- Email: [email protected]
The influence of kinetin on tobacco ringspot virus infectivity and the effect of virus infection on the cytokinin activity in intact leaves of Nicotiana glutinosa L
Physiotogical Plant Path&~
(1979) 14,227-233
The influence of kinetin on tobacco ringspot virus infectivity and the effect of virus infection on the cytokinin activity in intact leaves of Nicotiana gluti’nosa L. S. M. TAVANTZIS,
S. H. SMITH
Department of Plant Patholoa, The Petvlrylvania Stat4 UniversiQ, lJnivcrsity Park, PA 16802, U.S.A.
and F. H. WITHAM Department of BioloD, Tlvz Pennsylvania Statc University, University Park, PA 16802, U.S.A. (Acceptedfor publication September1978)
Intact tobacco leaves sprayed daily with kinetin (0.1 mg 1-l) begining 9 days prior to inoculation with tobacco ringspot virus (TRSV) exhibited high levels of virus infectivity whereas leaves sprayed as above with higher levels of kinetin (1.0 mg 1-l and 10.0 mg 1-l) exhibited appreciably lower levels of virus than the control plants. Conversely, the extent of viral infectivity in tobacco leaves, sprayed daily with kinetin at the three different concentrations (0.1, 1 .O and 10.0 mg 1-r) beginning 3 days prior to inoculation, showed no appreciable difference in the pattern of virus infectivity from that of non-sprayed inoculated leaves. Extracts of infected and non-infected tobacco leaves were assayed for cytokinin activity by the radish cotyledon expansion test. Leaf extracts of TRSV-infected plants contained less cytokinin activity than leaf extracts from non-infected plants.
INTRODUCTION
The synthetic cytokinins, 6-benzylaminopurine (6-BAP) and 6-furfurylaminopurine (kinetin) are known to influence the production of viruses in certain host systems [I, 7, 9, 15, IS, 221. Kiraly & Szirmai [7] reported that tobacco mosaic virus (TMV) production was inhibited in .Nicotiana glutinosa L. leaf discs when they were placed in kinetin (50 mg 1-r) immediately after inoculation whereas no inhibition was apparent when discs of TMV-infected leaves of N. glutinosa were incubated first in water and then 18 or 24 h after inoculation in kinetin solutions. Pre-treatment of the whole leaf with kinetin prior to leaf disc preparation seemed to be necessary for maximum inhibition. Kiraly & Szirmai [7] reported that kinetin did not influence infectivity in leaves of intact IV. glutinosa plants but only in the detached leaf discs. Opel [16] observed that kinetin pre-treatment of bean leaf discs inhibited necrosis and tobacco necrosis virus production after inoculation. The development of fewer and smaller lesions was observed also when petunia leaf strips were floated on kinetin solution for 16 h prior to inoculation with tomato spotted wilt virus [22]. Aldwinckle & Selman [I] showed that 6-BAP was as effective as kinetin in decreasing Ithe number of local lesions in petunia leaf strips inoculated with tomato spotted wilt 0048-4059/79/020227
+ 07 $02.00/O
@ 1979 Academic Press Inc. (London)
Limited
228
S. M. Tavantzis, S. H. Smith and F. H. Witham
virus (TSWV) and that 6-BAP was more effective than kinetin when supplied through the petioles of excised whole leaves. The number of local lesions and infectivity of TSWV produced in detached leaves of Mcotiana rustica decreased when 6-BAP was applied before and after inoculation, Lesions and infectivity decreased in attached tobacco leaves treated 9 days prior to inoculation while increased infectivity was observed in attached leaves treated with 6-BAP after inoculation. Daft [3] previously reported that kinetin either stimulated or inhibited tomato aucuba mosaic virus (TAMV) synthesis in attached leaves of N. glutinosa depending upon the amount of cytokinin applied and the age of the leaves. The above-mentioned results, and the findings of Kuriger & Agrios [9] that root exudates of TRSV-infected cowpea plants contained less cytokinin activity than root exudates of non-infected plants, provide evidence for the involvement of cytokinins in virus infectivity. The results reported herein provide further information concerning the influence of kinetin on TRSV infectivity and the effect of viral infection on the levels of naturally occurring cytokinins in intact leaves of N. glutinosa. MATERIALS
AND
METHODS
Determination of virus infectiv$y
Twelve-day-old Nicotiana glutinosa L. seedlings, planted in 1000 ml pots containing a steam-treated loam : perlite : peat (1 : 1 : 1) soil mixture, were sprayed with water or an aqueous solution of kinetin at the following concentrations: 0.1, 1-O or 10.0 mg 1-i. Depending upon the experiment, five plants per treatment were sprayed either 3 or 9 days prior to TRSV inoculation. The infectivity of the inoculum used to infect the test plants was about 25 local lesions per half leaf. After the initial application of water or kinetin solution, the plants were sprayed daily with the respective solutions. The entire leaf surface area of each plant was sprayed to avoid the preferential translocation of nutrients to “kinetin loci” [13]. Cowpea (Vigna sinews [Torner Savi.] “Big boy”) served as the local lesion host. Inoculum consisted of leaf discs, 1 cm in diameter, taken from the same leaf of each tobacco plant for the entire duration of the experiment, and ground in 1 ml of 0.05 M potassium phosphate buffer pH 7.2. Greenhouse temperature and relative humidity were maintained at approximately 21 “C and 40%, respectively.
The extraction and bioassay of naturally occurring cytokinins
Cytokinins were extracted from TRSV-infected leaves 10 days after infection and non-infected leaves of Jv. glutinosa using the methods of Krasnuk et al. [8]. The 1-butanol extracts were applied as a streak on Whatman No. 1 paper. The chromatograms were developed in an ascending fashion in water-saturated set-butanol (WSB). Preliminary investigations indicated that the use of 1-butanol extracts equivalent to 20 g fresh wt of leaf tissue extracted, produced the best resolution and that the cytokinin activity was localized within the O-60 to O-90 R, regions of the developed chromatograms. Cytokinin activity was determined by the radish cotyledon expansion test following the basic methods of Letham [IO]. Uniform cotyledons, approximately 7 to 9 mg fresh wt, were placed in Petri dishes containing strips of paper cut from
Tobacco ringspot virus infectivity
229
developed chromatograms on which the naturally occurring cytokinins were separated and which corresponded to specific R, regions. The strips were previously moistened with 3 ml of 0.002 M potassium phosphate buffer pH 6.0. Cotyledons placed in the same manner on filter paper strips of the same size as those taken from the chromatograms and moistened with 3 ml of buffer were used as controls for the detection of growth inhibitors possibly present in the chromatographed extracts. The covered Petri dishes containing the cotyledons were placed in a growth chamber under continuous fluorescent lighting of approximately 450 lx at 25 “C. After 72 h the c:otyledons were removed from the dishes, blotted and weighed immediately. RESULTS
Figure 1 summarizes the effects of kinetin (0.1 or 1-O mg 1-l) beginning 9 days prior to inoculation on TRSV infectivity. Plants sprayed with kinetin (0.1 mg l-1) exhibited a substantial increase in TRSV infectivity whereas in plants sprayed with 100
SO .F
70
s =
60
z % x-
40
50
$33
Days after incculotion FIG. 1. Local lesion bioassay. Viral infectivity expressed as a number of local lesions per half leaf of each cowpea test plant inoculated with extracts, taken at the indicated times, from intact tobacco leaves. Tobacco leaves were sprayed with water (controls, l ) or kinetin [O-1 mg 1-r (0) or 1*O mg 1-r ( q I)] ; daily beginning 9 days prior to inoculation and continued during the course of the experiment. These graphs represent data obtained in one experiment. Similar results were obtained in the other three replications.
kinetin at higher concentrations (1-O mg 1-l) the virus infectivity was considerably lower than the controls. In plants sprayed with kinetin at 10-O mg l-l, 9 days prior to inoculation, TRSV infectivity was similarly suppressed. Although not illustrated here, tobacco plants sprayed daily with kinetin (O-1, 1-O or 10-O mg 1-l) 3 days prior IO inoculation and for the remainder of the experimental time did not appear to influence virus infectivity as evidenced by similar patterns of virus infectivity in both the treated and non-treated inoculated plants. Kinetin did not activate or inactivate the virus per se. If the various kinetin solutions were incubated with virus suspensions prior to inoculation there was no effect on the virus infectivity (Table 1). Kinetin had no phytotoxic effects, nor did it inhibit the growth of plants (Table 2).
S. M. Tavantzis, S. H. Smith and F. H. Witham
230 TABLE
Effect of kin&
1
on Tobacco Ringskot Virus infectivity aftir mixing and incubating kin&in and virus solutionsfor 10 min prior to inoculation Number of local lesions per half leaf of Vigna sinensis Kin&n Kinetin Kinetin Control (10.0 mg 1-r) (0.1 mg 1-r) ( 1 .O mg 1-l)
Replication 1 2 3 Means
26.83 25.33 17.16 23.11
21.66 29.00 18.33 23.00
TABLE
24.50 32.16 16.50 24.39
23.33 30.66 17.16 23.72
2
Effects of kinetin treatments on the growth of N. glutinosa plants
Concentration of kinetin (w l-9 Control 0.1 1.0 10.0 0.1 1.0 10.0
Time of spraying prior to inoculation (days) 3 3 3 9 9 9
Dry wt of roots” (9)
Dry wt of stems and leave9 (9)
Height of stems” (4
Number of flowers per planta
3.06 3.24 3.26* 2.98 2.84 2,36b 2.96
9.34 10.54c 10.26c 10.34c 10.5oc 9.74 9.40
47.20 50.60 46.00 46.60 48.20 44.20 40.60
12.40 12.80 13.20 12.80 12.80 12.20 10.80
a Means of four replications, five plants per treatment. Two-way conducted; means were compared by the L.S.D. method. * Significantly lower than control at P = 0.05. c Significantly higher than control at P = 0.05.
analysis of variance was
Chromatographic separation of the cytokinin(s) occurring naturally in the leaves of virus-infected and non-infected tobacco plants and subsequent bioassays indicated that the cytokinin activity migrated to R, regions 0.60 to 0.90 in the WSB solvent (Fig. ‘2). Although the cytokinins from the two sets of plants were chromatographically similar, leaf extracts of infected plants contained less cytokinin activity than did those of non-infected plants. An approximate 26% reduction in fresh wt of cotyledons placed on leaf extracts of virus-infected plants was evident when 12 similar but separate extracts were bioassayed before and after chromatography.
DISCUSSION The results clearly indicate that the stimulation or inhibition of TRSV infectivity in intact leaves of N. glutinosa depends upon the kinetin concentration of solutions applied daily 9 days prior to and continued after inoculation. The lowest concentration of kinetin (0.1 mg 1-l) stimulated TRSV infectivity whereas kinetin concentrations of 1.0 and 10.0 mg 1-r inhibited TRSV infectivity in intact tobacco leaves (Fig. 1). Similarly, Reunov et al. [19] observed that kinetin (10 or 25 mg 1-l) significantly inhibited the replication of tobacco mosaic virus (TMV) and potato
‘Tobacco ringspot virus infectivity
231
32 30 28 26 -Z 8 24 5
22
5 i
20 18
r : ,’
16 14 12
tE
IO 8 6 4 2 I.
R, Regions
;I ‘-9
I .’
FIG. 2. Radish cotyledon bioassays of chromatograms of 1-butanol tobacco leaf extracts equivalent to 20 g fresh wt of tissue. The solvent used was water-saturated set-butanol. The bioassay was replicated 12 times using different sets of plants. The fresh wt per cotyledon varied from 22.07 to 25.71 (average 23.9 mg, s.e. 3.30) and from 31.56 to 34.44 mg (av. 32.45 mg, s.e. 2.71) for the cotyledons placed on extracts from infected and healthy plants respectively. The above values refer to RF region 0.7 to 0.8. The average fresh weight per cotyledon was 18.15 mg (s.e. 1.67) for cotyledons placed on unused paper and 17.82 (s.e. 2.86) for cotyledons placed on chromatogram strips cut from up to 2 c m below the starting line. H, Non-infected plants; q , infected plants.
virus X (PVX), evaluated in terms of infectivity of tissue homogenates, in upper leaves of N. tabacum and Datura stramonium, respectively. In their experiments, kinetin (10 mg 1-r) did not affect the virus inhibiting properties of extracts thus, the kinetin:induced decrease of infectivity in tobacco leaves apparently reflects the inhibition of virus replication by the phytohormone. Daft [3] originally observed that kinetin stimulated the production of tomato aucuba mosaic virus (TAMV) in detached tobacco leaves and that in attached leaves kinetin either stimulated or inhibited TAMV synthesis depending upon the quantity applied and the age of the leaves. YKiraly & Szirmai [7] found that previous daily spraying of the leaves of N. glutinosa Iplants with kinetin (50 mg 1-r) for 5 days did not result in inhibition of virus infection. We, as well as other investigators [19, 221 found kinetin (50 mg 1-l) to be phytotoxic. ‘Thus past results based upon the use of highly concentrated kinetin solutions (30 to 100 mg 1-l) may be quite erroneous due to the low solubility of kinetin in water and the difficulties encountered in keeping the kinetin in solution. Also, daily spraying of inoculated plants seems to be necessary for inhibition of virus replication. Future studies designed to assess the rate of uptake of radioactively labeled kinetin by intact tobacco leaves prior to and after inoculation should indicate the amount of kinetin incorporated that is necessary to enhance or inhibit virus replication. 16
232
S. M. Tavantris, S. H. Smith and F. H. Wltham
It has been shown that kinetin induces increases in cell protein and nucleic acid levels [4, 17, 211. Murai et al. [II] have found that kinetin was incorporated as N”-furfuryladenosine(frsA) in the rRNA and tRNA preparations from tobacco callus. The rRNA contained over 90% of these fr6A moieties. They suggest that incorporation by this route may involve either specific transcriptional or posttranscriptional errors. Berridge et al. [2] as well as other workers [5] have found that kinetin binds to plant ribosomes. According to Berridge et al. [2], in other systems binding of small molecules to ribosomes has been shown to have functional significance. Streptomycin, tetracycline, actidione, puromycin and many other drugs bind to ribosomes and modify ribosomal processes. Frequently this type of binding, as in the case of kinetin, appears to be reversible and concentration dependent. These findings, in conjunction with our observation that daily kinetin (1 to 10 mg 1-r) spraying starting 9 days prior to inoculation and throughout the duration of the experiment is necessary for suppression of virus infectivity (Fig. l), would suggest a working hypothesis which might be considered in future experimentation. The continuous “occupation” or modification of a large number of virus replication sites, such as ribosomes, by kinetin might hinder virus replication or infectivity to a considerable extent. In plants sprayed with kinetin (1 or 10 mg 1-l) starting 3 days prior to inoculation, no inhibition of TRW infectivity was observed possibly because an adequate number of unaffected virus replication sites still existed in the cell at that time. On the contrary, low kinetin concentrations (i.e. 0.1 mg 1-l) not only would be insufficient to cause blockage or modification of a large number of virus replication sites but would favor virus replication (Fig. 1) by inducing accumulation ofmetabolites, such as amino acids [13], essential for virus synthesis. The possibility of kinetin affecting virus synthesis or infectivity indirectly cannot be overlooked since it is possible that kinetin can interact with DNA [S, 11, 18, 20, 241, thus inducing transcriptional changes or permitting initiation and transcription of new DNA. Another indicator of the cytokinin relationship in TRSV infection in tobacco leaves is that leaf extracts of infected plants contained less cytokinin activity than did those of non-infected plants (Fig. 2). One assay of leaf extracts at 10 days after inoculation, when TRSV is approaching its maximum concentration, was considered to provide reliable information as far as the relationship of TRSV infection to cytokinin activity. Kuriger & Agrios [9] also demonstrated cytokinin levels were lower in extracts from root exudates and root tissue of TRSV-infected cowpeas than in those of non-infected plants. Although we have not identified the cytokinins present in tobacco leaves, they appear to be chromatographically similar to zeatin and/or zeatin ribonucleoside [S, 231. Additional information concerning the chemical nature of the endogenous cytokinins, their metabolism and translocation in TRSV-infected and non-infected tobacco leaves should provide greater insights into the role of these phytohormones and the control of virus replication and infectivity in intact plants. REFERENCES 1. ALDWINCKLE, H. S. & SELMAN, I. W. (1967). Some effects of supplying benzyladenine to leaves and plants inoculated with viruses. Annals of Applied Biology 60, 49-58. 2. BERRIDGE, M. V., F~ALPH, R. K. & LETHAM, D. S. (1970). The binding of kmetin to plant ribosomes. Biochemistry Journal 119, 75-84.
Tobacco ringspot virus infectivity
233 3. DAFT, M. J. (1965). Some interactions of kinetin and temperatures on tobacco leaves infected with tomato aucuba mosaic virus. Annals of Applied Biology 55, 51-56. Die Veranderung des Stiirke-, Protein- und RNS-geshaltes von bmna minor L. unter dem Einlluss von Kinetin (6-Furfurylaminopurin). Experimentia 23, 621-622. Fox, J. E. & ERION, J. L. (1975). A cytokinin binding protein from higher plant ribosomes. Biochemistry and Biophysics ResearchCommunications64, 694-700. HENDRY, L. B., WITHAM, F. H. & CHAPMAN, 0. L. (1977). Ge ne regulation: the involvement of stereochemical recognition in DNA-small molecule interactions. PerspcGtiuesin Bioloo and Medicine 21, 120-130. KIRALI, Z. & SZIRMAI,J. (1964). The influence of kinetin on tobacco mosaic virus production in Nicotiana glutirwsa leaf discs. Virology 23, 286-288. KRASNUK,M., WITHAM, F. H. & TEGLEY, J. R. (1971). Cytokinins extracted from pinto bean fruit. Plant Physiology48, 320-324. KURIGER, W. E. & AGRIOS,G. N. (1977). Cytokinm levels-and kinetin-virus interactions in tobacco ringspot virus-infected cowpeas. Phytojathology 67, 604-609. LETHAM, D. S. (1971). A cytokinin bioassay using excised radish cotyledons. Physiologia Plantarum
4. ERISMANN, K. H. & FANKHAUSER, M. (1967). 5.
6. 7. 8. 9. 10.
25, 391-396. 11. MATTHYSSE,A. G. & A~RAMS,M. (1970). A factor mediating interaction of kinins with the genetic material. Biochima et Biophysics Acta 190, 511-518. 12. MILLER, C. 0. & WITHAM, F. H. (1964). A kinetin-like factor from maize and other sources. Colloques internationaux Centn national recherchescientifique123, I-VI. 13. MOTHES, K. & ENGELBRECHT,L. (1961). Kinetin-induced directed transport of substances in excised leaves in the dark. Phytochemistry1, 58-62. 14. MURAI, N., TALLER, B. J., ARMSTRONG,D. J. & SKOOG, F. (1977). Kinetin incorporated into tobacco callus ribosomal RNA and transfer RNA preparations. Plant Physiology60, 197-202. 15. NAKAZAKI, Y. (197 1). Effect of kinetin on local lesion formation on detached bean leaves inoculated with tobacco mosaic virus or its nucleic acid. Annals of the Phytopathological Society (Japan) 37, 307-309. 16. OPEL, H. (1964). Wirkungen von Kinetin auf Virus-turd Mehltauinfizierte Pflanzen. Monatsberichte der Deutschen Akademie der Wissenschaftenzu Berlin 6, 276. 17. OSBORNE,D. (1962). Effect of kinetin on protein and nucleic acid metabolism in Xanthium leaves during senescence. Plant Physiolor3,37, 595-602. 18. PIATTELLI, M. M. GIIJDICI DE NICOLA & CASTROGIOVANNI,V. (1971). The effect of kmetin on amaranthin synthesis in Amaranthus tricolor in darkness. Phytochemistry10, 289-293. 19. RECNOV, A. V., REUNOVA, G. D., VASILYEVA, L. A. & REIFMAN, V. G. (1977). Effect of kinetin on tobacco mosaic virus and potato virus X replication in leaves of systemic hosts. Phytopathologische
r6 *
(1979) 14,227-233
The influence of kinetin on tobacco ringspot virus infectivity and the effect of virus infection on the cytokinin activity in intact leaves of Nicotiana gluti’nosa L. S. M. TAVANTZIS,
S. H. SMITH
Department of Plant Patholoa, The Petvlrylvania Stat4 UniversiQ, lJnivcrsity Park, PA 16802, U.S.A.
and F. H. WITHAM Department of BioloD, Tlvz Pennsylvania Statc University, University Park, PA 16802, U.S.A. (Acceptedfor publication September1978)
Intact tobacco leaves sprayed daily with kinetin (0.1 mg 1-l) begining 9 days prior to inoculation with tobacco ringspot virus (TRSV) exhibited high levels of virus infectivity whereas leaves sprayed as above with higher levels of kinetin (1.0 mg 1-l and 10.0 mg 1-l) exhibited appreciably lower levels of virus than the control plants. Conversely, the extent of viral infectivity in tobacco leaves, sprayed daily with kinetin at the three different concentrations (0.1, 1 .O and 10.0 mg 1-r) beginning 3 days prior to inoculation, showed no appreciable difference in the pattern of virus infectivity from that of non-sprayed inoculated leaves. Extracts of infected and non-infected tobacco leaves were assayed for cytokinin activity by the radish cotyledon expansion test. Leaf extracts of TRSV-infected plants contained less cytokinin activity than leaf extracts from non-infected plants.
INTRODUCTION
The synthetic cytokinins, 6-benzylaminopurine (6-BAP) and 6-furfurylaminopurine (kinetin) are known to influence the production of viruses in certain host systems [I, 7, 9, 15, IS, 221. Kiraly & Szirmai [7] reported that tobacco mosaic virus (TMV) production was inhibited in .Nicotiana glutinosa L. leaf discs when they were placed in kinetin (50 mg 1-r) immediately after inoculation whereas no inhibition was apparent when discs of TMV-infected leaves of N. glutinosa were incubated first in water and then 18 or 24 h after inoculation in kinetin solutions. Pre-treatment of the whole leaf with kinetin prior to leaf disc preparation seemed to be necessary for maximum inhibition. Kiraly & Szirmai [7] reported that kinetin did not influence infectivity in leaves of intact IV. glutinosa plants but only in the detached leaf discs. Opel [16] observed that kinetin pre-treatment of bean leaf discs inhibited necrosis and tobacco necrosis virus production after inoculation. The development of fewer and smaller lesions was observed also when petunia leaf strips were floated on kinetin solution for 16 h prior to inoculation with tomato spotted wilt virus [22]. Aldwinckle & Selman [I] showed that 6-BAP was as effective as kinetin in decreasing Ithe number of local lesions in petunia leaf strips inoculated with tomato spotted wilt 0048-4059/79/020227
+ 07 $02.00/O
@ 1979 Academic Press Inc. (London)
Limited
228
S. M. Tavantzis, S. H. Smith and F. H. Witham
virus (TSWV) and that 6-BAP was more effective than kinetin when supplied through the petioles of excised whole leaves. The number of local lesions and infectivity of TSWV produced in detached leaves of Mcotiana rustica decreased when 6-BAP was applied before and after inoculation, Lesions and infectivity decreased in attached tobacco leaves treated 9 days prior to inoculation while increased infectivity was observed in attached leaves treated with 6-BAP after inoculation. Daft [3] previously reported that kinetin either stimulated or inhibited tomato aucuba mosaic virus (TAMV) synthesis in attached leaves of N. glutinosa depending upon the amount of cytokinin applied and the age of the leaves. The above-mentioned results, and the findings of Kuriger & Agrios [9] that root exudates of TRSV-infected cowpea plants contained less cytokinin activity than root exudates of non-infected plants, provide evidence for the involvement of cytokinins in virus infectivity. The results reported herein provide further information concerning the influence of kinetin on TRSV infectivity and the effect of viral infection on the levels of naturally occurring cytokinins in intact leaves of N. glutinosa. MATERIALS
AND
METHODS
Determination of virus infectiv$y
Twelve-day-old Nicotiana glutinosa L. seedlings, planted in 1000 ml pots containing a steam-treated loam : perlite : peat (1 : 1 : 1) soil mixture, were sprayed with water or an aqueous solution of kinetin at the following concentrations: 0.1, 1-O or 10.0 mg 1-i. Depending upon the experiment, five plants per treatment were sprayed either 3 or 9 days prior to TRSV inoculation. The infectivity of the inoculum used to infect the test plants was about 25 local lesions per half leaf. After the initial application of water or kinetin solution, the plants were sprayed daily with the respective solutions. The entire leaf surface area of each plant was sprayed to avoid the preferential translocation of nutrients to “kinetin loci” [13]. Cowpea (Vigna sinews [Torner Savi.] “Big boy”) served as the local lesion host. Inoculum consisted of leaf discs, 1 cm in diameter, taken from the same leaf of each tobacco plant for the entire duration of the experiment, and ground in 1 ml of 0.05 M potassium phosphate buffer pH 7.2. Greenhouse temperature and relative humidity were maintained at approximately 21 “C and 40%, respectively.
The extraction and bioassay of naturally occurring cytokinins
Cytokinins were extracted from TRSV-infected leaves 10 days after infection and non-infected leaves of Jv. glutinosa using the methods of Krasnuk et al. [8]. The 1-butanol extracts were applied as a streak on Whatman No. 1 paper. The chromatograms were developed in an ascending fashion in water-saturated set-butanol (WSB). Preliminary investigations indicated that the use of 1-butanol extracts equivalent to 20 g fresh wt of leaf tissue extracted, produced the best resolution and that the cytokinin activity was localized within the O-60 to O-90 R, regions of the developed chromatograms. Cytokinin activity was determined by the radish cotyledon expansion test following the basic methods of Letham [IO]. Uniform cotyledons, approximately 7 to 9 mg fresh wt, were placed in Petri dishes containing strips of paper cut from
Tobacco ringspot virus infectivity
229
developed chromatograms on which the naturally occurring cytokinins were separated and which corresponded to specific R, regions. The strips were previously moistened with 3 ml of 0.002 M potassium phosphate buffer pH 6.0. Cotyledons placed in the same manner on filter paper strips of the same size as those taken from the chromatograms and moistened with 3 ml of buffer were used as controls for the detection of growth inhibitors possibly present in the chromatographed extracts. The covered Petri dishes containing the cotyledons were placed in a growth chamber under continuous fluorescent lighting of approximately 450 lx at 25 “C. After 72 h the c:otyledons were removed from the dishes, blotted and weighed immediately. RESULTS
Figure 1 summarizes the effects of kinetin (0.1 or 1-O mg 1-l) beginning 9 days prior to inoculation on TRSV infectivity. Plants sprayed with kinetin (0.1 mg l-1) exhibited a substantial increase in TRSV infectivity whereas in plants sprayed with 100
SO .F
70
s =
60
z % x-
40
50
$33
Days after incculotion FIG. 1. Local lesion bioassay. Viral infectivity expressed as a number of local lesions per half leaf of each cowpea test plant inoculated with extracts, taken at the indicated times, from intact tobacco leaves. Tobacco leaves were sprayed with water (controls, l ) or kinetin [O-1 mg 1-r (0) or 1*O mg 1-r ( q I)] ; daily beginning 9 days prior to inoculation and continued during the course of the experiment. These graphs represent data obtained in one experiment. Similar results were obtained in the other three replications.
kinetin at higher concentrations (1-O mg 1-l) the virus infectivity was considerably lower than the controls. In plants sprayed with kinetin at 10-O mg l-l, 9 days prior to inoculation, TRSV infectivity was similarly suppressed. Although not illustrated here, tobacco plants sprayed daily with kinetin (O-1, 1-O or 10-O mg 1-l) 3 days prior IO inoculation and for the remainder of the experimental time did not appear to influence virus infectivity as evidenced by similar patterns of virus infectivity in both the treated and non-treated inoculated plants. Kinetin did not activate or inactivate the virus per se. If the various kinetin solutions were incubated with virus suspensions prior to inoculation there was no effect on the virus infectivity (Table 1). Kinetin had no phytotoxic effects, nor did it inhibit the growth of plants (Table 2).
S. M. Tavantzis, S. H. Smith and F. H. Witham
230 TABLE
Effect of kin&
1
on Tobacco Ringskot Virus infectivity aftir mixing and incubating kin&in and virus solutionsfor 10 min prior to inoculation Number of local lesions per half leaf of Vigna sinensis Kin&n Kinetin Kinetin Control (10.0 mg 1-r) (0.1 mg 1-r) ( 1 .O mg 1-l)
Replication 1 2 3 Means
26.83 25.33 17.16 23.11
21.66 29.00 18.33 23.00
TABLE
24.50 32.16 16.50 24.39
23.33 30.66 17.16 23.72
2
Effects of kinetin treatments on the growth of N. glutinosa plants
Concentration of kinetin (w l-9 Control 0.1 1.0 10.0 0.1 1.0 10.0
Time of spraying prior to inoculation (days) 3 3 3 9 9 9
Dry wt of roots” (9)
Dry wt of stems and leave9 (9)
Height of stems” (4
Number of flowers per planta
3.06 3.24 3.26* 2.98 2.84 2,36b 2.96
9.34 10.54c 10.26c 10.34c 10.5oc 9.74 9.40
47.20 50.60 46.00 46.60 48.20 44.20 40.60
12.40 12.80 13.20 12.80 12.80 12.20 10.80
a Means of four replications, five plants per treatment. Two-way conducted; means were compared by the L.S.D. method. * Significantly lower than control at P = 0.05. c Significantly higher than control at P = 0.05.
analysis of variance was
Chromatographic separation of the cytokinin(s) occurring naturally in the leaves of virus-infected and non-infected tobacco plants and subsequent bioassays indicated that the cytokinin activity migrated to R, regions 0.60 to 0.90 in the WSB solvent (Fig. ‘2). Although the cytokinins from the two sets of plants were chromatographically similar, leaf extracts of infected plants contained less cytokinin activity than did those of non-infected plants. An approximate 26% reduction in fresh wt of cotyledons placed on leaf extracts of virus-infected plants was evident when 12 similar but separate extracts were bioassayed before and after chromatography.
DISCUSSION The results clearly indicate that the stimulation or inhibition of TRSV infectivity in intact leaves of N. glutinosa depends upon the kinetin concentration of solutions applied daily 9 days prior to and continued after inoculation. The lowest concentration of kinetin (0.1 mg 1-l) stimulated TRSV infectivity whereas kinetin concentrations of 1.0 and 10.0 mg 1-r inhibited TRSV infectivity in intact tobacco leaves (Fig. 1). Similarly, Reunov et al. [19] observed that kinetin (10 or 25 mg 1-l) significantly inhibited the replication of tobacco mosaic virus (TMV) and potato
‘Tobacco ringspot virus infectivity
231
32 30 28 26 -Z 8 24 5
22
5 i
20 18
r : ,’
16 14 12
tE
IO 8 6 4 2 I.
R, Regions
;I ‘-9
I .’
FIG. 2. Radish cotyledon bioassays of chromatograms of 1-butanol tobacco leaf extracts equivalent to 20 g fresh wt of tissue. The solvent used was water-saturated set-butanol. The bioassay was replicated 12 times using different sets of plants. The fresh wt per cotyledon varied from 22.07 to 25.71 (average 23.9 mg, s.e. 3.30) and from 31.56 to 34.44 mg (av. 32.45 mg, s.e. 2.71) for the cotyledons placed on extracts from infected and healthy plants respectively. The above values refer to RF region 0.7 to 0.8. The average fresh weight per cotyledon was 18.15 mg (s.e. 1.67) for cotyledons placed on unused paper and 17.82 (s.e. 2.86) for cotyledons placed on chromatogram strips cut from up to 2 c m below the starting line. H, Non-infected plants; q , infected plants.
virus X (PVX), evaluated in terms of infectivity of tissue homogenates, in upper leaves of N. tabacum and Datura stramonium, respectively. In their experiments, kinetin (10 mg 1-r) did not affect the virus inhibiting properties of extracts thus, the kinetin:induced decrease of infectivity in tobacco leaves apparently reflects the inhibition of virus replication by the phytohormone. Daft [3] originally observed that kinetin stimulated the production of tomato aucuba mosaic virus (TAMV) in detached tobacco leaves and that in attached leaves kinetin either stimulated or inhibited TAMV synthesis depending upon the quantity applied and the age of the leaves. YKiraly & Szirmai [7] found that previous daily spraying of the leaves of N. glutinosa Iplants with kinetin (50 mg 1-r) for 5 days did not result in inhibition of virus infection. We, as well as other investigators [19, 221 found kinetin (50 mg 1-l) to be phytotoxic. ‘Thus past results based upon the use of highly concentrated kinetin solutions (30 to 100 mg 1-l) may be quite erroneous due to the low solubility of kinetin in water and the difficulties encountered in keeping the kinetin in solution. Also, daily spraying of inoculated plants seems to be necessary for inhibition of virus replication. Future studies designed to assess the rate of uptake of radioactively labeled kinetin by intact tobacco leaves prior to and after inoculation should indicate the amount of kinetin incorporated that is necessary to enhance or inhibit virus replication. 16
232
S. M. Tavantris, S. H. Smith and F. H. Wltham
It has been shown that kinetin induces increases in cell protein and nucleic acid levels [4, 17, 211. Murai et al. [II] have found that kinetin was incorporated as N”-furfuryladenosine(frsA) in the rRNA and tRNA preparations from tobacco callus. The rRNA contained over 90% of these fr6A moieties. They suggest that incorporation by this route may involve either specific transcriptional or posttranscriptional errors. Berridge et al. [2] as well as other workers [5] have found that kinetin binds to plant ribosomes. According to Berridge et al. [2], in other systems binding of small molecules to ribosomes has been shown to have functional significance. Streptomycin, tetracycline, actidione, puromycin and many other drugs bind to ribosomes and modify ribosomal processes. Frequently this type of binding, as in the case of kinetin, appears to be reversible and concentration dependent. These findings, in conjunction with our observation that daily kinetin (1 to 10 mg 1-r) spraying starting 9 days prior to inoculation and throughout the duration of the experiment is necessary for suppression of virus infectivity (Fig. l), would suggest a working hypothesis which might be considered in future experimentation. The continuous “occupation” or modification of a large number of virus replication sites, such as ribosomes, by kinetin might hinder virus replication or infectivity to a considerable extent. In plants sprayed with kinetin (1 or 10 mg 1-l) starting 3 days prior to inoculation, no inhibition of TRW infectivity was observed possibly because an adequate number of unaffected virus replication sites still existed in the cell at that time. On the contrary, low kinetin concentrations (i.e. 0.1 mg 1-l) not only would be insufficient to cause blockage or modification of a large number of virus replication sites but would favor virus replication (Fig. 1) by inducing accumulation ofmetabolites, such as amino acids [13], essential for virus synthesis. The possibility of kinetin affecting virus synthesis or infectivity indirectly cannot be overlooked since it is possible that kinetin can interact with DNA [S, 11, 18, 20, 241, thus inducing transcriptional changes or permitting initiation and transcription of new DNA. Another indicator of the cytokinin relationship in TRSV infection in tobacco leaves is that leaf extracts of infected plants contained less cytokinin activity than did those of non-infected plants (Fig. 2). One assay of leaf extracts at 10 days after inoculation, when TRSV is approaching its maximum concentration, was considered to provide reliable information as far as the relationship of TRSV infection to cytokinin activity. Kuriger & Agrios [9] also demonstrated cytokinin levels were lower in extracts from root exudates and root tissue of TRSV-infected cowpeas than in those of non-infected plants. Although we have not identified the cytokinins present in tobacco leaves, they appear to be chromatographically similar to zeatin and/or zeatin ribonucleoside [S, 231. Additional information concerning the chemical nature of the endogenous cytokinins, their metabolism and translocation in TRSV-infected and non-infected tobacco leaves should provide greater insights into the role of these phytohormones and the control of virus replication and infectivity in intact plants. REFERENCES 1. ALDWINCKLE, H. S. & SELMAN, I. W. (1967). Some effects of supplying benzyladenine to leaves and plants inoculated with viruses. Annals of Applied Biology 60, 49-58. 2. BERRIDGE, M. V., F~ALPH, R. K. & LETHAM, D. S. (1970). The binding of kmetin to plant ribosomes. Biochemistry Journal 119, 75-84.
Tobacco ringspot virus infectivity
233 3. DAFT, M. J. (1965). Some interactions of kinetin and temperatures on tobacco leaves infected with tomato aucuba mosaic virus. Annals of Applied Biology 55, 51-56. Die Veranderung des Stiirke-, Protein- und RNS-geshaltes von bmna minor L. unter dem Einlluss von Kinetin (6-Furfurylaminopurin). Experimentia 23, 621-622. Fox, J. E. & ERION, J. L. (1975). A cytokinin binding protein from higher plant ribosomes. Biochemistry and Biophysics ResearchCommunications64, 694-700. HENDRY, L. B., WITHAM, F. H. & CHAPMAN, 0. L. (1977). Ge ne regulation: the involvement of stereochemical recognition in DNA-small molecule interactions. PerspcGtiuesin Bioloo and Medicine 21, 120-130. KIRALI, Z. & SZIRMAI,J. (1964). The influence of kinetin on tobacco mosaic virus production in Nicotiana glutirwsa leaf discs. Virology 23, 286-288. KRASNUK,M., WITHAM, F. H. & TEGLEY, J. R. (1971). Cytokinins extracted from pinto bean fruit. Plant Physiology48, 320-324. KURIGER, W. E. & AGRIOS,G. N. (1977). Cytokinm levels-and kinetin-virus interactions in tobacco ringspot virus-infected cowpeas. Phytojathology 67, 604-609. LETHAM, D. S. (1971). A cytokinin bioassay using excised radish cotyledons. Physiologia Plantarum
4. ERISMANN, K. H. & FANKHAUSER, M. (1967). 5.
6. 7. 8. 9. 10.
25, 391-396. 11. MATTHYSSE,A. G. & A~RAMS,M. (1970). A factor mediating interaction of kinins with the genetic material. Biochima et Biophysics Acta 190, 511-518. 12. MILLER, C. 0. & WITHAM, F. H. (1964). A kinetin-like factor from maize and other sources. Colloques internationaux Centn national recherchescientifique123, I-VI. 13. MOTHES, K. & ENGELBRECHT,L. (1961). Kinetin-induced directed transport of substances in excised leaves in the dark. Phytochemistry1, 58-62. 14. MURAI, N., TALLER, B. J., ARMSTRONG,D. J. & SKOOG, F. (1977). Kinetin incorporated into tobacco callus ribosomal RNA and transfer RNA preparations. Plant Physiology60, 197-202. 15. NAKAZAKI, Y. (197 1). Effect of kinetin on local lesion formation on detached bean leaves inoculated with tobacco mosaic virus or its nucleic acid. Annals of the Phytopathological Society (Japan) 37, 307-309. 16. OPEL, H. (1964). Wirkungen von Kinetin auf Virus-turd Mehltauinfizierte Pflanzen. Monatsberichte der Deutschen Akademie der Wissenschaftenzu Berlin 6, 276. 17. OSBORNE,D. (1962). Effect of kinetin on protein and nucleic acid metabolism in Xanthium leaves during senescence. Plant Physiolor3,37, 595-602. 18. PIATTELLI, M. M. GIIJDICI DE NICOLA & CASTROGIOVANNI,V. (1971). The effect of kmetin on amaranthin synthesis in Amaranthus tricolor in darkness. Phytochemistry10, 289-293. 19. RECNOV, A. V., REUNOVA, G. D., VASILYEVA, L. A. & REIFMAN, V. G. (1977). Effect of kinetin on tobacco mosaic virus and potato virus X replication in leaves of systemic hosts. Phytopathologische
r6 *