Ribofuranosyl triazolone: A natural product herbicide with activity on adenylosuccinate synthetase following phosphorylation

Article abstract of DOI:10.1021/np990590i

2,4-Dihydro-4-(β-D-ribofuranosyl)-1,2,4(3H)-triazol-3-one (2) was identified as the principal phytotoxic component of a fermentation broth derived from an Actinomadura. The compound is a new natural product, but known by synthesis. Bread-spectrum herbicidal activity was demonstrated in greenhouse tests. Metabolite reversal studies suggested the target site was adenylosuccinate synthetase, which was confirmed by direct measurement of the activity of the 5'-phosphorylated derivative on the isolated enzyme.

Full text of DOI:10.1021/np990590i

J . Nat. Prod. 2000, 63, 777-781
777
Ribofu r a n osyl Tr ia zolon e: A Na tu r a l P r od u ct Her bicid e w ith Activity on
Ad en ylosu ccin a te Syn th eta se F ollow in g P h osp h or yla tion
Paul R. Schmitzer, Paul R. Graupner, Eleanor L. Chapin, Steven C. Fields, J eff R. Gilbert, J im A. Gray,
Cathy L. Peacock, and B. Clifford Gerwick*
Dow AgroSciences, 9330 Zionsville Road, Indianapolis, Indiana 46268
Received November 24, 1999
2,4-Dihydro-4-(â-D-ribofuranosyl)-1,2,4(3H)-triazol-3-one (2) was identified as the principal phytotoxic
component of a fermentation broth derived from an Actinomadura. The compound is a new natural product,
but known by synthesis. Broad-spectrum herbicidal activity was demonstrated in greenhouse tests.
Metabolite reversal studies suggested the target site was adenylosuccinate synthetase, which was
confirmed by direct measurement of the activity of the 5′-phosphorylated derivative on the isolated enzyme.
The recent commercialization of sulcotrione, a synthetic
optimization of the plant-derived natural product lep-
tospermone, has further validated the importance of natu-
ral products as starting points for herbicide discovery.1
Sulcotrione is unique in combining high levels of activity
with a novel mode of action, inhibition of quinone biosyn-
thesis.2 The large number of patent applications on this
and closely related areas of chemistry suggest this family
of compounds, known as the triketones, will have a major
role in shaping the future of weed-control technology.
unusual ene-diyne madurapeptin,15 and a series of xan-
thone antibiotics, the simaomicins.16,17 No previous reports
of a ribofuranosyl triazolone exist from any biological
source.
MS and UV data had suggested that 2 was a nucleoside.
This was confirmed by NMR data, which also indicated a
â-D-ribofuranose sugar. LC/MS analysis of 2 revealed a [M
+ H]+ peak at m/z 218. Negative ion LC/MS produced the
[M - H]- and [M + acetate - H]- adduct ions at m/z 216
and 276. LC/MS/MS of the [M + H]+ peak at m/z 218
produced a neutral loss of 132 daltons, consistent with loss
of a ribose ring. With the ribose ring accounting for 133
amu, a base unit of 84 amu remained. The most likely
formula arising from this is C2H2N3O, consistent with a
triazole ring system. The proton NMR indicated a single
C-H bond, implying the existence of a single carbonyl
group in the molecule. This molecular formula may be
assembled in three different ways: 2, 3, and 4.
Another natural product with excellent herbicidal activ-
ity and a target site not represented in commercial
herbicides, hydantocidin (1), has also been the subject of
intense research. This compound was first described from
Streptomyces hygroscopicus SANK 63584 by Sankyo3 but
has subsequently been identified in a number of different
Streptomyces strains at Novartis,4 Mitsubishi,5 and Dow
AgroSciences (unpublished). It has been pursued syntheti-
cally by several agricultural chemical companies including
Sandoz,6 Ciba Geigy,7 and American Cyanamid.8 The mode
of action was examined by three groups, including ours,
and found to be inhibition of adenylosuccinate synthetase
(AdSS) following bioactivation by phosphorylation of the
2′-hydroxyl group.9-12 Commercialization of hydantocidin
and synthetic analogues is uncertain, with at least part of
the barrier being attributed to the cost of synthesis and to
the difficulty of identifying active analogues that are more
easily accessible.13 In the course of our screening program
for new phytotoxins, we discovered a ribofuranosyl tri-
azolone (2) that shares the same target site as hydantoci-
din, is a new natural product but known by synthesis, is
readily approachable synthetically, and also appears bio-
activated by phosphorylation of the primary hydroxyl. In
this report we describe the discovery of this compound as
a natural product and detail its synthesis, herbicidal
activity, and mode of action.
Two of these molecules (2 and 3) have been described,18
the third (4) is unknown. To distinguish these molecules,
the long-range coupling from the anomeric proton to the
protonated carbon atom was measured using 1D INEPT
giving a value of 4 Hz, thus discounting structure 3 as the
identity of the natural product. To distinguish between 2
and 4, the chemical shift of the protonated carbon atom
was predicted (Advanced Chemical Development, Toronto).
For 2, the predicted chemical shift was 134 ppm; whereas,
for 4 it was 148 ppm. The actual value of the chemical shift
was 136 ppm. Thus, the structure of the natural product
was assigned as 2.
Resu lts a n d Discu ssion
The organism was identified using PCR amplification of
a 357 base pair region of DNA encoding 16s ribosomal RNA
and indicated the producing strain closely resembled
Actinomadura madurae. The Actinomadurae are known to
produce a range of antibiotics, including specifically the
carbazole antibiotics carbazomadurins A and B,14 an
To confirm this structure and to provide more of the
compound for biological assessment, 2 was synthesized
according to the procedure of Haines et al.18 In this TMS
* To whom correspondence should be addressed. Tel.: (317) 337-3119.
Fax: (317) 337-3252. E-mail: cgerwick@dowagro.com.
10.1021/np990590i CCC: $19.00
© 2000 American Chemical Society and American Society of Pharmacognosy
Published on Web 05/03/2000

778 J ournal of Natural Products, 2000, Vol. 63, No. 6
Schmitzer et al.
Ta ble 1. Phytotoxicity of Ribofuranosyl Triazolone (2) to Several Plant Species in the Greenhouse
visual injury (%)
application
rate kg ha-1
HELANa
IPOHE
ECHCG
AVEFA
ABUTH
AMARE
SETFA
ALOMY
postemergence
4.0
2.0
1.0
0.5
4.0
2.0
1.0
0.5
95
95
100
90
60
20
10
0
90
80
80
75
40
40
25
15
90
85
85
70
0
0
0
0
60
70
60
50
0
0
0
0
80
80
70
85
65
30
30
15
70
50
50
0
0
0
85
60
70
50
0
0
0
0
80
70
70
40
40
35
30
0
preemergence
0
0
a
HELAN, Helianthus annuus; IPOHE, Ipomoea hederacea; ECHGG, Echinochloa crusgalli; AVEFA, Avena fatua; ABUTH, Abutilon
theophrasti; AMARE, Amaranthus retroflexus; SETFA, Setaria faberi; ALOMY, Alopecurus myosuroides.
F igu r e 1. Root-growth inhibition of Arabidopsis elicited by ribofura-
nosyl triazolone (2) in the presence and absence of adenine.
procedure, the bis-TMS-protected triazole is coupled with
protected R-D-ribofuranose-1-acetate. This reaction gives
a number of products, which were separated by liquid
chromatography (Scheme 1, Supporting Information). Depro-
tection of the benzoyl groups furnished the appropriate
nucleosides, one of which was shown to be identical with
the natural product 2 both by LC and NMR analyses.
The ribofuranosyl triazolone 2 demonstrated broad-
spectrum phytotoxicity in the greenhouse, with activity on
all test species (Table 1). Symptoms included stunting,
systemic chlorosis, and necrosis of apical meristems. In the
F igu r e 2. Dose-response curves for ribofuranosyl triazolone (2) (rtz)
and phosphoribofuranosyl triazolone (p-rtz) on (A) mammalian AdSS
and (B) plant AdSS; reaction time, 5 min.
postemergence test, the ribofuranosyl triazolone had high
levels of phytotoxicity, with activity on all species except
Amaranthus retroflexus at 500 g ha-1, the lowest rate
tested. The activity was much less after preemergence
applications, most likely due to poor soil stability.
Root growth of Arabidopsis thaliana was inhibited by
ribofuranosyl triazolone with an I50 of 0.04 ppm, or 0.2 µM
(Figure 1). When the medium was supplemented with a
combination of adenine and guanine, the effect of the
herbicide on Arabidopsis declined markedly. The I50 for the
inhibition of Arabidopsis roots in the presence of adenine
and guanine increased to 2.5 ppm, or 11.5 µM. A test of
the individual purines indicated that adenine reversed the
effects of the ribofuranosyl triazolone, with an I50 of 1.8
ppm, or 8.3 µM (Figure 1), while guanine had no effect
(data not shown). Alleviation of the herbicidal activity of
the ribofuranosyl triazolone by adenine is the same me-
tabolite reversal reported by Heim et al.9 for hydantocidin
F igu r e 3. Time course of the plant AdSS reaction (A) without any
treatment and (B) with 0.1 ppm phosphoribofuranosyl triazolone (2).
(1). Therefore, the reversal data implicated AdSS as a
potential target site of 2.
to the required bioactivation of 1. The 5′-phosphate deriva-
tive of 2 was prepared synthetically and found to be a
potent inhibitor of both mammalian and plant AdSS in a
time-dependent manner (Figure 3). The time-dependent
nature of the inhibition was also observed in inhibition of
AdSS by phosphohydantocidin.10 Using a linear part of the
inhibition curve, the phosphorylated 2 demonstrated I50
Direct measurement of the effect of 2 on isolated AdSS
isolated from both maize cell culture and rabbit-muscle
acetone powder indicated no inhibition (Figure 2). Given
the Arabidopsis reversal data and the structural similari-
ties of 2 to 1, the hypothesis was made that 2 required
phosphorylation to inhibit AdSS in a manner analogous

Ribofuranosyl Triazolone Herbicide
J ournal of Natural Products, 2000, Vol. 63, No. 6 779
g), and hexaglycerol dioleate (15.0 g),per liter of deionized H2O,
and incubated for 8 days at 28-30 °C with shaking at 195
rpm. After 8 days, the culture was harvested and processed
as described below.
values of 0.18 µM and 0.98 µM for rabbit-muscle AdSS and
maize cell culture AdSS, respectively (Figure 2).
Ribofuranosyl triazolone (2) could represent an impor-
tant new lead for herbicide discovery. Its synthetic ap-
proachability and apparent overlap with the structure-
activity relationships governing hydantocidin (1) suggest
opportunities for further optimization/hybridization of
these structures. It is the second reported compound that
must be bioactivated by phosphorylation in order to inhibit
the activity of AdSS. Compound 2 is a new natural-product
phytotoxin that is herbicidal via inhibition of AdSS, a target
site not represented among commercial herbicides today.
Isola t ion a n d Id en t ifica t ion of R ib ofu r a n osyl Tr i-
a zolon e (2). The material from two 13.5-mL fermentations
were combined, centrifuged at 10000g for 30 min, and the
supernatant transferred to a 100-mL separating funnel. The
supernatant was extracted three times with an equal volume
of n-BuOH, and the combined extracts were evaporated in
vacuo. Compound 2 was isolated by liquid chromatography
(Hypercarb S graphite) using 5% aqueous acetonitrile contain-
ing 0.05% trifluoroacetic acid, to give an off-white solid (ca.
0.5 mg). Two separate chromatography runs were required to
isolate 2 at >98% purity: colorless solid; 1H NMR (D2O) δ 7.9
(1H, s, H-2), 5.4 (1H, d, H-1′), 4.4 (1H, dd, H-2′), 4.2 (1H, dd,
H-3′), 4.0 (1H, ddd, H-4′), 3.7 (H, dd, H-5′a), 3.6 (1H, dd, H-5′b);
13C NMR (D2O) δ 136 (d, C-2), 86 (d, C-1′), 85 (d, C-4′), 73 (d,
C-2′), 70 (d, C-3′), 61 (d, C-5′); positive ion ESIMS m/z 218 [M
+ H]+; negative ion ESIMS m/z 216 [M - H]- 276 [M + acetate
- H]-.
Syn th esis of Ribofu r a n osyl Tr ia zolon e (2) a n d th e
Cor r esp on d in g P h osp h or ibofu r a n osyl Tr ia zolon e. Struc-
ture confirmation was achieved by independent synthesis of
2 following the method of Haines et al.18 (see Schemes 1 and
2, Supporting Information). Experimental variations are noted
below.
4-Im id a zolin -2-on e. Semicarbazide hydrochloride (10 g)
was refluxed in an excess of triethyl orthoformate (100 mL)
for 2 h. The resultant homogeneous solution was concentrated
in vacuo to give the desired product, which was used without
any further purification.
1-(Tr im eth ylsilyl)-2-[(tr im eth ylsilyl)oxy]im idazole. The
reaction was run on 4-imidazolin-2-one prepared above as
described by Haines et al.18 After the solvent was removed in
vacuo, the residue was sublimed at 60 °C, 0.01 mmHg, to give
the title compound as a white solid.
1-(2,3,5-Tr i-O-b en zoyl-â-D-r ib ofu r a n oyl)-4-t r ia zol-3-
on e. According to the published procedure,18 tin tetrachloride
was added last over 1 h to a mixture of imidazole and
ribofuranose at 0 °C. After 3 h of stirring at 0 °C, the aqueous
workup was performed as described. Reversed-phase gradient
HPLC (C18 fully end-capped ODS) using H2O and acetonitrile
containing 0.05% trifluoroacetic acid, gave five products that
were identified by NMR analysis as alkylation on N5 (5%),
alkylation on N2 (30%), acylated N2 derivative (15%), the
ribose starting material (15%), and bis-ribosylated material
(35%).
Depr otection of O-Ben zoylated Ribon u cleosides. Depro-
tection of both the second and third products by the published
procedure of Haines et al.18 yielded 2, which was identical in
all respects to the material produced by fermentation.
P h osp h or yla tion of 2. To a solution of 2 (8.1 mg, 0.04
mmol) in DMF (125 µL) and THF (1.25 mL) was added
tetrazole (7 mg, 0.085 mmol) and dibenzyl diethyl phosphora-
midite (32 µL, 0.1 mmol). The reaction was stirred for 4 h and
quenched with oxone (93 mg, 0.15 mmol) in H2O (1 mL). The
reaction was stirred for an additional 30 min and partitioned
between EtOAc and H2O. The organic fraction was evaporated
and the residue purified by reversed-phase gradient HPLC
(C18), using H2O and acetonitrile containing 0.05% trifluoro-
acetic acid, to produce the protected phosphate derivative (2
mg, 10%); ESIMS, m/z 477.
The protected phosphate (2 mg) was dissolved in EtOH (1.5
mL) and cyclohexene (2.5 mL). After addition of palladium-
on-carbon catalyst (1 mg), the reaction was heated to reflux
for 5 h. The reaction was filtered, evaporated, and chromato-
graphed by reversed-phase HPLC as described above to
produce the desired phosphorylated ribofuranosyl triazolone
(ca. 1.0 mg, 76%); ESIMS, m/z 297.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. NMR spectra were
recorded on a Bruker DRX400 spectrometer, operating at
400.13 MHz (1H) and 100.62 MHz (13C), and equipped with
either a 3-mm micro inverse probe or a 3-mm micro dual probe
(Nalorac). Samples were dissolved in 140 µL of D2O. All spectra
were referenced to the residual proton solvent resonance at
4.7 ppm. Carbon data were externally referenced to sodium
3-trimethylsilylpropionate. LC/MS analyses were conducted
using a Hewlett-Packard 1050 HPLC coupled to a Finnigan
LCQ mass spectrometer. Mass spectra were obtained using
both electrospray ionization (ESI) and atmospheric pressure
chemical ionization (APCI).
Id en tifica tion of Or ga n ism by DNA Sequ en cin g. DNA
was isolated from 0.25 mL of liquid vegetative culture using
the QIAamp Tissue Kit (Quiagen, Valencia, CA) and eluted
in a final volume of 400 µL. The 16s rRNA gene was amplified
by PCR using a Gene Amp Kit (PE Applied BioSystems, Foster
City, CA). Each 100-µL reaction contained 10 µL of the
bacterial DNA and 20 pmol each of the 16s sense (5′>AGAGTT-
TGATCCTGGCTCAG<3′) and 16s anti-sense (5′>AAGGAG-
GTGATCCAGCCGCA<3′) primers. Amplification was carried
out in a DNA Thermal Cycler 480 (PE Applied Biosystems),
after a 5-min soak at 94 °C, 25 cycles of 94 °C for 1 min, 50 °C
for 1 min, and 72 °C for 3 min, followed by a 7-min extension
at 72 °C. The reaction product was purified using Quiagen’s
QIAquick Kit and eluted in 30 µL of H2O. DNA-sequencing
reactions were carried out using a Perkin-Elmer ABI PRISM
Dye Terminator Ready Reaction Kit supplemented with 10%
DMSO. The eluted PCR product (1 µL) was sequenced in a
20-µL reaction using 1 pmol of the 519 primer (5′>GWAT-
TACCGCGGCKGCTG<3′). Unincorporated nucleotides were
removed using Centriflex Gel Filtration Cartridges (Edge
BioSystems, Inc., Gaithersburg, MD) and then analyzed on
an Applied Biosystems 373A DNA sequencer.
The resulting sequence was compared with the public
database at the National Center for Biotechnology Information
using the search tool BLAST 2.0 (Washington, DC). The query
sequence consisted of 357 bases. There was no identical
sequence in the public database (Genbank Database, May
1998). However, it was similar to members of the genus
Actinomadura (98% identity). Within the Actinomadura, the
sequence most closely matched A. madurae (Vincent 1894)
Lechevalier and Lechevalier 1970 (349 out of 357 bases).
Cu ltu r e a n d F er m en ta tion of Or ga n ism . The culture
(M05053-G8) was isolated from a soil sample according to
standard methods19 and deposited at Dow AgroSciences (9330
Zionsville Road, Indianapolis, IN 46268). The culture was
fermented for 72 h in a vegetative medium (50 mL) containing
glucose (5.0 g), yeast extract (Difco, 2.5 g), soluble starch (10.0
g), N-Z Amine (casein, 2.5 g), and CaCO3 (0.5 g) per liter of
deionized H2O. Incubation was maintained at 28-30 °C with
shaking at 195 rpm for 3 days. This seed inoculum (30 µL)
was then added to small rectangular bottles with vented cap
closures each containing 14 mL of production medium. The
production medium was composed of corn steep powder (5.0
g), dextrose (5 g), lactose (50 g), CaCO3 (3 g), soybean flour
nutrisoy (10 g), bacto peptone (5 g), NH4SO4 (2 g), FeCl2‚4H2O
(0.1 g), ZnCl2 (0.1 g), MnCl2‚4H2O (0.1 g), MgSO4‚7H2O (0.5
Eva lu a tion of Her bicid a l Activity. Whole plant activity
was assessed on several plant species using foliar-applied
(postemergence) and soil-applied (preemergence) applications.
Seeds of sunflower (Helianthus annuus, HELAN), morning-
glory (Ipomoea hederacea, IPOHE), velvetleaf (Abutilon theo-

780 J ournal of Natural Products, 2000, Vol. 63, No. 6
Schmitzer et al.
phrasti, ABUTH), pigweed (Amaranthus retroflexus, AMARE),
barnyardgrass (Echinochloa crusgalli, ECHCG), giant foxtail
(Setaria faberi, SETFA), wild oats (Avena fatua, AVEFA), and
blackgrass (Alopecurus myosuroides, ALOMY) were planted
in mineral soil (sandy clay loam, 51% sand, 26% silt, 23% clay,
2.8% O. M., pH 7.8) and Metro-mix (Bulk Sak, Inc., Malvern,
AR) for preemergence and postemergence treatments, respec-
tively. Pots receiving preemergence treatments were watered
immediately after application. Prior to postemergence treat-
ment, plants were grown in the greenhouse (16-h photoperiod,
27 °C) and thinned to a density of 2-15 plants per pot,
depending on species. At the time of postemergence applica-
tions, plants were 8-12 days old, 3-10 cm in height, and in
the one- to three-true-leaf stage. Natural light in the green-
house was supplemented with metal halide lights that pro-
vided an average photosynthetic photon flux of 500 µmol m-2
Ma m m a lia n Ad SS Extr a ction . Rabbit-muscle acetone
powder was extracted by gently shaking 3-5 g in 20 mL of
AdSS extraction buffer containing 20 mM Tris-HCl, pH 7.4, 1
mM EDTA, and 5 mM DTT. This suspension was filtered
through four layers of cheesecloth. The residue was rinsed
twice with 10 mL of extraction buffer. The combined filtrate
was centrifuged for 20 min at 25000g. Protamine sulfate (88
µL of a 5% solution) was added to each milliliter of the
supernatant while it was stirring at 4 °C. The precipitate was
removed by centrifugation for 10 min at 10000g. Saturated
ammonium sulfate (pH 7.0) was added to the supernatant to
35% saturation and stirred for 30 min at 4 °C. The precipitate
was removed by centrifugation for 10 min at 10000g. The
saturated ammonium sulfate solution was added to the
supernatant to 60% saturation and stirred for 30 min at 4 °C.
The precipitate was collected by centrifugation at 10000g for
10 min. The resulting pellet was resuspended in 8 mL of
dialysis buffer containing 10 mM Tris-HCl, 1 mM EDTA, and
0.5 mM DTT and dialyzed overnight against 1 L of the same
buffer.
Ma ize Ad SS Extr a ction . Maize (Zea mays) AdSS was
extracted from stationary-phase BMS cells (day 7). A 20 mL
packed-cell volume was added to a Waring blender with 80
mL of AdSS extraction buffer (see above) and blended on high
for 2 min. The ground tissue was filtered through four layers
of cheesecloth. The filtrate was centrifuged for 20 min at
25000g. Protamine sulfate (88 µL of a 5% solution) was added
to each milliliter of the supernatant while it was stirring at 4
°C. The precipitate was removed by centrifugation for 10 min
at 10000g. Saturated ammonium sulfate (pH 7.0) was added
to the supernatant to 35% saturation and stirred for 30 min
at 4 °C. The precipitate was removed by centrifugation for 10
min at 10000g. The saturated ammonium sulfate solution was
added to the supernatant to 60% saturation and stirred for
30 min at 4 °C. The precipitate was collected by centrifugation
at 10000g for 10 min. The resulting pellet was resuspended
in 8 mL of dialysis buffer containing 10 mM Tris-HCl, 1 mM
EDTA, and 0.5 mM DTT and dialyzed overnight against 1 L
of the same buffer.
The dialyzed sample was centrifuged for 10 min at 10000g
before being placed on a monoQ 10/10 column that had been
equilibrated with 10 mM Tris-HCl, pH 7.4. The column was
subjected to a 0-300 mM KCl gradient to remove the AdSS
activity. Fractions containing AdSS activity were pooled and
concentrated.
Ad SS Assa y. The AdSS assay was conducted as described
by Heim et al.9 The AdSS reaction mixture contained 50 mM
tricine, pH 8.0, 5 mM MgCl2, 0.04 mM GTP, 0.15 mM IMP,
0.04 mM ATP, 0.4 mM phosphoenolpyruvate, 4 U/ mL pyru-
vate kinase, and enzyme extract. The reaction was initiated
with the addition of aspartate (2 mM final concentration).
Reaction volumes were 1 mL each. The treatments were
diluted into the reaction mixture from concentrated solutions
in DMSO. DMSO concentration never exceeded 0.1%. The
assays were followed in a Beckman diode-array spectropho-
tometer at 30 °C. The formation of adenylosuccinate was
monitored as a difference in absorbance between 282 and 320
nm. All rates were calculated from the linear part of the
reaction curves (Figures 2 and 3).
s-1
.
A sample of ribofuranosyl triazolone was dissolved in 4 mL
of distilled H2O followed by 10 mL of formulation stock “B”
[200 mL of 2-propanol, 20 mL of crop oil concentrate, 0.4 g of
Triton X-155 (Sigma Chemical Co., St. Louis, MO 62718), and
78 mL of H2O]. This was followed by three 1:1 (v/v) serial
dilutions using formulation stock “C” (100 mL of 2-propanol,
10 mL of crop oil concentrate, 0.2 g of Trition X-155, 390 mL
of H2O, 485 mL of acetone, and 15 mL of DMSO).
Treatments were made at four different doses (4000, 2000,
1000, and 500 g ha-1). For preemergence treatments, 1.75 mL
of spray solution was applied to the soil surface in each pot
using a glass Cornwall syringe fitted with a Teejet SS8001E
flat-fan nozzle (Spraying Systems Co., Wheaton, IL 60189-
7900). For postemergence applications, the foliage of the test
plants was sprayed using a DeVilbiss atomizer (DeVilbiss
Health Care, Inc., Somerset, PA 15501) driven by compressed
air at a pressure of 22 kPa. Untreated controls were included.
Pots were placed in the greenhouse after compound applica-
tion. Pots from postemergence and preemergence treatments
were sub-irrigated and top-watered, respectively, as needed
for the duration of the test. Visual injury ratings were taken
9 and 16 days after treatment for postemergence and preemer-
gence applications, respectively, on a 0-100 scale, where 0
represents no injury and 100 represents complete necrosis.
Results are summarized in Table 1.
Meta bolite Rever sa l Stu d ies. Metabolite reversal studies
were conducted on A. thaliana as previously described by D.
R. Heim et al.9 with minor changes. The Arabidopsis growth
medium was supplement with 0.8% sucrose and 0.45% agar-
ose. A 10-mL aliquot of this melted medium, maintained at
62 °C, was used for each treatment. The inhibitors and/or
metabolites were added to the medium as concentrated solu-
tions in either H2O or DMSO. Controls included the appropri-
ate amount of DMSO added without the inhibitor and/or
metabolite. The final concentration of DMSO varied but never
exceeded 0.8%. We established that DMSO concentrations in
this range did not alter Arabidopsis growth, development, or
response to common herbicidal agents (data not shown).
Adenine was added to a final concentration of 185 µM, and
guanine was added to a final concentration of 165 µM. After
thoroughly mixing, 8 mL of the treated medium was trans-
ferred into a 60 × 15-mm sterile Petri dish (Falcon 1007;
Becton-Dickinson, Lincoln Park, NJ ) and allowed to solidify.
Approximately 25 µL of sterilized seed was then applied onto
the solidified medium and spread evenly over the plate using
the remaining 2 mL of the treatment medium. These plates
were incubated for 7 days at 23 °C under continuous fluores-
cence lighting (50 µE m-2 s-1).
Treatment effects were recorded as root measurements.
Roots of Arabidopsis, when grown as above, characteristically
elongate as a single tap root with little or no branching. Root
measurements involved carefully extracting individual plants
from the medium and measuring the length of the tap root.
Each reported value is the average of five such measurements.
All values were rounded to the nearest millimeter, and
averages are reported as percentages of control. Visual mea-
surements of shoot development were recorded as percentages
of control (Figure 1).
Su p p or tin g In for m a tion Ava ila ble: This material is available
free of charge via the Internet at http://pubs.acs.org.
Refer en ces a n d Notes
(1) Stonard, R. J .; Miller-Wideman, M. A. In Agrochemicals from Natural
Products; Godfrey, C. R. A., Ed.; Marcel Dekker: New York, 1994;
pp 285-310.
(2) Lee, D. L.; Prisbylla, M. P.; Cromartie, T. H.; Dagarin, D. P.; Howard,
S. W.; Provan, W. M.; Ellis, M. K.; Fraser, T.; Mutter, L. C. Weed
Sci. 1997, 45, 601-609.
(3) Haneishi, T.; Nakajima, M.; Torikata, A.; Okazaki, T.; Tohjigamori,
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