
Endogenous levels of IAA after TMV infection
375
had increased only 30 Ofo by day 3, IAA levels in leaves covered with lesions had risen up to 270 Ofo by that time. Seven days after TMV-inoculation when lesions stopped to enlarge, the IAA content of these leaves was no longer significantly higher than that of corresponding leaves from water-inoculated plants (Table 2). Another 7 days later, the inoculated leaves had withered and died. At this stage, IAA levels were determined in the leaves directly above, which had acquired systemic resistance. Such leaves possessed IAA levels slightly, but not significantly lower than those of corresponding leaves of water-inoculated plants that had not developed resistance. These observations demonstrate that a reduction in IAA levels accompanying virus infection is nOt a general phenomenon. In contrast to tobacCo:> varieties reacting to TMV with systemic mosaic symptoms, those developing local lesions show an increase in endogenous IAA in the tissues where the virus is active. This increase may well result from the tissue necrotization accompanying lesion enlargement. Since both the inoculated leaves with increased IAA levels and non-inoculated leaves with slightly decreased IAA content have acquired induced resistance to further infection, final lesion size cannot be connected with the level of IAA present at the time of inoculation. Therefore, small lesions on old leaves and on young, systemic resistant leaves, both indicative of enhanced virus localization (VAN LOON, 1976), are not a consequence of low auxin levels. However, application of exogenous auxins does influence lesion size, as will be discussed in a separate communication (VAN LOON, 1978). Acknowledgements The authors thank Dr. E. KNEGT for help and advice on the IAA determination and Ms.]. H. YFF-WOLTERS for careful technical assistance.
References BEAUCHAMP, CH.: Rev. gen. Bot. 65, 477 (1958). KNEGT, E. and]. BRUINSMA: Phytochemistry 12, 753 (1973). PAVILLARD,].: Congr. Sci. Internatl. Tabac 1, 658 (1955). RAJAGOPAL, R.: Z. Pflanzenphysiol. 83,403 (1977). Ross, A. F.: Virology 14, 329 (1961 a). - Virology 14, 340 (1961 b). VAN LOON, L. c.: Physiol. PI. Path. 8, 231 (1976). - Physiol. PI. Path. (1978), in press. VAN LOON, L. C. and A. VAN KAMMEN: Virology 40,199 (1970). WIGHTMAN, F.: In: PILET, P. E. (Ed.): Plant Growth Regulation, Proc. 9th Int. Conf. Plant Growth Substances, Lausanne, 77, Springer-Verlag, Berlin, 1977. L. C. VAN LOON, Department of Plant Physiology, Agricultural University, Arboretumlaan
4, Wageningen, The Netherlands.
Z. P/lanzenphysiol. Ed. 89. S. 373-375. 1978.
Endogenous Levels of Indoleacetic Acid in Leaves of Tobacco Reacting Hypersensitively to Tobacco Mosaic Virus
1. C. VAN LOON and A. TH. BERBEE Received 10 April 1978 . Accepted 24 May 1978
Summary The formation of necrotic local lesions on Samsun NN tobacco leaves after infection with tobacco mosaic virus was accompanied by an increase in the IAA content of the inoculated leaves. After lesion growth had stopped, IAA levels in both inoculated and in non-inoculated, symptomless, virus free leaves did not significantly differ from those in leaves of water-inoculated plants. No connection could be established between IAA level and extent of virus localization. Key words: T MV injection, tobacco leaves, hypersensitive reaction, indoleacetic acid.
Tobacco varieties carrying the NN genes react to infection with tobacco mosaic virus (TMV) with the formation of necrotic local lesions on the inoculated leaves. At 20°, lesions on young expanding leaves attain sizes up to 1 mm in diameter, whereas on leaves ranging from mature to senescent they remain progressively smaller (VAN LOON, 1976). However, when after lesion development such leaves are inoculated for a second time with TMV, no lesions develop in the immediate vicinity of the already existing lesions and lesions that are formed further away remain considerably smaller (Ross, 1961 a). Such acquired resistance is not confined to the originally inoculated leaves, but is likewise manifested in non-inoculated young leaves that neither show symptoms, nor contain demonstrable virus (Ross, 1961 b). In tobacco leaves endogenous levels of auxins are high during rapid expansion and fall gradually to low levels during leaf senescence (WIGHTMAN, 1977). In varieties that develop systemic mosaic symptoms as a result of TMV infection a reduction in IAA content is well established (PAVILLARD, 1955; BEAUCHAMP, 1958; RAJAGOPAL, 1977). Therefore, it became of interest to investigate whether in a tobacco variety reacting with local lesions, this hypersensitive reaction is also accompanied by a decrease in IAA level and whether the slower rate of lesion enlargement resulting in small local lesions in older leaves and in leaves with acquired resistance might be connected with lower levels of endogenous auxin. Z. Pjlanzenphysiol. Bd. 89. S. 373-375. 1978.
374
L. C. VAN LOON and A. TH. BERBEE
Tobacco plants (Nicotiana tabacum L. cv. Samsun NN) were grown from seed in a growth chamber under controlled conditions of 18 to 20 DC temperature, 16 h daylength, 25,000 erg sec-1 cm-2 fluorescent light at plant height and 65 to 80 Ofo relative humidity. After 9 to 11 weeks the leaves were dusted with carborundum and inoculated with purified TMV W VI (100 ,ug/ml) using a gauze pad (VAN LOON and VAN KAMMEN, 1970), followed by a water rinse. Control leaves were similarly inoculated with water only. For IAA determination, three consecutive, almost to just full-grown leaves per plant were harvested, deribbed and frozen in liquid nitrogen. Immediately after liquid evaporation, 5 g was weighed and extracted with methanol, as described by KNEGT and BRUINSMA (1973). Following their procedure, IAA, after purification, was converted to indolo-a-pyrone and determined spectrophotofluorimetrically at 490 nm. Each determination was performed at least in duplicate, and each experiment was repeated at least twice.
When IAA was determined daily after inoculation, the amount in water-inoculated leaves tended to decline, in accordance with the increase in mean leaf age. Except at day 1 after inoculation, the values for TMV-infected leaves were always higher than those .:::Jf water-inoculated leaves (Table 1). Since local lesions became visible about 2 days after inoculation, the appearance of lesions and their subsequent enlargement up to day 7 are accompanied by an increase in IAA content of the leaves. Whereas in leaves developing few lesiQns, IAA content Table 1: Changes in IAA content (ng/g fro wt.) of tobacco leaves after inoculation with water or TMV. Lesions appeared on the leaves 2 days after inoculation with TMV and enlarged up to day 7. Mean values from three experiments. Water-inoculated leaves
Days after inoculation
o
10.2 13.7 9.5 5.7 6.1 8.1 2.8 3.2
1 2
3 4 5 6 7
TMV- inoculated leaves 10.7 12.0 12.4 8.0 20.4 14.8 4.7
Table 2: IAA content (ng/g fro wt.) of leaves from water-inoculated and TMV-inoculated plants. Leaf condition
Water-inoculated plants IAA Number of content experiments
TMV-inoculated plants IAA Number of content experiments
Inoculated leaves 7 days after inoculation
6.3
± 1.9
7
7.0± 1.0
4
Non-inoculated upper leaves 14 days after inoculation
5.3 ± 2.4
6
4.7 ± 2.4
5
Z. Pjlanzenphysiol. Ed. 89. S. 373-375.1978.
Endogenous levels of IAA after TMV infection
375
had increased only 30 Ofo by day 3, IAA levels in leaves covered with lesions had risen up to 270 Ofo by that time. Seven days after TMV-inoculation when lesions stopped to enlarge, the IAA content of these leaves was no longer significantly higher than that of corresponding leaves from water-inoculated plants (Table 2). Another 7 days later, the inoculated leaves had withered and died. At this stage, IAA levels were determined in the leaves directly above, which had acquired systemic resistance. Such leaves possessed IAA levels slightly, but not significantly lower than those of corresponding leaves of water-inoculated plants that had not developed resistance. These observations demonstrate that a reduction in IAA levels accompanying virus infection is nOt a general phenomenon. In contrast to tobacCo:> varieties reacting to TMV with systemic mosaic symptoms, those developing local lesions show an increase in endogenous IAA in the tissues where the virus is active. This increase may well result from the tissue necrotization accompanying lesion enlargement. Since both the inoculated leaves with increased IAA levels and non-inoculated leaves with slightly decreased IAA content have acquired induced resistance to further infection, final lesion size cannot be connected with the level of IAA present at the time of inoculation. Therefore, small lesions on old leaves and on young, systemic resistant leaves, both indicative of enhanced virus localization (VAN LOON, 1976), are not a consequence of low auxin levels. However, application of exogenous auxins does influence lesion size, as will be discussed in a separate communication (VAN LOON, 1978). Acknowledgements The authors thank Dr. E. KNEGT for help and advice on the IAA determination and Ms.]. H. YFF-WOLTERS for careful technical assistance.
References BEAUCHAMP, CH.: Rev. gen. Bot. 65, 477 (1958). KNEGT, E. and]. BRUINSMA: Phytochemistry 12, 753 (1973). PAVILLARD,].: Congr. Sci. Internatl. Tabac 1, 658 (1955). RAJAGOPAL, R.: Z. Pflanzenphysiol. 83,403 (1977). Ross, A. F.: Virology 14, 329 (1961 a). - Virology 14, 340 (1961 b). VAN LOON, L. c.: Physiol. PI. Path. 8, 231 (1976). - Physiol. PI. Path. (1978), in press. VAN LOON, L. C. and A. VAN KAMMEN: Virology 40,199 (1970). WIGHTMAN, F.: In: PILET, P. E. (Ed.): Plant Growth Regulation, Proc. 9th Int. Conf. Plant Growth Substances, Lausanne, 77, Springer-Verlag, Berlin, 1977. L. C. VAN LOON, Department of Plant Physiology, Agricultural University, Arboretumlaan
4, Wageningen, The Netherlands.
Z. P/lanzenphysiol. Ed. 89. S. 373-375. 1978.