Riboflavin Nullification of Inhibitory Actions of
3-Amino-1,2,4-Triazole on Seedling Growth'
J. L. Hilton
United States Department of Agriculture, Agricultural Research Service,
Crops Research Division, Beltsville, Maryland
The
propionic acicl (Dalapon ) toxicity to plants. The
seed-treatmiient slurry is prepared as follows: A, Add
2 g of metlhyl cellulose ( 15 cps) to 10 ml of hot water:
add 30 ml of cold water. B, Add metabolite (usuially
2 g) to 30 nil of water anid stir until dissolvecl or uintil
a homogeneous suspension suitable for pipetting is
obtained. C, Mfix three parts of the appropriate
metabolite solution to four parts (by volume) of the
methyl cellulose slurry. D, Apply 2.5 ml of the miiixture 50 g of see(ls in 8-oz bottles aInd roll to coat
the seeds thoroughly. E;, Spread the seeds on paper
towels andl allow- themii to dry.
Barley (Hordomn viulgare L. cv. Wong) seeds
coated witlh the various cellulose-metabolite miixtures
were planted in 8-oz polyethylene cups containitng
perlite saturate(d with Hoagland's solution. Amitrole wvas applie(d to the perlite surface in 10 ml of
solution at rates equivalent to 1. 2, and 4 lb "A
(resp. 0.43, 0.87. & 1.73 mig per cup). Eaclh treatment was replicated six times with five see(ds
planted per replication. Planits wvere growxv in conItrolled-environmient rooms set for a 12-hotur alternating temperatul-e cycle (27-18 C) aild a 16-lhour
photoperiod. Light intensity at plant level xwas ca.
1,000 ft-c. High to low temperature shifts coincided
wvith the end of the liglht periods.
The preparation of chemiiical solutions ansd all
other procedures involve(d in setting up thle petri
dish experiments were carried out in the laboratory
in subdued daylight. These conditions were adopted
after chemlical anid biological evidence showed that
no measurable destruction of amitrole in solution
xwith flaviins occurred (luring the short time spent
in setting up the experinments. A dark room equiipped with a ruby-red safety liglht was used for preparing materials for experiments on the effect of
light aindl (larkniess. Tlle amlitrole-flavin solutionis
were exposed for 24 hours to complete darkness or
to a light intensity of 250 ft-c. The source of illumination was overlhea(l laboratory fluorescent lights
supplemeinted witlh one 1 50-xv incandescent lamp and
two 1 5-w fluorescent lamps placed 12 inchees above
the solutions. Teimiperatutre at thle soltution level
phy.siological meclhanismls involved inl the
toxic actions of 3-amino-1 ,2,4-triazole (aamitrole)
have been actively investigated since the chemical was
first initroduce(d as a potential herbicide in 1954 (2).
Much of the researlch has been guided by hypotheses
involving one of three major metabolic processes: A,
porphyrin syntlhesis. B, cation functions, and more
recently C, the metabolism of purines and their derivatives. The extensive literature associated xvith the
first two categories xvill not be considlered hlereini.
Amlitrole interferences in some of the biochemiiical
processes of the thiir( category noxv appear to be the
actions of major plhysiological importance in the response of plants to the herbicide. Much of the pertinent research deals with attempts to find physiological compounds capable of re(lucing the toxicity of
amitrole to various organisms. The most effective
compouinds are purines (23, 24) or histidine (13. 23)
for unicellular plants and riboflavin or riboflavin (lerivatives (22, 24) for higlher plants. The investigations reporte(d herein wvere initiate(d as a result of nmy
observations that the riboflavin effect could Inot be
dlemlonstratedl with barley seedlings by techniques that
had been used successfully witlh other herbicidemetabolite combinations (9. 14.,18).
The objectives of this study xvere: A, to develop
teclhniques by 'whiclh the riboflavin nullification of
amlitrole toxicity coul(d be observed with highler l)lants.
and to coiimpare adenine an(d histidline with riboflavin
by these techniques: B, to consider the possibility
that the riboflavin effect -vas the result of non-phy0siological processes: C, to comlpare the effectiveness ot
riboflavin in species of higher I)lants that differ in
their response to amitrole, an(l D, to determine if certain other nietabolites of theoretical interest mig-ht
gi ye a(lditional redtuctions in amitrole toxicitv.
Materials & Methods
The seed-treatmnent methtod( in whichl metabolites
weere applie(d to seeds
was modified fromii a
in
a
coating of metlhyl cellulose
proce(lure originally developed
by J. K. Leasure (personal commllluniication). The
Dow Chemical Co.. -Midland, Mich.. for investigations
on calcium pantotheniate nutllificatioin of 2.2-dichloro-
1
2 The experimental sample of recrystallized 3-aiiiio1,2,4-triazole, 99+ ° pure, xvas supplied by research personnel of Amchem Produicts, Inc., Ambler, Pa.
Received Sept. 22, 1961.
238
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1962 American Society of Plant Biologists. All rights reserved.
239
HILTON-RIBOFLAVIN NULLIFICATION OF A'MITROLE INHIBITION
24 C in anl air-conditioned
returned to the dark room,
sufficient water added to compensate for evaporation,
and 10-ml aliquots added to petri dishes prepared for
growth experiments. From other solutions treated
in the same manner, 2.0 ml of solution (50.4 Ag of
amitrole) were chemlically analyzed for amitrole by
the colorimetric methodl of Green an(l Feinstein (10).
A Klett-Summerson colorinmeter equipped with a No.
54, green filter (500-570 m/L) was used for photometric determinations. Riboflavin or isoriboflavin
in solutions did not interfere w-ith amitrole determina-
was maiintainie(d at ca.
room. Solutions w-ere
tionls.
Seeds of the ninle (lifferent plant species were
germinated and grown in petri dishes on three 9-cm
disks of Whatman No. 2 filter paper, soaked about
in 10-ml, pH 5.7 solutions of chemicals.
The molar concentrations reported for chemicals refer to concentrations of the 10-ml solutions. Excess
solution was poure(l off prior to the addition of seeds
one hour
except
in
the
experiments
with
corn
or
soybeans.
Plants used in these experinments included barley
(Hordeunt vilgare L. cv. \Wong), wheat (Triticumn
aesetivum L. cv. Taylandl), oats (Avena sativa L. cv.
Clinton), alfalfa (Mledicago sativca L. cv. Atlantic),
buckwheat (Fagopyr ion esculenhturnt Moench), flax
L. cv. Arny), cucumber
(Linun usitatissiinnu
corn (Zea tmays
Marketer),
cv.
L.
sativus
(Ciicumis
L. cv. US-13). and soybeans (Gltcine witax (L.)
}{err. cv. Lee). W'heat, oats, and barley (20 seeds
per dish) were prechilledl in (larkness at 5 C for 24
hours prior to tranisfer to dark incubators maintained
at 20 C. Growth periods were 5 (lavs for oats and
4 days for all other species. Alfalfa, buckwheat, and
flax (20 seeds per dlish) an(d corn, soybeans, and
cucunmber (10 seeds per (lish) were placed in incubators maintained at 18 C for alfalfa, 20 for buckwlheat, 25 for flax, corn, and soybeans, and 28 for
cucumber. Seedling (levelopment x-as arrested by
freezing at the end( of the growvth period. Immediately after thawing of individual dishes the lengths
of the coleoptile an(d roots were (determined for monocots. Growth miieasurenients for all dicots were taken
from the cotyledlons to the tip of the root. The
graphical data for individual species were obtained lby
taking the average value of three replications expressed as a percentage of the untreated control and
averaging these values for the three independent experiments.
the root and riboflaviin to a leaf or by foliar application of anmitrole and root application of riboflavin.
These observations might be expected if riboflavin
is not readily absorbed or trainslocated through mature
cells.
During the first week or two after planting, seedlings grown from riboflavin treate(d seeds were little
affected by amiiitrole applicatioils that caused severe
chlorosis andl inhibition of growth to other seedlings.
In some experiments. histidinie or a(lenine appeared
to cause a slight reduction in aamitrole inhibitions
of groNvth: but the effects were not consistently observed and were, therefore, considered fortuitous.
The method of applicationi of metabolites does not
permlit exact calculations of herbicide-nmetabolite ratios
in the experiment reported (table I). However, calculations based on the mla;ximiunm amiiount of chemicals
available to plants in the indivicidual cups revealed
that on a molar basis amlitrole exceedled riboflavin by
approximiately four to one in the low-amitrole, highriboflavin treatment and by at least 66 to 1 in the
high-amiiitrole. low-riboflavin treatment. All three
riboflavin concentrations were comlpletely effective
in protecting growtth against inhibition by the
lowest amitrole concentration. At higher herbicide concentrations complete protection was not
obtained with the metaolxoite, but reduction of
amitrole inhibition increased with increasing riboflavin. For all three amlitrole concentrations chlorosis was delayed in seedllings treated with riboflavin
but finally appearedl during the second or the third
week after planting. The effectiveness of a given
amount of riboflavin in the prevention of chlorosis
and growth inhibitions varied from experiment to
Table I
Growth Response of Barley Seedlings*
Height*** of seedlings at indicated
amitrole ratest, cm
4 lb/A
1 lb/A 2 lb/A
0 lb 'A
3.9
12.1
23.2
33.4
None
5.9
10.5
18.0
33.4
0 miig
14.2
21.0
32.4
32.5
36 mg
17.5
30.5
33.7
33.2
71 mg
23.9
28.7
31.9
32.8
143 Ilmg
3.0
LSD 5 % level
4.0
1 % level
* Tlree weeks after planting and treating with amitrole, riboflavini, and amitrole-riboflavin combinatiolns.
Riboflavin was applied to seeds in a methyl cellulose
(57 mg/50 g of seed) coating prior to planting.
The methyl cellulose coating was omitted from treat(5
ment "None". Maximum riboflavin per cup0.13,
seeds/cup) was calculated to be approximately
0.26, and 0.52 mg, respectively, for the 36-, 71-, and
Riboflavin
applied per
50 g of
seed**
Experimental Results & Discussion
Treatment Techniques. Four different techniques for applying chemicals to barley plants were
used in these investigations. Nullification of amitrole toxicity by riboflavin was observed in two
different types of experiments in which both amitrole
an(l riboflavin were available to the seed at the time
of planting. However, amitrole toxicity vas not affected by riboflaviin wlhen establishe(d seedlings were
treated by simultaneous applications of amitrole to
-
143-mg
*
Average
rates.
heighit
for five plants per
replication,
six
replications per treatment.
± Amitrole rates equivalent to 1, 2, and 4 lb/A were
0.43, 0.87, and 1.73 mg per cup. respectively, applied
to the perlite surface immediately after planiting.
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1962 American Society of Plant Biologists. All rights reserved.
PLANT PHYSIOLOGY
240
Table II
Growth Response of Barley Seedlings*
Root length**
Coleoptile length**
Without
With
With
Amitrole Without
ribo- riboflavin***
riboflavin***
vriboconc
flavin
flavin
m
mmmm
mm
27
28
0
30
35
28
29
33
31
3 X 10 5 At
26
6
28
9
10 4 M
24
1
24
3
10-3 M
14
1
17
3
10-2 Mr
9
1
16
2
3 X 10 ' AI
1
1
3
1
10 1 M\
* After 4-day exposure to solutioIns of amitrole, riboflavin, and amitrole plus riboflavin.
** Average lengths obtained from one coleoptile and
five roots per seed, 20 seeds per replication, three
replications.
*** Riboflavin supplied in a suspenision containing 3.76
mg of riboflavin per 10 ml of treatment solution.
experiment. This v ariability \ as probably associated with differences in the loss of metabolite from
the methyl cellulose seed coating to the surrounding
me(liumii. The mletabolite diffused readily from the
seed coating and \vas observed on the surface of
the perlite above the seeds in some experiments.
The protective effect of riboflavin was apparent
as early as 5 days after planting; therefore, a laboratory method seemed feasible for short-terml experiments with seedlings germinated and grown in petri
dishes. Results obtained (table II) by this methodI
showed that riboflavin reduced amitrole inhibition
of both coleoptile and root growth over a herbicide
concentration range of 10-4 to 3 x 10-2 Im. Histidine, adenine, or cytosine had little or no effect in
subsequent experiments with wvheat, oats, and barley.
Evidence for Physiological and Non-physiological
Mechanisms. The techniques used in my investigations and the techniques used by others (22, 24) have
involved simultaneous application of riboflavin alndI
amitrole to plants. In some of these techniques pnotochemical reactions were possible and could cause inactivation of the herbicide molecule through nonphysiological mechanisms. Isoriboflavin was used in
experiments to investigate this possibility. Isoriboflavin is an analog of riboflavin which differs in
chemical structure from the physiological flavin in
the position of a single methyl group on the isoalloxazine ring (fig 1). This compound would not be
expectedl to replace riboflavin in physiological systems, but its chemical activities in solution with amitrole might be expected to be sinmilar to those of riboflavin. Preliminary experimiients reveale(d that both
isoriboflavin and riboflavin w-ere effective in inactivation of the biological activity of 2,4-dichlorophenoxyacetic aci(l (2.4-D) in the light; but neither flavin
had any effect against 2.4-D in the (lark. Inactivation of 2,4-D by photoactivate(d-riboflavin has been
reporte(l by others (3, 11,12).
Experiments xvith amitrole revealed that both
riboflavin and isoriboflavin nullified the toxicity of
3 X 10-4 WI amitrole to wheat seedlings grown in
petri dishes under illumination; but only riboflavin
was effective in nullification of amitrole damage to
seeds grown in complete darkness. Subsequent experiments (table III) demonstrated that illumination
of the amitrole-flavin treatment solutions prior to
treatment and growth of seedlings in the (lark was also
effective in nullification of amitrole toxicity. These
results established the hypothesis that (lestruction of
amitrole by photoactivated flavins had occurred. A
Table III
Growth of Wheat Roots*
Avg.** length of roots
on solutions exposed to
solutionih
Light***
Treatment
Darko
mm
mnm
43
38
38
8
\Water control
43
45
Isoriboflavin 10-4 M
44
Riboflavin 10-4 M\
13
Amitrole 3 X 10-4 Al
14
34
Amitrole + isoriboflavin
36
38
Amitrole + riboflavin
* Seedlings grown in petri dishes on filter paper saturated with solutionis exposed to 24 hours of light
or darkness prior to germination of seeds and growth
of seedlings in the dark.
** Average length per root, three roots per seedling,
20 seedlings per replication, three replications per
treatment.
2250 ft-c
rescent
inltenlsity, froml inicanidescenit
total
and fluo-
lights.
CH2(CHOH)3C H20H
I
H
-
HC'
C°~
c CNc8
CH3
0
ISORIBOFLAVIN
CH2 (CHOH)3CH20H
H3C
I
II
-Ct
-C
ZCH ICI
0
RIBOFLAVIN
Fig.
1.
Chemlical structures of isoriboilavin and ribo-
flavin.
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1962 American Society of Plant Biologists. All rights reserved.
241
HILTON-RIBOFLAVIN NULLIFICATION OF AMITROLE INHIBITION
Table IV
Amitrole Content of Solutions Exposed to 24 Hours of
Liglit or Darkness With & Without Riboflavin
or Isoriboflavin
Initial solution
Amitrole content (Ag/2 ml)
after exposure to
Light*
Dark
Amitrole 3 x 10-4 5
50
50
(50.4 ,ug/2 ml)
Amitrole 3 x 10-4 M
8
50
+ isoriboflavin 10-4 M
Amitrole 3 X 10-4 M
50
<5
+ riboflavin 10-4 M
* 250 ft-c total intensity from incandescent and fluorescent lights.
chemical analysis of the amitrole-flavin solutions after
exposure to 24 hours of light or dlarkness demonstrated
(table IV) that the herbicide was partially destroyed
bv either of the two flavins un(ler illumination but
was unaffected in darkness. The observed nullification of amitrole toxicity in the absence of light by
riboflavin but not by isoriboflavin (table III) and
the absence of any detectable alteration in the chemical structure of amitrole (table IV) may be interprete(l as evidence that a physiological mechanism
was involve(d in the reduction of amitrole toxicity
to seedlings.
Response of Representative Plant Species. A
comparison of species was undertaken to determine
whether the physiological nullification of amitrole
toxicity by riboflavin could be implicated in a variety
of plants that differ in their sensitivity to amitrole
and in their metabolism of the herbicide. Experiments were conducted first to determine the amount
of riboflavin required for maximum reduction of
amitrole toxicity. Wheat, oats, and barley were
treated with riboflavin concentrations of 10-5 Ar and
10-4 m and a riboflavin suspension equivalent to
10-3 M. Each riboflavin concentration was examined
in combination with amitrole concentrations ranging
from 3 X 10-5 MA to 10-1 alr. The lowest concentration of riboflavin was partially effective but only
against the lowest concentrations of amitrole inhibitory to growth of the various species. The 10-4 M1
concentration was much more effective, and little
additional effect was obtained with the riboflavin
suspension. The 10-3 M suspension and a true
10-3 Nf riboflavin solution (prepared by dissolving the
metabolite in a minimum amount of 10 % NaOH before dilution with water and HCI to pH 5.7) were
equally effective in experiments with wheat. The
riboflavin suspension was selected for use in further
studies on the maximum riboflavin-reduction of amitrole toxicity to other plant species (fig 2).
Wheat and alfalfa, representative monocot and
dicot species considered to be sensitive to amitrole
under field conditions, were tested for the riboflavin
effect. Oats and buckwheat were included in these
studies as representative monocot and dicot species
that tolerate relatively high rates of amitrole in field
experiments. Corn was selected to represent a species
that degrades amitrole without accumulating large
quantities of amitrole-metabolites (26). Soybeans
were included to represent a species that rapidly
metabolizes amitrole and accumulates relatively large
quantities of non-toxic triazole metabolites (26)).
Flax and cucumber were examined as additional dicot
species after the two legumes (alfalfa & soybeans)
were observed to respond to amitrole in a manner
unlike other species. Growth of the two legumes
was partially inhibited by low rates of amitrole, but
little additional inhibition was observed until the
herbicide concentration was increased approximately
100-fol(d (fig 2).
The action and interactions of riboflavin and amitrole on growth of the eight species (Fi,g 2) were
evaluated separately in three independent experiments
and the data combined for presentation. Tlle control
growth values, averaged from the three experi rents
and representing 100 % in figure 2, were as follows:
wheat roots, 40 mm; oat roots, 24 mm: alfalfa seed-
-
_
D
oI
_-I. _o
10-4 10-3 1 0-
io-aoo4
10-1 0
o
10-4 10-3
a
o-1
1 Cr2 I 0-
AMITROLE CONC. (M)
Fig. 2. Effect of amitrole on growth of various seedlings during a 4-day (5-day for oats) germination and
growth period in petri dishes with (open circles) and
without (closed circles) riboflavin. Seedlings were
grown in darkness on filter papers pre-soaked in solutions
containing amitrole at the indicated concentrations without or with riboflavin in suspension in amounts equivalent
to a 10-s3 .r concentration (see text for discussion).
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1962 American Society of Plant Biologists. All rights reserved.
242
PLANT PHYSIOLOGY
lings. 28 mnm: buckwheat seedlings, 63 mm; corn
roots, 53 mm; soybean seedlings, 53 mm; flax seedlings, 49 mm, and cucumber seedlings, 46 mm. The
comparison of root growth for monocots and seedling growth for dicots is valid because the effects
of amitrole, riboflavin, and amitrole-riboflavin combinations appeared to be similar for roots and shoots
of both types of plants. Measurements from monocots
were quantitatively more reproducible for roots than
for coleoptiles. Growth measurements for the entire
seedling length were used for all dicots because the
transition region between roots and hypocotyls was
not clearly distinguishable on some species.
The differences in sensitivity of the species to
amitrole are apparent in the data (fig 2). In the
absence of riboflavin, 50 % inhibition (ED 50) of
growth for the different species was obtainedl at
amitrole concentrations ranging from 3 X 10-5 M to
ca. 10Riboflavin alone had no real effect on
seedling growth, but partial nullification by riboflavin
of the inhibitory action of amitrole to seedling growth
of balley (table II), wheat, oats, alfalfa, corn, soybeatns flax. and cucumber (fig 2) is clearly demonstr;itedl. Experiments with buckwheat, the species
considered most resistant to amitrole under field conditions, revealed little evidence of riboflavin reductiolis in toxicity of the herbicide. In th2 presenec
of riboflavin, the other species seemed to approach
a level of insensitivity to amitrole approximately
equal to that of buckwheat (ED 50 values of ca.
2
10-2 r).
The response of the various species to amitrole
andl to amitrole-riboflavin combinations leads one to
consider the possibility that differences in riboflavin
content of species might be important in determining
the selective herbici(dal action of amitrole. Published
values (17) of the amount of ribloflavin in the various
crop seeds and foliage reveal no clear correlation between riboflavin content and species sensitivity.
§ uch a correlation would not necessarily be expected
if A, amitrole controls growth through inhibition of
an early step in riboflavin synthesis, as suggested by
Sund (20), and if B. riboflavin is not readily translo)ated. The relative capacities of the various plants
to produce flavoprotein at or near the sites of amitrole accumulation in young tissue would be more
important than the amlount of riboflavin present at the
time of herbicide treatment. However, failure to
find a clear correlation would seem to argue against
the assignment of an important role for flavin-med1iatedl inactivation of amlitrole as a determining factor
in the selective action of the herbicide.
0 Effects of Other- MAetabolites.
Amitrole concentrations of 10-2 Mr a;nd( higheir caused inhibitions of
growtlh of all species (fig 2) even in the presence
of riboflavin. Therefore, an effort was miiade to find
other metabolites capable of producing additional
reductions in the toxicity of 10-2 AI, 3 X 10-2 M, and
10-1 M concentrations of amitrole. Riboflavin, riboflavin-5-phosphate (FMN), and flaviin adenine dinucleotide (F\D) wvere comlpared in growth experi-
Table V
Nullification of Amitrole Inhibition of Wheat Root
Growth by Riboflavin, FMN, & FAD
Avg. length* of wheat
roots on 10-3 M
solutions of: (mm)
conc
None Riboflavin FMN
FAD
0
41.1
46.6
38.2
37.6
10-2 M
1.0
19.5
18.3
20.8
10.6
11.1
3 X 10-2 M
9.8
0.7
10 1M
1.0
1.6
1.3
0.5
* Average length per root, three roots per seedling, 20
seedlings per replication, three replicationis per treat-
Aniitrole
ment.
mlents witlh wheat seedliings. Both FAIN anid FAD
were
effective in reducing amiiitrole inhibition of
growsth but neither chemiiical was any mlor-e effective
thani riboflavin (table V). Simiiilar results wvere
obtained with buckwheat seedlings. Otlher metabolites were then examine(d in combination with 10-3Ni
FMN and each of the three herbicide concentrations
in the wheat bioassay test. The metabolites investigated were selected from the literature and evidence
has been presented for possible amitrole effects on
the metabolism of each. The mletabolites evalua-ted,
the concentrations usedl in the evaluation, and the
literature citations are as follows: adenine, 10-3 Nt
(1, 13, 22, 23, 24) ; histidine, 10-3 M (13, 15, 23); glvcine, 10- a (5, 6): serine, 10 31 (5, 6), and glucose, 10-1 AI (7, 8). In Ino instance di(d any of these
metabolites at the selecte(d conicentr-ationis produce
definite redtncLions in amiiitrole toxicity over an(d above
the re(luctions obtained with FM1\,N.
Conclusion
The results reported herein provide substalitiatinig
evidence that the inhibition of plant growth by aiiiitrole can be nullified by riboflavin, FMN, and FAD.
This effect can result from chemiical inactivation
of amitrole with photoactivated-flavins. However.
evidence is also presented for a physiological meclhanism which can nullify the toxic actionl of a:mitrole
with natural flavins. The physiological mechanismll
was observed wvith a number of plant species that
vary greatly in their sensitivity to amitrole an'! in
their metabolism of the herbicide. Sund and Little
(21) demonstrated that amiitrole inhlibits the Crlluir tion of riboflavin in vivo; therefore, exogenouis riboflavin may overcome amiiitrole iinhlibition of growtlh
by circumvention of a mletabolic block in riboflavin
biosynthesis. If this explanationi is correct, the iresults reported hereiin in(licate that inhibition of riboflavin synthesis is a nmajor meclhanism for the lherbicidal action of amitrole. However, the amitroleriboflavin interactions in vivo may be much more
complicated. The rapid disappearance of amiiitr-ole
from plant tissues might involve (legradationi tlhrough
flavin-mediated svstems.
Investigations on growtlh of higher an(d lower
forms of plaints seemii to suggest that anmitrole lpro-
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1962 American Society of Plant Biologists. All rights reserved.
HILTON-RIBOFLAVIN NULLIFICATION OF AMITROLE INHIBITION
duces mietabolic aberrations ultimately expressed as
deficiencies of different growth-essential cellular constituents in the various classes of plants. Riboflavin
nullified the toxicity of amitrole to higher plants but
it was not effective against amitrole in experiments
witlh Chlorella pyrenoidosa (24) or in my own experiments with bakers' yeast. Histidine nullified the
toxicitv- of amitrole to three species of yeast (13. 23)
and. in combination with adenine. to Escherichia coli
(23). But, with higher plants, histidine was not
effective (13, 16, 22. 24) except for a slight reduction
in amitrole inhibition of root hair growth (15).
Both histidine and riboflavin are derived metabolically
from the purine. adenine. Reduction of amitrole
toxicity by purines is usually small in comparison to
reductions obtained with riboflavin or histidine but
theylhave been reported for three species of higher
plants (1, 22) and one species each of yeast (13),
algae (24), and bacteria (23). These observations
may be interpreted as evidence either A, that amitrole
is a general inhibitor of purine utilization or B. that
amitrole inhibits the biosynthesis of purines. The
first hypothesis draws support from the evidence that
amitrole probably inhibits biosynthesis of histidine
(13), riboflavin (21), and possibly nucleic acids (25)
and from the inhibition of 4-aminoimidazole hydrolase. an enzyme involved in purine catabolism (19).
Sund (20) has suggested that the block in riboflavin
biosynthesis occurs at the step involving breakage
of the five-membered ring of adenine. The second
hypothesis draws some support from the demonstration by Carter and Navlor (5, 6) that glycine and
serine are utilized in the formation of a non-toxic
metabolite of amitrole which is produced in higher
plants (4). Since glycine is a purine precursor, the
detoxification of aniitrole might restrict purine svnthesis if the glycine content is reduced below levels
needed for nornmal growth and development. Fither
of the two proposed mechanisms of amitrole action
could account for the nullification of amitrole toxicitv
by different nmetabolites in different organisms.
VTariations in the relative capacities of higher and
lower forms of plants to produce histidine, riboflavin,
or other products of purine metabolism may determine which metabolite becomes growth limiting after
amitrole treatment.
Summary
I. Reductions in the toxicity of 3-amino-1.2,4triazole (amitrole) to barley seedlings treated with
riboflavin were observed by using techniques involving treatments with both riboflavin and amitrole at
time of planting. Riboflavin nullification of amitrole
toxicity was not observed by techniques involving isolated applications of the two chemicals to different
organs of established seedlings. Histidine or adenine had no effect on growth of amitrole-treated
barley seedlings.
-
243
II. Inactivation of amitrole in illuminated solutions containing riboflavin or isoriboflavin Avas
demonstrated by chemical analysis for amitrole and
bioassay for amitrole toxicity. Riboflavin, but not
isoriboflavin, was effective in the partial reduction
of amitrole inhibition of growth in the dark. Flavinmediated destruction of amitrole did not occur in solutions stored in the dark.
ITT. Growth of seedllings of eight amitrole-sensitive species of higher plants was relatively insensitive
to the herbicide in the presence of riboflavin under
conditions that did not produce chemical inactivation
by riboflavin. Little reduction of amitrole inhibition
of growth by riboflavin was observed with seedlings
of buckwheat, a species sensitive only to relatively
high rates of amitrole. All nine species were equally
insensitive to amitrole in the presence of riboflavin.
IV. Riboflavin, FMN. and FAD were equally
effective in partial nullification of amitrole toxicity
to wheat seedlings. Histidine. adenine. glycine,
serine. and glucose didi not produce additional re(ductions in toxicity.
-
-
Acknowledgment
I am grateful to R. M. Hayes for his able technical
assistance.
Literature Cited
1. ALDRICH, F. D. 1958. Some indications of antagonism hetween 3-amino-1,2,4-triazole & purine &
pyrimidine bases. (Abstr.) Weed Soc. Am., 1958
Meeting, p. 32.
2. AMERICAN CHEMICAL PAINT CO. 1954. Technical
service data sheet H-52, 16 n.
3. BELL, G. R. 1956. On the photochemical degradation of 2,4-dichlorophenoxyacetic acid & structurally related compounds in the presence & absenice of
riboflavin. Botan. Gaz. 118: 133-136.
4. CARTER, M. C. & A. W. NAYLOR. 1960. Metabolism
of 3-amino-1,2,4-triazole-5-Cl4 in plants. Botan.
Gaz. 122: 138-143.
5. CARTER, M. C. & A. W. NAYLOR. 1961. Studies on
an unknown metabolic product of 3-amino-1,2,4triazole. Physiol. Plantarum 14: 20-27.
6. CARTER, M. C. & A. W. NAYLOR. 1961. The effect
of 3-amino-1,2,4-triazole upon the metabolism of
carbon labeled sodium bicarbonate, glucose, succinate, glycine, & serine by bean plants. Physiol.
Plantarum 14: 62-71.
7. FREDRICK, J. F. & A. C. GENTILE. 1960. The formation of the glucose derivative of 3-amino-1,2,4triazole under physiological conditions. Physiol.
Plantarum 13: 761-765.
8. FREDRICK, J. F. & A. C. GENTILE. 1961. The structure of the D-glucose adduct of 3-amino-1,2,4triazole: Infrared absorption studies. Arch. Riochem. Biophys. 92: 356-359.
9. GENTNER, W. A. & J. L. HILTON. 1960. Effect of
sucrose on the toxicity of several phenylurea herbicides to barley. Weeds 8: 413-417.
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1962 American Society of Plant Biologists. All rights reserved.
244
P'LANT PHYSIOLOGY
10. GREEN, F. O., & R. N. FEINSTEIN. 1957. Quantitative estimation of 3-amino-1,2,4-triazole. Anal.
Chem. 29: 1658-1660.
1 1. HA-MILTON, R. H. & R. J. ALDRICH. 1953. Failure
of light activated riboflavin to counteract the
growth inhibition of 2 benzoic acid derivatives.
Weeds 2: 202-203.
12. HANSEN, J. R. & K. P. BUCHHOLTZ. 1952. Inactivation of 2,4-D by riboflavin in light. Weeds
1: 237-242.
13. HILTON, J. L. 1960. Effect of histidine on the
inhibitory action of 3-amino-1,2,4-triazole. Weeds
8: 392-396.
14. HILTON, J. L., J. S. ARiD, L. L. JANSEN, & W. A.
GENTNER. 1959. The pantothenate-synthesizing
enzyme. a metabolic site in the herbicidal action
of chlorinated aliphatic acids. Weeds 7: 381-396.
15. JACKSON, W. T. 1961. Effect of 3-amino-1,2,4triazole & L-histidine on rate of elongation of root
hairs of Agrostis alba. Weeds 9: 437-442.
16. JANSEN, L. L. 1960. Effects of controlled environmental factors on the absorption, translocation, &
mode of expression of 3-amino-1,2,4-triazole in the
soybean plant. (Abstr.) Weed Soc. Am. 1960
Meeting, p. 36.
17. MILLER, D. F. 1958. Composition of cereal grains
& forages. National Academy of Sciences-National Research Council, Washington 25. D.C.
Publication No. 585, 663 p.
18. MORELAND, D. E., W. A. GENTNER, J. L. Hli.TON,
& K. L. HILL. 1959. Studies on the mechanism
of herbicidal action of 2-cliloro-4.6-bis (ethylamin.-)
s-triazine. Plant Physiol. 34: 432-435.
RABINOWITZ, J. C. & W. E. PRICER, JR. 1956. Purine fermentation by Clostridi)um cylindrosporunm.
V. Formiminoglycine. J. Biol. Chem. 222: 537
554.
SUND, K. A. 1961. The effect of 3-amino-1,2,4triazole on certain plant tissues grown in vitro.
Physiol. Plantarum 14: 260-265.
SUND, K. A. & H. N. LITTLE. 1960. Effect of 3amino-1,2,4-triazole on the synthesis of riboflavin.
Science 132: 622.
SUND, K. A., E. C. PUTALA, & H. N. LIrrLE. 1960.
Reduction of 3-amino-1,2,4-triazole phytotoxicity in
tomato plants. J. Agric. & Food Chem. 8: 210212.
WVEYTER, F. W. & H. P. BROQUIST. 1960. Interference with adenine & histidine metabolism of
microorganisms by aminotriazole. Biochim. Biophvs. Acta 40: 567-569.
WOLF, F. T. 1961. Mechanism of action of 3amino-1,2,4-triazole. Plant Phvsiol. 36 suppl:
-
19.
20.
21.
22.
23.
24.
xxxix.
WN ORT, D. J. & B. C. LOUGHMAN. 1961. The effect
of 3-amino-1,2,4-triazole on the uptake, retention,
distribution, & utilization of labelled phosphorus by
young barley plants. Can. J. Botany 39: 339-351.
26. YOST, J. F. & E. P. WILLIA-MS. 1958. Some residue & metabolism findings in the development of
3-amino-1,2,4-triazole. Proc. NE Wreed Conitrol
Conf. 12th Meeting, p. 9-15.
25.
Downloaded from on July 31, 2017 - Published by www.plantphysiol.org
Copyright © 1962 American Society of Plant Biologists. All rights reserved.