Plant Physiol. (1973) 52, 660-662
T-2 Toxin Decreases Logarithmic Growth Rates
of Tobacco Callus Tissues
Received for publication June 21, 1973
JOHN P. HELGESON AND G. T. HABERLACH
Pioneering Research Laboratory, Agricultural Research Service, United States Department of Agriculture,
Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin 53706
LARRY N. VANDERHOEF
Department of Botany, University of Illinois, Urbana, Illinois, 61801
ABSTRACT
T-2 toxin, a mycotoxin produced by Fusarium tricinctum,
decreases logarithmic growth rates of tobacco (Nicotiana
tabacum L.) pith callus tissues. Toxin concentrations as low
as 0.003 ,uM will decrease growth rates; a concentration of
0.081 aM will halt growth completely. Additional exogenous
cytokinin will reduce the inhibition by toxin only when the
initial cytokinin and toxin concentrations are quite low (about
0.01 jaM). When inhibited tissues are transferred to media lacking toxin, they assume the faster, control rates ahnost immediately. Maximal yields of tissue (yields at the point at
which no sugar was detected in the medium) are not affected
by toxin concentrations of 0.01 to 0.036 ,uM.
Logarithmic growth rates of tobacco pith callus tissues can
be controlled by the concentration of exogenous cytokinin
supplied in the growth medium (5, 6). Maximal yields of
callus, on the other hand, are dependent on the sugar concentration (7, 11). A reversible inhibitor that decreases growth
rates in log phase, without decreasing maximal yields, could be
valuable in determining the mechanism by which cytokinins
control growth rates. Such an inhibitor would make it possible
to vary growth rates without changing cytokinin concentration.
We now report that T-2 toxin, a naturally occurring mycotoxin,
is such an inhibitor.
T-2 toxin, 4,15-diacetoxy-8-(3-methylbutyrloxy)-12,13 expoxy-A'-trichothecene-3-ol, is produced by Fusarium tricinctum (cda.) Snyd. & Hans. This toxin is one of more than
twenty 12, 13-epoxytrichothecene toxins that affect both plants
and animals (1, 3, 9). For example, Marasas et al. (9) reported
that growth (fresh weight and length) of pea seedlings was depressed in comparison to controls 24 hr after treatment with
1.34 uMm T-2 toxin. Our data reported below indicate that
tobacco callus tissues are even more sensitive to this mycotoxin.
MATERIALS AND METHODS
Stock callus tissues of Nicotiana tabacum L. were maintained on Linsmaier and Skoog's medium (8) containing 11.5
/iM IAA and 0.2 uMm 6-(3-methyl-2-butenylamino)purine. The
callus tissues were derived from the North Carolina clone
52-23 supplied by J. L. Apple, North Carolina State University.
Tissues from these plants exhibited the same dependence on
cytokinin for their control of logarithmic growth rates as does
the line of Wisconsin No. 38 used in our previous studies.
Growth kinetics (5, 6) and sugar in callus medium (7, 11) were
measured as previously reported. Growth substances and T-2
toxin were autoclaved in the medium; their concentrations are
reported for each experiment. Crystalline T-2 toxin, prepared
by the method of Bamburg et al. (2) was obtained from Dr.
E. B. Smalley, University of Wisconsin. This preparation exhibited a single spot upon TLC, a single peak upon gas-liquid
chromatography, and a clean mass spectrum from T-2 toxin
(E. B. Smalley, personal communication).
RESULTS
Initial experiments indicated that T-2 toxin decreased callus
yields when supplied at concentrations ranging from 0.003 to
0.081 ,uM (Fig. 1). At low 2iP' and T-2 concentrations, 2iP
can decrease toxin inhibition to a limited degree.
When the growth of the tissues was followed with standard
kinetic measurements, tissues supplied with 11.5 Mm IAA, 0.1
Mm 2iP, and 0, 0.018, or 0.036 Mm T-2 toxin grew logarithmically with doubling times of 3.3, 5.5, and 7.55 days, respectively (Fig. 2).
The reversibility of T-2 toxin inhibition is illustrated in
Figure 3. Tissues were planted initially on medium containing 11.5 ,uM IAA and 0.1 puM 2iP, with or without 0.018 ,uM
T-2 toxin. After 14 days, some tissues treated with toxin were
transferred to fresh medium containing 11.5 uM and 0.1 uM
2iP, but without toxin. Almost immediately after transfer from
toxin medium, tissues assumed the same growth rate as the
tissues that had never been treated with toxin.
Measurements of medium sugar for the experiment shown in
Figure 2 indicated that 0.68, 0.74, and 0.65 g dry weight of
tissue was produced/g sucrose during log phase from tissues
treated with 0, 0.018, or 0.036 uM toxin, respectively. In a
subsequent experiment, toxin-treated and control tissues were
allowed to go to maximal yield (the point at which no sugar was
detected in the medium). Dry weight yields were 168 mg (0
toxin) and 163 mg (0.018 [kM toxin) per piece. Thus, although
the growth rate was depressed by the toxin, the maximal yields
for the toxin-treated and control tissues were nearly the same.
The slightly decreased yield for the toxin-treated tissue might
be attributable to experimental variability or the increased need
for maintenance energy during nearly 2 additional weeks required for the toxin-treated tissues to reach maximal yield.
'
Abbreviation: 2iP: 6-(3-methyl-2-butenylamino)purine.
660
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Plant Physiol. Vol.
52, 1973
EFFECT OF T-2 TOXIN ON TOBACCO CALLUS
110
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72 CONCENTRATION (MM)
FIG. 1. Fresh weight yields (per cent of controls) of tobacco
callus tissues after 22 days' growth on Linsmaier and Skoog medium
(8) containing 11.5 AM IAA, 0.009 ,M (0) or 0.081 ,M (0) 2iP and
various concentrations of T-2 toxin. Control yields were 1114 mg/
piece (0.009 AM 2iP) and 2126 mg/piece (0.081 ,AM 2iP).
(DAYS)
FIG. 3. Reversibility of T-2 toxin inhibition. Tissues planted on
Linsmaier and Skoog medium (8) with (A) or without (0) 0.018 gM
T-2 toxin. At day 14 some tissues (0) were transferred from
medium containing toxin to medium lacking toxin. All media also
contained 11.5 ,.M IAA and 0.1 lM 2iP.
10,000
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FIG. 2. Growth of tobacco callus tissues on Linsmaier and Skoog medium (8) containing 11.5
(A) or 0.036 (o) ,AM T-2 toxin. A: fresh weight basis; B: dry weight basis.
AGE
(DAYS)
/AM IAA, 0.1 /AM 2iP and either 0 (0), 0.018
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662
HELGESON, HABERLA(UC.H, AND VANDERHOEF
DISCUSSION
Logarithmic growth rates of tobacco callus tissues are,
within certain limits, controlled by exogenous cytokinin rather
than by inorganic minerals or sugars (5-7, 11). These logarithmic growth rates, programmed by the cytokinin concentration, appear to be decreased reproducibly, quickly and reversibly by low concentrations of T-2 toxin. Because the concentrations of toxin that give these effects are very low (3-81
X 10-' M), this system may be highly useful as a toxin bioassay.
In addition, the toxin is potentially useful for basic studies of
hormone-controlled plant growth. We now have another means
for varying log phase growth rates without, it appears, appreciable effect on the efficiency of carbohydrate utilization. Comparisons of metabolism of tissues controlled at the same rate,
but by different substances, may demonstrate that the toxin
acts quite specifically on some metabolic component that is
essential for maintaining cytokinin-controlled optimal growth
rates.
Toxins of the 12,13-epoxytrichothecene type have been reported to inhibit protein synthesis (10) without affecting codonanticodon recognition (4). E. Cundliffe, M. Cannon, and J.
Davies report (personal communication) that T-2 toxin (2-200
,uM) inhibits polypeptide chain initiation in H-Hela cells and
yeast spheroplasts. These effects, however, have been noted
with concentrations that will prevent any growth of tobacco
callus tissue. It will be of considerable interest to determine if
there is appreciable inhibition of protein synthesis (either in
general or of a specific protein species) in tobacco callus tissues
treated with toxin concentrations that merely decrease growth
rates.
Plant Physiol. Vol. 52, 1973
Acknowledgments-We wish to thank Dr. E. B. Smalley, University of Wisconsin, for the gift of T-2 toxin and Dr. J. L. Apple of North Carolina State
University for seeds of NC 52-23 tobacco. We greatly appreciate the kindness of
Dr. Julian Davies, University of Wisconsin, in supplying a copy of his manuscript in preparation.
LITERATURE CITED
1. BAMBURG, J. R. AND F. M. STRONG. 1971. 12,13-Epoxytrichothecenes. In: S.
Kadis, A. Ciegler and A. Ajl, eds., Microbial Toxins, Vol. VII. Academic
Press, New York. pp. 207-292.
2. BAMBURG, J. R., N. V. RIGGS, AND F. M. STRONG. 1968. The structures of
toxins from two strains of Fusarium tricinctum. Tetrahedron 24: 3329-3336.
3. BRIAN, P. W., A. W. DAWKINS, J. F. GROVE, H. G. HEMMING, D. LOEWE,
A'ND G. L. NORRIS. 1961. Phytotoxic compounds produced by Fusarium
equiseti. J. Exp. Bot. 12: 1-12.
4. CARRASCO, L., M. BARBACID, AND D. VAZQI.EZ. 1973. The trichodermin group
of antibiotics, inhibitors of peptide bond formation by eukaryotic ribosomes. Biochim. Biophys. Acta 312: 368-376.
5. HELGESON, J. P., S. Ml. KRUEGER, AND C. D. UPPER. 1969. Control of logarithmic growth rates of tobacco callus tissue by cytokinins. Plant Physiol.
44: 193-198.
6. HELGESON, J. P. AND C. D. UPPER. 1970. Modification of logarithmic growth
rates of tobacco callus tissue by gibberellic acid. Plant Physiol. 46: 113-117.
7. HELGESON, J. P., C. D. UPPER, AND G. T. HABERLACH. 1972. Medium and
tissue sugar concentrations during cytokinin-controlled growth of tobacco
callus tissue. In: D. J. Carr, ed., Plant Growth Substances, 1970. Springer
Verlag, Berlin. pp. 484-492.
8. LINSMAIER, E. M. AND F. SKOOG. 1965. Organic growth factor requirements of
tobacco tissue cultures. Physiol. Plant. 18: 100-127.
9. MARASAS, W. F., E. B. SMALLEY, J. R. BAMBURG, AND F. M. STRONG. 1971.
Phytotoxicity of T-2 toxin produced by Fusarium tricinctum. Phytopathology 61: 1488-1491.
10. UENO, Y., M. HoSOYA, AND Y. ISHIKAWA. 1969. Inhibitory effects of mycotoxins on protein synthesis in rabbit reticulocytes. J. Biochem. 66: 419-422.
11. UPPER, C. D., J. P. HELGESON, AND G. T. HABERLACH. 1970. Limitation of
tobacco callus tissue growth by carbohydrate availability. Plant Physiol. 46:
118-122.
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