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Lipid Biosynthesis Inhibitors
Author(s): John W. Gronwald
Source: Weed Science, Vol. 39, No. 3 (Jul. - Sep., 1991), pp. 435-449
Published by: Weed Science Society of America and Allen Press
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Weed Science, 1991. Volume 39:435-449
Lipid BiosynthesisInhibitors1
JOHN W. GRONWALD2
Abstract. Five classes of herbicides (carbamothioates,
chloroacetamides, substituted pyridazinones, cyclohexanediones, and aryloxyphenoxypropionic acids) have been
reported to inhibit lipid biosynthesis in higher plants.
Carbamothioates impair the synthesis of surface lipids
(waxes, cutin, suberin). These effects have been attributed
to the ability of this herbicide class to inhibit one or more
acyl-CoA elongases. Though as yet poorly characterized,
these enzymes are associated with the endoplasmic
reticulum and catalyze the condensation of malonyl-CoA
with fatty acid acyl-CoA substrates to form very longchain fatty acids used in the synthesis of surface lipids.
There is contradictory evidence regarding the effects of
chloroacetamide herbicides on de novo fatty acid biosynthesis. Selected substituted pyridazinones decrease the
degree of unsaturation of plastidic galactolipids. This
effect is attributed to the ability of selected members of
this herbicide class to inhibit fatty acid desaturases which
are thought to be located in the chloroplast envelope.
Aryloxyphenoxypropionic acid and cyclohexanedione herbicides inhibit de novo fatty acid biosynthesis in grasses.
The target site for these herbicide classes is the enzyme
acetyl-CoA carboxylase which is found in the stroma of
plastids. In most cases, selectivity between grasses and
dicots is expressed at this site. Aryloxyphenoxypropionic
acids and cyclohexanediones are reversible, linear, noncompetitive inhibitors of acetyl-CoA carboxylase from
grasses. Both classes are also mutually exclusive inhibitors
of grass acetyl-CoA carboxylase which suggests that they
bind at a common domain on the enzyme. Nomenclature:
Acetyl-coenzyme A carboxylase (EC 6.4.1.2).
Additional index words. Fatty acid, carbamothioate, chloroacetamide, substituted pyridazinone, cyclohexanedione,
aryloxyphenoxypropionate, elongase, desaturase, acetylCoA carboxylase.
INTRODUCTION
During the past 12 yr, there have been several reviews of
lReceived for publicationJuly 30, 1990, and in revised form January11,
1991. Publ. of U.S. Dep. Agric., Agric. Res. Serv. and Minnesota Agric.
Exp. St. Paper No. 18,462, Sci. J. Ser., MinnesotaAgric. Exp. Stn., St. Paul,
MN.
2PIantPhysiol., Plant Sci. Res. Unit, Agric. Res. Serv., U.S. Dep. Agric.
and Assoc. Prof., Dep. Agron. and Plant Genet., Univ. Minnesota, St. Paul,
MN 55108.
3Abbreviations: ACCase, acetyl-CoA carboxylase; ATP, adenosine
triphosphate;BMS, Black Mexican Sweetcorn; CoA, coenzyme A; DGDG,
digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol;Mr relative molecular mass; PG, !Phosphatidylglycerol;VLCFA, very long-chain
fatty acids; 16:1(t), trans-A -hexadecenoic acid.
the effects of herbicides on various aspects of lipid
metabolism in plants (29, 39, 54, 60, 109). Two recent
reviews are those of Harwood et al. (54) and Hoppe (60).
In this review, five classes of herbicides that have been
reportedto interfere with certain aspects of lipid (fatty acid)
metabolism will be discussed. These classes are: a) carbamothioates, b) chloroacetamides, c) substituted pyridazinones, d) cyclohexanediones, and e) aryloxyphenoxypropionic acids. Carbamothioates, chloroacetamides, and
substitutedpyridazinones are older classes of chemistry that
have been reportedto inhibit multiple target sites, including
sites other than those involved in lipid biosynthesis (29, 39,
54, 60, 80, 115, 123, 145). For these herbicide classes, the
literatureconcerningtheir effects on lipid biosynthesis will be
briefly reviewed with an emphasis on recent reports. The
major focus of this review will concern the effects of two
relatively new herbicide classes, the cyclohexanediones and
aryloxyphenoxypropionicacids, on lipid biosynthesis. Within
the past 4 yr, considerable advances have been made in
elucidating the mechanism of action of these two herbicide
classes which are potent inhibitors of de novo fatty acid
biosynthesis in grasses (14, 15, 54, 60, 72, 73, 106, 107, 113,
135).
A discussion of the details of fatty acid biosynthesis in
higher plants will not be provided in this review. The reader
is referredto recent reviews by Stumpf (129) and Harwood
(50, 51).
CARBAMOTHIOATES
Representatives of the carbamothioates are shown in
Figure 1. These herbicides are applied preplant incorporated
and provide selective control of annual grasses and some
annualbroadleafweeds duringgerminationand early seedling
growth. Symptoms of herbicidal activity in grasses include
stunting of early seedling growth and interference with the
emergence and unfolding of leaves from the coleoptile (3, 39,
145).
The primary site of action of the carbamothioateshas not
been identified. The literaturesuggests that the two metabolic
processes most sensitive to inhibition by carbamothioatesare
gibberellin and lipid biosynthesis (39). Inhibitory effects of
carbamothioates on gibberellin biosynthesis have been
describedelsewhere (143, 144, 150) and will not be discussed
in this review.
There is considerable evidence that carbamothioates
inhibit the synthesis of surface lipids such as waxes, cutin,
and suberin(3, 7, 29, 39, 44, 53, 54, 75, 109, 121, 141, 145,
146, 147). This effect has been attributedto the ability of
carbamothioatesto inhibit the biosynthesis of very long-chain
fatty acids (VLCFA)3 (7, 39, 53, 54, 75, 121, 145), which are
defined as fatty acids of chain length greater than eighteen
435
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GRONWALD: LIPID BIOSYNTHESIS NHIBITORS
CH3
CH3CH2CH2
~CH
0
?
N-C
-S-CH2
-CH3
CH3CH2CH2
CH3
C
CH3
EPTC
CH3
OH
CH3
CH3
CH3
0
C CH
3 I
,N-C
CH
-S-CH2
3.5 /
CH
I I
Cl CII
-C=
Diallate
CI
0
N-C
= C
-S-CH2-C
I
cI
C-
I
Cl
%-3.1
UPPER
n\
Triallate
Figure 1. Chemical structures of selected carbamothioates.
Z 2.9 _X
carbons (50). VLCFA are components of surface lipids and
they also serve as precursors for such surface lipid
components as alkanes, aldehydes, alcohols (primary and
secondary), and ketones (74).
The first report that carbamothioatesinterfered with the
synthesis of surface lipids was that of Gentner (44) who
found that EPTC4 (S-ethyl dipropylcarbamothioate)inhibited
wax formation on developing leaves of cabbage (Brassica
oleracea L.). Since then, the inhibitory effects of carbamothioates on surface lipid biosynthesis have been
corroboratedby others. Wilkinson and Hardcastle(147) found
that EPTC reduced cuticle thickness on the upper and lower
surfaces of sicklepod [Cassia obtusifolia (L.) #5 CASOB]
leaflets. The inhibitory effect of increasing EPTC concentration on cuticle thickness in sicklepod is shown in Figure 2.
Other studies (75, 121) have demonstrated the inhibitory
effect of carbamothioateson the formation of cuticular and
epicuticular wax on pea (Pisum sativum L.) leaves. EPTC
preferentiallyinhibited the incorporationof [14C]acetateinto
surface lipids as opposed to internal lipids in pea (75).
Carbamothioateswere also selective inhibitorsof [14C]acetate
incorporationinto VLCFA in germinatingpea seedlings (53).
In additionto being utilized in the synthesis of epicuticular
wax and cutin, VLCFA are also used in the synthesis of
suberin found on stem, root, and wound surfaces. If
carbamothioatesact as general inhibitors of VLCFA synthesis, they should also inhibit the synthesis of suberin. To test
this hypothesis, Bolton and Harwood (7) examined the effect
of carbamothioateson the synthesis of suberin in excised
potato (Solanum tuberosum L.) discs during aging. This
tissue was utilized because it exhibits rapid rates of suberin
synthesis (6). EPTC, diallate [S-(2,3-dichloro-2propenyl)bis(I-methylethyl)carbamothioate],and triallate [S-
4Mention of a trademark, vendor, or proprietary product does not
constitute a guaranteeor warrantyof the productby the U.S. Dep. Agric. or
the Univ. Minnesota, and does not imply its approval to the exclusion of
other products or vendors that may also be suitable.
5Letters following this symbol are a WSSA-approved computer code
from Composite List of Weeds, Revised 1989. Available from WSSA, 309
West Clark Street, Champaign, IL 61820.
436
UU2.4
/
W2.2
LOWER
2.0
0.5 1
2
3
EPTC (kg/ha)
4
Figure 2. Effect of EPTC on cuticle thickness on the upper and lower leaf
surface of sicklepod. From Wilkinson and Hardcastle (147).
(2,3 ,3-trichloro-2-propenyl)
bis( 1-methylethyl)carbamothioate]were selective inhibitorsof fatty acid elongation
in excised potato discs. At concentrationsin the range of 10
FM, these herbicides severely reduced the proportion of
[14C]acetate incorporated into VLCFA (20:0, 22:0, 24:0)
while having little or no effect on the incorporation of
radiolabel into palmitic and stearic acid.
The evidence is ratherclear that carbamothioatesinterfere
with the biosynthesis of surface lipids. It has been proposed
that this effect is due to the ability of this herbicide class to
inhibit acyl-coenzyme A (CoA)3 elongases (7, 54, 75). These
enzymes, found in epidermalcells (1, 4, 83, 84) and cells at
wound surfaces (6, 7, 74, 134), are integral membrane
proteins associated with the endoplasmic reticulum. AcylCoA elongases have proven to be difficult to isolate and
characterize in vitro and only recently has information
regardingtheir propertiesbecome available. Most researchon
this enzyme system has been conducted with epidermal
preparationsfrom leek (Alliumporrum L.) (1, 4, 83, 84) and
aged slices of potato tubers (134). Nicotinamide adenine
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WEED SCEENCE
dinucleotide phosphate (NADPH)-dependent elongation of
selected acyl-CoA substrates using malonyl-CoA as the
condensing agent has been demonstrated in a microsomal
preparationisolated from leek epidermal cells (1, 4, 83, 84).
Microsomes from leek containedtwo acyl-elongases: one that
elongated stearoyl-CoA or palmitoyl-CoA to arachidoyl-CoA
and the other that elongated arachidoyl-CoA to very longchain homologs (1). In a study conducted with potato tuber
slices, Walker and Harwood (134) obtained evidence for the
presence of three separate chain-specific elongases. After
slicing potato tubers, there was a sequentialinductionof acylCoA elongases that catalyzed the synthesis of 20:0, 22:0, and
24:0 fatty acids.
Recently, the effects of carbamothioateherbicides on in
vitro incorporationof [14C]malonyl-CoAinto VLCFA were
examined in a microsomal fraction isolated from germinating
pea seeds (54). The microsomal fraction isolated from
untreated, germinating pea seeds catalyzed the synthesis of
VLCFA (20:0, 22:0), but the microsomal fraction isolated
from seeds pretreatedwith 100 FM triallate or diallate was
not able to synthesize VLCFA. These results indicated that
the herbicides (or metabolites such as sulfoxide derivatives)
prevented the synthesis of acyl-CoA elongases or inhibited
their activity in vivo (54). The in vitro effects of
carbamothioates were also examined. Diallate and triallate
inhibited elongation of fatty acids in a microsomal preparation from germinating pea seeds. However, rather high
concentrations(100 [iM) were requiredand the effect was not
selective for the synthesis of VLCFA. Total fatty acid
biosynthesis was also strongly inhibited. Two interpretations
were suggested to explain the selective effect of carbamothioates on in vivo synthesis of VLCFA in pea (53, 75, 121) but
lack of selective effect in vitro (54). It was suggested that a
metabolite of the carbamothioate, such as its sulfoxide
derivative, could be generated in vivo and act as a selective
and potent inhibitor of acyl-CoA elongases. Alternatively,
under in vivo conditions, carbamothioatesmay be rapidly
metabolizedor poorly translocated,and hence are only able to
exert their inhibitory effect on lipid biosynthesis at the plant
surface where VLCFA are being synthesized.
There are conflicting reports concerning the effects of
carbamothioateson de novo fatty acid biosynthesis. EPITC
had no effect on the incorporationof [14C]malonicacid into
lipids of sesbania [Sesbania exaltata (Raf.) # (SEBEX)]
hypocotyl segments (90). However, EPTC and other carbamothioates inhibited the incorporation of radiolabeled
precursors ([14C]acetate, [14C]malonate)into fatty acids in
isolated spinach (Spinacia oleracea L.) chloroplasts (148,
149). EPIC also inhibited the incorporationof [14C]acetate
into lipids of maize (Zea mays L.) cell suspension cultures
(38). As discussed above, high concentrations of carbamothioates inhibited de novo fatty acid biosynthesis in a
membranefraction isolated from germinatingpea seeds (54).
Two hypotheses have been proposed to explain the
inhibitory effects of carbamothioateson lipid biosynthesis. A
common assumption of both hypotheses is that the sulfoxide
derivatives of carbamothioates, formed in vivo, act as
alkylating (carbamoylating) agents. Carbamothioates are
metabolizedin vivo by a two-stepprocess.The first step,
catalyzedby a cytochromeP-450 or a peroxidase,resultsin
the formationof a sulfoxidederivative(77, 112).The second
stepinvolvesthe conjugationof the sulfoxidederivativewith
glutathione(77, 78, 112). One hypothesisproposes that
sulfoxidederivativesof carbamothioates
alkylate(carbamoylate)key enzymesinvolvedin fattyacidbiosynthesis(43, 78,
112). There is, however, no evidence to support this
hypothesis.The second hypothesisproposesthat the carbamothioate-sulfoxide
derivative can alkylate CoA and
therebyinterferewith CoA metabolism(43, 78). Alkylation
of CoA could depletecells of this cofactorwhich plays an
role in the transferof acyl groupsduringfattyacid
important
biosynthesis.Alternatively,the CoA conjugates of carbamothioatesulfoxides could interferecompetitivelywith
reactionsutilizing acetyl-CoAor other CoA intermediates.
The sulfoxidederivativeof EPIC can alkylateCoA in vitro
(78), but it is not knownwhetherthis conjugateis formedin
vivo. It is interestingto note thatxenobiotic-CoAconjugates
canbe formedin vivo in certainmammalian
tissuesandit has
been hypothesizedthat these conjugates could inhibit
enzymesusing acetyl-CoAor otheracyl-CoAsas substrates
(18).
Recently,it has been proposedthat the carbamothioate
molecule itself (as opposed to its sulfoxide derivative)
inhibitskey enzymesinvolvedin the synthesisof acetyl-CoA.
Wlkinson and Oswald (151) reported that EPTC, at
submicromolar
actedas a reversibleinhibitor
concentrations,
of threeenzymesthat catalyzethe synthesisof acetyl-CoA:
the plastidic and mitochrondrialpyruvate dehydrogenase
complexes, and the plastidic acetyl-CoAsynthetase.This
hypothesisdiffers from the alkylationhypothesisdescribed
above in that the carbamothioate,
ratherthan its sulfoxide
derivative,is the herbicidallyactive form of the molecule,
and the interactionis reversibleas opposedto irreversible.
CHLOROACETAMIDES
The chloroacetamides
are appliedpreemergenceand are
used primarily for control of grass weeds (80, 115).
Representative
structures
of this herbicideclass are shownin
Figure3. Chloroacetamide
herbicideshave many properties
in commonwith carbamothioates.
Bothherbicideclasses are
consideredto be generalgrowthinhibitorsthat impairearly
seedlinggrowthof grasses(80, 115). Chloroacetamides
and
carbamothioatesare also similar in their spectrum of
selectivityand the injurysymptomswhich they induce in
grasses (80, 115). Furthermore,
chloroacetamides,
like carbamothioates,
inhibitseveralmetabolicprocesses(43, 69, 80,
115) but the primarysite of actionhas not been identified.
Chloroacetamides
inhibitlipidmetabolism(20, 90, 139, 140)
as well as the synthesisof proteins(26, 31, 80, 93, 115),
gibberellins(80, 115, 142), andproductsof the phenylpropanoid pathway(lignin, anthocyanin)(97). This review will
only discuss the purportedeffects of chloroacetamides
on
lipid metabolism.For informationregardingthe effects of
chloroacetamides
on othermetabolicprocesses,the readeris
referredto two recentreviews (80, 115).
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437
GRONWALD: LIPID BIOSYNTlIESIS INHIBITORS
CO -CH2C1
H=C-O2N
-CO - CH2CI
/\N
C/H'
/NO-H2I
H2C=CH-CH2
CH-CH3
-
I
CH3
Propachlor
CDAA
CF3
CO-CH2CI
2
"CH OCH3C
CH200H3
C2H5
Alachlor
NH
N2
0
/ ~~ NN
o
Ci
NCH3
/
0
ci
SAN 6706
Pyrazon
NQ
~CH3
N /,CH3
H
N
Ci
BASF 13-338
SAN 9785
CF3
2
2H5 CO-CH2CI
CO-CH2CI
AN
AN
CH -CH2 -OCH3
OH, OH3I
Metolachlor
/CH3
N
g
\
CH2-OC2H5
OH3
Acetochlor
CO-CH2C
CH2- N -N
-=
0
CI
OH3
Metazachlor
Figure 3. Chemical structures of selected chloroacetamides.
There are contradictoxyreports regarding the effects of
chloroacetamideson lipid biosynthesis. Some reportssuggest
that chloroacetamides have little or no effect on lipid
metabolism. Neither alachlor [2-chloro-N-(2,6-diethylphenyl)N-(methoxymethyl)acetamide]nor metolachlor [2-chloro-N1(2-ethyl-6-methylphenyl)-N-(2-methoxyinhibited incorporation of
methylethyl)acetamide]
[14C]acetateor [14C]malonicacid into different lipid classes
in excised cotton (Gossypium hirsutum L.) root tips (94).
Alachlor (8.2 pM) did not alter total lipid biosynthesis or
fatty acid composition of sorghum(Sorghumbicolor L.) roots
(138). Furthermore,metolachlor (10 p) had no effect on
acetate incorporation into total lipids or individual lipid
classes of sorghum roots (158).
In conflict with the above, there are reportswhich suggest
that chloroacetamides inhibit lipid biosynthesis. Butachlor
-2 -chloro-N- (2, 6 -diethyl[(N- (buthoxymethyl)
phenyl)acetamide] (20) inhibited the incorporation of
[14C]acetateinto lipids of isolated leaf cells of red kidney
bean (Phaseolus vulgaris L.), while CDAA [2-chloro-N,N-diinhibited incorporation
of
2-,propenylacetamide]
[14C]malonateinto lipids in excised hypocotyls of sesbania
(90). Incorporation of [14C]acetate into lipids of sorghum
protoplasts was strongly inhibited by 1 pM metolachlor
(159). In the green alga (Scenedesmus acutus), metazachlor
[N-(2,6-dimethylphenyl)-N-(1 -pyrazolyl-methyl)-chloroacetamide] inhibited the incorporation of [14C]acetate into
polar lipids (139). In further studies with Scenedesmus
acutus, Weisshaer et al. (140) reportedthat treating this alga
with 5 FM alachlor or metazachlordecreased linolenic acid
content but increased the levels of palmitic and oleic acid.
The authors concluded that chloroacetamides inhibit fatty
acid biosynthesis in Scenedesmus at some point between the
elongation of palmitic acid and the desaturationof oleic acid.
They speculated that chloroacetamidesmay inhibit plastidic
desaturases.
In addition to their effects on de novo fatty acid
biosynthesis, chloroacetamidesalso inhibit the synthesis and
alter the composition of epicuticularwax on the primaryleaf
of developing sorghum seedlings (33, 34).
The mechanisms proposed to explain the inhibitoryeffects
of chloroacetamideson lipid metabolism are similar to those
438
/C~H
-
Norflurazon
Figure 4. Chemical structures of selected substituted pyridazinones.
describedabove to explain the inhibitoryeffects of carbamothioateson lipid metabolism.Chloroacetamides,
like
have been hypothesizedto interferewith
carbamnothioates,
fatty acid metabolismeither by alkylatingkey enzymes
involvedin fattyacidbiosynthesisor by alkylatingCoA and
thereby interferingwith CoA metabolism(43, 68, 69).
Jaworski(68) firstproposedthatthe mechanismof actionof
chloroacetamides
mightrelateto their activityas alkylating
agents.He postulatedthatCDAA interferedwith respiration
and other metabolicprocesses by alkylatingcertain sulfhydryl-containingenzymes. Later work by Hamm (49)
demonstrateda strong correlationbetween the herbicidal
activity of chloroacetamidesand their ability to act as
alkylatingagents. As of yet, there is no evidence that
chloroacetamides
alkylatespecificenzymesin vivo. However, it has been shown, both in vivo and in vitro, that
can alkylateplant proteins(93).
chloroacetamides
Since the report by Jaworski (68), there has been
speculationthat the diverseeffects of chloroacetamides
on
metabolismmay be relatedto their abilityto alkylateCoA
(43, 97). CoAplaysan important
rolein lipidmetabolismand
othermetabolicprocessesthat are inhibitedby chloroacetamides.The in vitroformationof a CoA conjugateof alachlor
has been reported(79). However,thereis no evidencethat
CoA conjugatesof the chloroacetamides
are formedin vivo.
SUBSTITUTEDPYRIDAZINONES
The structuresof representative
substitutedpyridazinones
areshownin Figure4. Minordifferencesin structure
of these
herbicidesresult in pronounceddifferencesin target site
specificity. Dependingon applicationrate and the plant
species to which they are applied,the substitutedpyridazinones shown in Figure4 can inhibit one or more of the
following:a) photosynthesis,b) carotenoidbiosynthesis,c)
fatty acid desaturation(35, 54, 60, 123, 124, 137).
The herbicidal effect of pyrazon [5-amino-4-chloro2-phenyl-3(2H)-pyridazinone]
is largelydue to its abilityto
inhibitphotosynthetic
electrontransport(35, 123). Veryhigh
concentrations
of pyrazonarerequiredbeforeeffectson fatty
acidcompositionor carotenoidbiosynthesisareobserved(35,
123). As indicatedby competitivebinding studies, this
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WEED SCIENCE
herbicide binds to D1, the quinone-binding protein on the
reducing side of photosystem 1I (123). However, comparedto
triazines and ureas, pyrazon is not a potent inhibitor at this
site. The I50 values for inhibition of photosystem II electron
transportare 7 pM for pyrazon and 0.14 pM for diuron [N'(3,4-dichlorophenyl)-N,N-dimethylurea](123).
Norflurazon
[4-chloro-5-(methylamino)-2-(3(trifluoromethyl)phenyl)-3(2H)-pyridazinone]causes bleaching in susceptible species (54, 60, 110, 111). It inhibits
carotenoidbiosynthesis which results in the photooxidationof
chlorophyll. Current evidence suggests that norflurazon
blocks carotenoid biosynthesis by inhibiting a desaturase,
located in chloroplastmembranes,that catalyzes the desaturation of phytoene. This hypothesis has been difficult to test
because this enzyme is unstable during isolation and hence is
not readily amenable to in vitro assay (111). However, a
recent study conducted with a chromoplast fraction isolated
from daffodil (Narcissus pseudonarcissus L.) flowers lends
credence to this hypothesis (92). In this system, norflurazon
was found to be a reversible, noncompetitive inhibitor of
phytoene desaturase.
To varying degrees, the substitutedpyridazinonesalter the
fatty acid composition of lipids (29, 54, 60). In particular,
they increased the degree of saturation of certain lipids,
primarilygalactolipids, by blocking the conversion of linoleic
acid to linolenic acid (29, 54, 60, 82, 122, 123, 124, 125,
137). Galactolipids [monogalactosyldiacylglycerol (MGDG)3, digalactosyldiacylglycerol(DGDG)3] make up a large
portion of chloroplast membranes, and the fatty acids
associated with these lipids exhibit a high degree of
unsaturation (70). Compared to other substituted pyridazinones,
BASF 13-338
(SAN 9785)
[4-chlorois rather se5(dimethylamino)-2-phenyl-3(2H)-pyridazinone]
lective in its mode of action. It inhibits desaturationof fatty
acids with little or no effect on photosynthesis or carotenoid
biosynthesis (29, 35, 54, 60, 123). Research by several
investigatorshas shown that this herbicide causes an increase
in the 18:2/18:3 ratio found in the galactolipid fraction of
plant tissues (10, 71, 81, 98, 101, 122, 125, 152). Data
illustratingthis effect on the MGDG fraction of wheat shoots
are shown in Table 1. Treatmentwith BASF 13-338 caused a
pronounced decrease in the level of linolenic acid with a
concomitant increase in the level of linoleic acid. The data in
Table 1 also show that pyrazon has little effect on the degree
of unsaturation of the MGDG fraction. As stated above,
pyrazon acts primarily as an inhibitor of photosynthesis (35,
54, 60, 123).
It is not surprisingthat BASF 13-338, being a relatively
selective inhibitorof fatty acid desaturationin plastids, is not
a potent herbicide. Treatment with this herbicide causes
minor changes in chloroplast ultrastructure which are
attributedto the decrease in linolenate content of plastidic
membranes (25, 81).
SAN 6706 [4-chloro-5-(dimethylamino)-2(a,a,a-trifluorom-tolyl)-3(2H)-pyridazinone] is a substituted pyridazinone
that inhibits both carotenoid biosynthesis and fatty acid
desaturation (35, 54, 60, 71, 123). In some species, SAN
6706 is metabolized to norflurazon(128), which explains its
Table 1. Effect of substituted pyridazinones on fatty acid composition of
MGDG of wheat shoots.
Herbicide
(0.1 mM)
C16:0
Control
Pyrazon
BASF 13-338
(SAN 9785)
Fatty acid composition
C18:0
C18:2
C18:1
15.0
17.2
3.3
5.3
21.2
5.0
% by weight
4.3
19.0
21.1
6.1
5.6
53.9
C18:3
58.9
50.2
14.1
From St. John (122).
inhibitoryeffect on carotenoidbiosynthesis.At high concentrations,SAN 6706 inhibits the desaturationof 18:2 in
galactolipids(25, 71). However,at lower concentrations,
it
appearsto be a selectiveinhibitorof the desaturation
of 16:0
to fonn trans-A3-hexadecenoic
acid [16:1(t)]3in the sn-2
position of phosphatidylglycerol,
the major phospholipid
found in plastids(25, 71).
The effects of BASF 13-338 and SAN 6706 on the
desaturation
of fattyacidshavebeenattributed
to theirability
to inhibitdesaturasesthat catalyzethese reactions(29, 54,
60). These enzymes,which utilize complexlipids [MGDG,
DGDG, phosphatidylglycerol(PG)3] as substrates, are
thoughtto be locatedin the chloroplastenvelope(70). It has
been hypothesizedthat the chloroplastenvelope contains
multipledesaturases(11, 25, 82) andthatthe selectiveeffect
of certainsubstitutedpyridazinones
is due to theirabilityto
selectivelyinhibitone or moreof theseenzymes(29, 54, 60).
The selectiveeffectof BASF 13-338on the 18:2/18:3ratioof
MGDG(Table1) has been attributed
to its abilityto inhibit
the A-15 desaturasethat catalyzesthe desaturation
bf 18:2
bound to MGDG (25, 29, 54, 60, 82, 98, 152). Likewise, it
has been proposedthat SAN 6706 (at low concentrations)is a
specific inhibitor of the desaturasecatalyzing the conversion
of 16:0 to 16:1(t) at the sn-2 position of phosphatidylglycerol
(71).
Testing the hypothesis that selected substituted pyridazinones inhibit fatty acid desaturaseshas been difficult because
these enzymes have proven to be refractory to in vitro
characterization.
However, Nornan et al. (102) recently
reportedthat a chloroplastmembranefractionisolatedfrom
soybeanleavescontaineda desaturase(s)
capableof desaturating 18:2to 18:3in eitherthe sn-i or sn-2 positionof MGDG.
BASF 13-338 inhibitedthis reaction.
ARYLOXYPHENOXYPROPIONIC
ACIDS AND
CYCLOHEXANEDIONES
Introduction.The aryloxyphenoxypropionates
andcyclohexanedionesare two relativelynew herbicideclasses that are
used for postemergencecontrol of annual and perennial
grassesin certainbroadleafcrops (30, 54, 60). Becauseof
their selectivity,these two herbicideclasses are sometimes
referredto as graminicides.
Representative
chemistriesof the
two classes are shown in Figures5 and 6.
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439
GRONWALD: LIPID BIOSYNTHESIS IHITORS
CI
Table2. Effect of haloxyfop (1 pM on precursoruptakeand incorporationinto
radicle tips of corn and soybeana.
N
CloaN;
e
?O-CHCOOC2H5
0
CF3
CH3
/
e
0
-CHCOO(CH2),CH,
CH,
Incubation period (h)
0
Fluazitop-butyl
Quizalofop-ethyl
4
8
% of control
Cl
/
\
/
O-CHCOOCH3
CH,
CF3
/
/
~~~~~-
Diclofop-methyl
O -CHCOOCH3
CH,
Haloxyfop-methyl
N
C
O-CHCOOC2H5
CH3
CI
Fenoxaprop-ethyl
Figure 5. Chemical structures of selected aryloxyphenoxypropionates.
Both herbicide classes are foliar applied. They are readily
absorbedand translocatedto meristematicregions where they
exert their herbicidalactivity imgrasses (8, 30, 46, 54, 58, 60,
64, 66, 86, 87, 130). The aryloxyphenoxypropionatesare
applied as esters which are rapidly converted to acids via
carboxylesteraseactivity upon entering the leaf (12, 30, 57).
The acid form of the herbicide, which is consideredto be the
active form, is translocatedto meristematicregions (12, 30,
57).
In addition to having a similar spectrumof selectivity, the
aryloxyphenoxypropionic acids and the cyclohexanediones
cause similar injury symptoms in grasses. Initially, treated
plants exhibit chlorosis in developing leaves and a cessation
of growth (2, 30, 85, 86, 87). Within a few days, necrosis of
the shoot apex and meristematicregions of leaves and roots is
apparent (2, 8, 30, 58, 65, 130).
Most evidence suggests that the selective action of both
the aryloxyphenoxypropionicacids and the cyclohexanediones is not due to differentialabsorptionor translocation(30,
54, 60). Differential metabolism contributesto the selectivity
of the aryloxyphenoxypropionicacid diclofop {(?)-2-[4-(2,4dichlorophenoxy)phenoxy]propanoicacid). Wheat (Triticum
aestivum L.) is tolerant to diclofop because it can metabolize
the herbicide (28, 118). However, for most grasses, the
selectivity of the cyclohexanediones and aryloxyphenoxypropionic acids cannot be attributedto differential absorption, translocation, or metabolism which suggests that
selectivity residues at the site of action (12, 30, 54, 60).
Mechanism of action. The fact that both the aryloxyphenoxypropionic acids and cyclohexanediones cause the same
injury symptoms in grasses suggested that they may have a
common site of action. Much of the early work regardingthe
mechanism of action of the aryloxyphenoxypropionicacids
was conducted by Hoppe and co-workers (58, 59, 61, 62). In
1981, Hoppe (58) demonstratedthat lipid biosynthesis in corn
(Zea mays L.) radicles was impaired by diclofop. This
herbicide strongly inhibited the incorporationof [14C]acetate
into fatty acids while having little or no effect on the
440
Corn
[14C]Leucine:
Uptake
Incorporation
93
95
94
97
109
97
[14C]Uracil:
Uptake
Incorporation
110
95
117
124
95
105
103
109
99
116
103
94
98
106
99
79
90
95
100
106
[14C]Thymine:
Uptake
Incorporation
[14C]Acetate:
Uptake
Incorporation
Soybean
[14C]Acetate:
Uptake
Incorporation
85*
42*
99
111
*Indicates value significantly different from control using the t-test
(0.05).
aRadicle tips (0.5 cm) were incubated in a buffer containing 1 Jim
haloxyfop for the period indicated, then transferredto a solution containing
the radiolabeled precursors. Uptake and incorporation of precursor were
measured after 30 win.
From Stoltenberg (126).
incorporation
of precursorsinto nucleicacids or proteins.In
subsequentstudies,Hoppeand co-workers(59, 61, 62) and
others (14, 15, 23, 72, 114, 126, 135) have shown that the
aryloxyphenoxypropionic acids are potent inhibitors of
[14C]acetateincorporationinto fatty acids of grasses while
having little or no effect on [14C]acetateincorporationinto
fatty acids of dicots. Data from Stoltenberg (126) illustrates
the selective effect of the aryloxyphenoxypropionic acid
{2- [4-[[3-chloro-5-(trifluoromethyl)-2haloxyfop
pyridinyl]oxy]phenoxy]propanoicacid} on lipid biosynthesis
in corn radicles (Table 2). As determined by precursor
OH
H3
H2CO
~s
OCH3
N -O - CH2 -CH3
OH
N -O - CH2 -CH,
C -CH2 -CH2 -H3
C -CH2 -CH2 -CH3
0
a
CH
~CH2
Sethoxydim
Cycloxydim
OH
I
N-O-CH2-CH=CH--CI
11H-
I
C - CH2 -CH3
CH3 CH3
I
I
H2C
CH
's
"-OH2
0
Clethodim
Figure 6. Chemical structures of selected cyclohexandiones.
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7
120
0
o0
6
o
o
N
~~~~~PEA
\
80
5
0
0
N
E
40
4
C)~~~~~~~~~~~1
\
220
CORN
0
(n
0 2
10-7
10-6
10-5
10-4
10-3
Herbicide Concentration (M)
0
Figure 7. Effect of haloxyfop (-) and sethoxydim (- -) on ACCase activity
measured in chloroplast fractions from com (--)
and pea (-0-). From
Burton et al. (15).
0.1
incorporation studies, lipid biosynthesis was much more
sensitive to haloxyfop than was nucleic acid or protein
synthesis. Furthermore,the data indicate that impaired lipid
biosynthesis in the presence of haloxyfop was not the result
of inhibited [14C]acetateuptake. The inhibition of acetate
uptake was much less than the inhibition of acetate
incorporationand most likely reflected the inhibition of fatty
acid biosynthesis.
Early studies conducted with the cyclohexanediones also
suggested that this herbicide class interfered with lipid
metabolism. Burgstahlerand Lichthenthaler(13) reportedthat
{ 2-[l -(ethoxyimino)butyl]-5-[2sethoxydim
(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one} reduced
glycolipid and phospholipid content of corn seedlings. The
authorsconcluded that sethoxydim inhibited an early stage in
lipid metabolism in grasses. Further evidence for this
hypothesis was provided by Ishiharaet al. (67) who reported
that lipid biosynthesis in corn root tips was more sensitive to
sethoxydim than was ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or protein synthesis. Several recent
reports have demonstratedthat sethoxydim and other cyclohexanediones are potent and selective inhibitors of
[14C]acetate incorporation into lipids of sensitive grass
species (14, 15, 42, 63, 73, 106, 114).
The above studies clearly showed that both classes of
graminicides were selective inhibitors of de novo fatty acid
biosynthesis (as measured by [14C]acetateincorporation)in
grasses. In order to more clearly define the target site of the
two herbicide classes, Burton et al. (15) examined the effect
of haloxyfop and sethoxydim on the incorporation of
[14C]acetate, [14C]pyruvate,and [14C]malonyl-CoAinto fatty
acids in isolated corn chloroplasts. Incorporation of
1.0
10.0
Herbicide Concentration (tM)
Figure 8. Effect of selected aryloxyphenoxypropionicacids and cyclohexanediones on ACCase activity isolated from BMS maize cell suspension
cultures. Fluazifop (-0-), sethoxydim (-A-), clethodim (-U-), cycloxydim
(-A-), haloxyfop (-4-), diclofop, (),
quizalofop (-*-). Burton and
Gronwald, unpublished results.
[14C]acetateand [14C]pyruvatewas inhibited 90% or greater
by 10 FM sethoxydim and 1 gM haloxyfop, while
incorporationof [14C]malonyl-CoAwas slightly stimulated.
On the basis of these results, Burton et al. (15) examined the
effects of sethoxydim and haloxyfop on acetyl-CoA carboxylase (ACCase)3 activity in chloroplastfractions isolated from
corn and pea. Both sethoxydim and haloxyfop were potent
inhibitorsof this enzyme from corn chloroplastsbut had little
or no effect on the enzyme from pea chloroplasts (Figure 7).
The I50 values for inhibition of corn ACCase by sethoxydim
and haloxyfop were 4.7 p.M and 0.5 juM, respectively.
Sethoxydim (1 mM) or haloxyfop (0.1 mM) did not inhibit
ACCase from pea chloroplasts. In further studies conducted
with ACCase isolated from Black Mexican Sweetcorn
(BMS)3 suspension cultures, it was shown that there were
differences in the relative efficacy of selected aryloxyphenoxypropionic acids and cyclohexanediones as inhibitors of this
enzyme (Figure 8). The lowest I50 value was obtained with
{ (?)-2-[4-[(6-chloro-2-quinoxquizalofop
alinyl)oxy]phenoxy]propanoic acid} and the highest with
sethoxydim and fluazifop {(?)-2-[4-[[5-trifluoromethyl)2-pyridinyl]oxy]phenoxy]propanoicacid}.
Within a period of several months during the latterpart of
1987 and the first half of 1988, there were several reports(14,
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441
GRONWALD: LIPID BIOSYNTHESIS INHIBITORS
20 -S-)
3 E 15 -
~~~0
S
cE 1o
_
5
0
0.1
1.0
10.0
100.0
[Haloxyfop] pM
Figure 9. Effect of the S(-) and R(+) enantiomers of haloxyfop on the
activity of ACCase isolated from maize leaves. From Secor et al. (114).
15, 42, 72, 73, 106, 107, 113, 136) describing the
aryloxyphenoxypropionicacids and the cyclohexanediones as
potent and selective inhibitors of ACCase in grasses. These
reports provide compelling evidence that ACCase is the site
of action of the aryloxyphenoxypropionicacids and cyclohexanediones and that the basis for selectivity between grasses
and dicots resides at this site in most cases.
It is not surprising that inhibition of ACCase by
aryloxyphenoxypropionic acids and cyclohexanediones is
lethal in grasses. These herbicides are translocatedto regions
of cell division and elongation (shoot and root apices,
intercalary meristems) where there is a high demand for
malonyl-CoA to support rapid rates of de novo fatty acid
biosynthesis. Inhibition of lipid biosynthesis by these two
herbicide classes would account for their disruptiveeffects on
plastid ultrastructure(8, 86, 87) and membranepermeability
(8), and the eventual necrosis that develops in meristematic
regions (2, 46, 66, 130). Lipid biosynthesis would not be the
only metabolic process disrupted by ACCase inhibitors.
Malonyl-CoA also serves as a key intermediate in the
synthesis of cuticular waxes, flavonoids, anthocyanins,
stilbenoids, and anthraquinones(100, 129).
Futher evidence that ACCase is the site of action of the
graminicides is provided by: a) the stereospecificity of the
activity of aryloxyphenoxypropionicacids at both the whole
plant and ACCase level (27, 30, 45, 62, 113, 114); and b) the
expression of graminicidetolerance in selected grasses due to
either the presence of a tolerantform of ACCase (48, 91, 105,
114) or overproduction of ACCase (104).
The aryloxyphenoxypropionicacids exist in two enantiomeric formnsbecause of the 2-substituted propionic acid
moiety of the molecule (30). When applied postemergence,
the R(+) enantiomers of diclofop (30), haloxyfop (30, 45),
quizalofop (133), or fluazifop (27) are herbicidally active on
grasses while the S(-) enantiomershave little or no herbicidal
activity. When applied to the soil, the S(-) enantiomers of
certain aryloxyphenoxypropionicacids (fluazifop, haloxyfop)
exhibit some herbicidal activity, but this may reflect
conversion of the S(-) form to the R(+) form by soil-borne
microorganisms (27, 45). The R(+) enantiomer of arylox442
yphenoxypropionic acids is also much more active as an
inhibitor of grass ACCase compared to the S(-) enantiomer
(62, 107, 113, 114, 136). The relative efficacy of the R(+)
and S(-) enantiomers of haloxyfop as inhibitors of ACCase
isolated from corn leaves (114) is shown in Figure 9.
Inhibition of ACCase by the S(-) form was attributedto
contaminationof this enantiomerform by a small amount of
the R(+) enantiomer (114).
A few grasses are tolerantto the aryloxyphenoxypropionic
acids and cyclohexanediones. With certain grasses, tolerance
is due to their ability to metabolize the herbicide (28, 30,
118). For example, wheat is tolerant to diclofop because it
can metabolize the herbicide (28, 30, 118). However, for
selected graminicide-tolerantgrasses, tolerance at the whole
plant level correlateswith tolerance at the level of ACCase.
This is observed in red fescue [Festuca rubra (L.) # FESRU]
(114, 127), a diclofop-resistant form of Italian ryegrass
(Lolium multiflorumLam. # LOLMU) (48) and graminicidetolerant maize lines selected in tissue culture (47, 91, 105).
Red fescue is tolerant to sethoxydim (17, 64, 88, 127),
tralkoxydim [2-(1-ethoxyimino)propyl]-3-hydroxy-5mesitycyclohex-2-enone] (114), and haloxyfop (114, 127)
while tall fescue [Festuca arundinacea (Schreb.) # FESAR],
a member of the same genus, is susceptible to these
herbicides. In these two fescue species, the differential
tolerance to haloxyfop, sethoxydim, and tralkoxydim expressed at the whole plant level paralleled tolerance at the
level of ACCase (114, 127).
Approximately 4 yr ago, a diclofop-resistant biotpe of
Italian ryegrass was found in an Oregon wheat field that had
been treatedwith diclofop for at least seven consecutive years
(120). Resistance to diclofop was due to the presence of a
tolerantform of ACCase (48). Breeding studies indicatedthat
resistance was controlled by a single, partially dominant
allele encoding a diclofop-insensitive form of ACCase (5). It
is interesting to note that the diclofop-resistant Italian
ryegrassbiotype from Oregon exhibits little or no toleranceto
sethoxydim at either the enzyme (ACCase) or whole plant
level (48).
Maize lines tolerant to sethoxydim and haloxyfop have
been selected from BMS nonregenerable(104) and embrogenic, regenerable cell cultures (47, 91, 105). In the BMS
system, the ACCase in tolerant cell lines exhibited no
differences in sensitivity to inhibition by sethoxydim or
haloxyfop (as determined by 150 values). However, the
tolerant lines exhibited higher ACCase activity measured in
the absence of the herbicides (104). As determined by
Westem blots, the selected cell lines had higher levels of a
220 000 relative molecular mass (Mr)3polypeptide, presumably the subunit of ACCase. These results suggested that
tolerancewas due to overproductionof ACCase in the variant
lines (104). In the regenerablecell culture system, lines were
selected for tolerance to sethoxydim or haloxyfop (47, 105).
Tolerance to the herbicides was expressed in the regenerated
plants (47, 105). Inheritancestudies indicated that tolerance
was controlled by a single, partially dominant allele that
encoded for a graminicide-tolerantform of ACCase (91, 105).
Breeding studies conducted with five regenerated granniVolume 39, Issue 3 (July-September) 1991
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cide-tolerant maize lines selected in tissue culture indicated
that resistance to sethoxydim and/or haloxyfop at the whole
plant level cosegregated with resistance at the level of
ACCase (91, 105).
The above reportsprovide strong evidence that ACCase is
the primary site of action of the graminicides. However, it
also has been proposed that the aryloxyphenoxypropionic
acids, and in particulardiclofop, exert their herbicidalactivity
in grasses by acting as protonophoresthat dissipate the proton
gradient across membranes (24, 116, 117, 154). This
mechanism has been referred to as the antiauxin hypothesis
(24, 117). Recently, Shimabukuro (116) described the
protonophoreeffect of diclofop as the biophysical mechanism
of action of this herbicide and suggested that it contributesto
the herbicidal activity of diclofop. Since this review concems
the effects of herbicides on lipid biosynthesis, the biophysical
or antiauxin hypothesis will not be discussed. For a
discussion regardingthis proposed mechanism of action, the
readeris referredto the recent review by Shimabukuro(116).
Properties of ACCase. ACCase is a multifunctional,
biotinylated protein located in the stroma of plastids. It
catalyzes the ATP-dependentcarboxylationof acetyl-CoA to
form malonyl-CoA. This reaction is the first committed step
in the de novo synthesis of fatty acids and there is speculation
that it might be the rate-limiting step in fatty acid
biosynthesis (50, 55, 132). The two partialreactions catalyzed
by the enzyme are shown below:
(1) Enzyme-biotin +HCO3- + ATP
+ ADP
*-4
Enzyme-biotin-COj-
+ Pi
(2) Enzyme-biotin-CO-
+
acetyl-CoA <- Malonyl-CoA
+ Enzyme-biotin.
These two reactions are thought to occur at distinct
topological sites on the enzyme with the biotin prosthetic
group serving as a mobile carboxyl carrierbetween the two
sites (50, 52, 129). The catalytic mechanism is described as a
hybrid two-site ping pong (41). The ATP-dependentcarboxylation of biotin occurs at the carboxylation site. The biotin
prosthetic group then moves to the carboxyltransferasesite
where the carboxyl group is transferred to acetyl-CoA
forming malonyl-CoA.
The ACCase in higher plants has not been as extensively
characterizedas the enzyme from mammals (103, 156, 157)
or yeast (95). Higher plant ACCase, like mammalianor yeast
ACCase, appears to be a multifunctional protein (50, 52).
Mammalian ACCase is a dimer with a subunit Mr of
approximately 260 000 (50, 52, 103, 156, 157). There is a
lack of agreement regardingthe Mr of the native enzyme in
higher plants. Reported Mr values for the native enzyme
range from approximately400 000 to 800 000 (21, 36, 37, 40,
50, 52, 96, 99, 129). There is also a lack of agreement
regardingthe Mr of the ACCase subunit.Reports by Nikolau
and colleagues (99, 100) indicated that maize leaf ACCase
had a subunit Mr of approximately62 000. However, other
reports suggest that the subunitMr of higher plant ACCase is
in the range of 200 000 to 240 000 (9, 16, 21, 36, 37, 40, 50,
52, 56, 96). It may be that the smaller subunit (62 000 Mr)
reportedfor plant ACCase reflects proteolysis of the enzyme
during isolation. Mammalian ACCase is known to be
susceptible to proteolysis during isolation (89, 131) and this
also appearsto be the case with the plant enzyme (36, 119).
Alternatively, the ACCase subunit of 62 000 Mr reportedin
maize leaves may representan isozyme of the enzyme (100,
155). It has been proposed that ACCase isozymes exist in
plants and that they may serve to regulate the utilization of
malonyl-CoA by different metabolic pathways (100, 129).
The regulation of higher plant ACCase has not been
extensively characterized.Similar to the mammalianACCase,
there is evidence that higher plant ACCase is inhibited by
malonyl-CoA (41, 99, 107, 108) and palmitoyl-CoA (99).
Malonyl-CoA was reported to act as a noncompetitive
inhibitor of ACCase from castor oil seed [Ricinus communis
(L.)] (41) and wheat leaves (107, 108). The ACCase from rat
liver is activated by CoA (156). CoA activated the ACCase
from spinach chloroplasts (76) and castor bean seeds (40),
had no effect on ACCase from soybean [Glycine max (L.)]
seed (21), and inhibited the enzyme from com leaves (99).
Like the ACCase from rat liver (157), plant ACCase is
regulated by adenine nucleotides. Adenosine diphosphate
(ADP) and adenosine monophosphate (AMP) inhibited the
activity of wheat germ ACCase by competing for adenosine
triphosphate(ATP)3 (32). In other respects, the regulationof
higher plant and mammalianACCase differs. In contrast to
mammalian ACCase, higher plant ACCase is not allosterically regulatedby phosphorylationor tricarboxylicacids (21,
22, 40, 52, 96, 107, 129). In addition, there is no evidence
that plant ACCase forrns polymers in situ as does the
mammalian enzyme (50, 52).
Mechanism of inhibition of ACCase. Kinetic analyses
revealed that aryloxyphenoxypropionicacids and cyclohexanediones are linear, noncompetitive inhibitors of grass
ACCase for all three substrates (MgATP, HCO3-, acetylCoA) of the enzyme (16, 106, 107, 108). Kinetic analyses
also indicated that the two herbicide classes are reversible
inhibitorsthat exhibit a high affinity for the enzyme (16, 106,
107, 108).
Of the two partial reactions catalyzed by ACCase, the
carboxyltransferasereaction is most sensitive to inhibition by
the aryloxyphenoxypropionicacids and cyclohexanediones
(16, 106, 107, 108). This is suggested by the fact that in vitro
inhibition of ACCase by the two herbicide classes is most
sensitive to the concentrationof acetyl-CoA, as opposed to
the other substrates of the enzyme (MgATP, HCO3-) (16,
106, 107, 108). Additional evidence for this hypothesis was
provided by Rendina et al. (108) who measuredthe effects of
diclofop and clethodim {(E,E)-(?)-2-[l-[[3-chloro-2propenyl)oxy]imino]propyl] -5 -[2-(ethylthio)propyl]-3hydroxy-2-cyclohexen-1-one} on the two partial reactions of
wheat ACCase. The partial reaction measured at the
carboxyltransferasesite (malonyl-CoA -['4C]acetyl-CoA exchange) was much more sensitive to these herbicides than
that measuredat the carboxylationsite (ATP-32Pi exchange).
For both the aryloxyphenoxypropionicacids and cyclohexanediones, the family of lines generated by kinetic analyses
of the inhibition of ACCase versus the three substratesof the
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443
GRONWALD: LIPID BIOSYNTHESIS INHIBITORS
Acetate
Pyruvate
PLASTID
:2-M
AryloxyphenoxyAcetyl-CoA
> '| ACCase
propionic Acids
Cyclohexanediones
Substituted
P
Syridazinones
esatu rase
1:3
Malonyl-CoA
-
MGDG
{FAS
16:0 - ACP .-
16:0 CoA
18:0 - CoA <
?18:0 - ACP -
o18:1 - ACP
0
M on
Very Long Chain
Wax Suberin Cutin
FattyAcidsWa,SbrnCui
CoA
'
ENDOPLASMIC
RETICULUM
Carbamothioates
Figure 10. Simplifiedschematicof fattyacidbiosynthesis
in higherplantsillustrating
proposedsitesof actionfor thecarbamothioate,
substituted
pyridazinone,
aryloxyphenoxypropionic
acid, andcyclohexanedione
herbicides.Takenin partfromHarwood(54). Abbreviations:
ACCase,acetyl-CoAcarboxylase;
ACP,
acyl carrierprotein;ACS, acetyl-CoAsynthetase;CoA, coenzymeA; FAS, fatty acid synthase;MGDG,monogalactosyldiacylglycerol;
PDC, pyruvate
dehydrogenase
complex.
enzyme are strikingly similar (16, 107, 108). This suggested
that the herbicides may bind at a common domain on the
enzyme. Multiple inhibition kinetic analyses have been used
to test this hypothesis. The results of two separate studies
demonstrated that the aryloxyphenoxypropionic acids and
cyclohexanediones are mutually exclusive inhibitors of grass
ACCase (16, 108). These results may indicate that the two
chemistries bind at a common site. However, it is not
necessary that two inhibitors bind at a common site in order
to be mutually exclusive. For two compounds to be mutually
exclusive simply means that the binding of one compound
prevents the binding of the other. It is possible that the
cyclohexanediones and the aryloxyphenoxypropionatesbind
at different sites on ACCase, with the binding of one
herbicide class inducing an allosteric change that preventsthe
binding of the other.
Additional multiple inhibition kinetic analyses conducted
with wheat ACCase indicated that both diclofop and
clethodim are mutually exclusive for malonyl-CoA but not
CoA (108). It was postulated that the aryloxyphenoxypropionic acids and the cyclohexanediones have a common
structuralfeature that overlaps with the thioester region of
acetyl-CoA and malonyl-CoA.
Recently, it has been proposed that the cyclohexanediones
may act as stable transition-stateanalogues of the complex
444
formed at the carboxyltransferase
site of ACCase (153).
Evidencein supportof this is basedsolely on modelingusing
moleculargraphics.It is interestingthat the cyclohexanediones (andaryloxyphenoxypropionic
acids)have propertiesas
inhibitors that would be expected for transition state
analogues;i.e., they arereversible,noncompetitive
inhibitors
that exhibit a high affinityfor a bindingsite.
SUMMARY
A simplifiedschematicof lipid biosynthesisin higher
plants(Figure10) will be used to summarizewhathas been
discussed regardingthe effects of the selected herbicide
classes on lipid biosynthesis.
Carbamothioates.Carbamothioates
inhibitthe synthesisof
surfacelipids presumablyby inhibitingacyl-CoAelongases
which catalyzethe synthesisof VLCFA(7, 54, 75). AcylCoA elongases have not yet been well characterizedbut
appearto be associatedwith the endoplasmicreticulumand
catalyzethe condensation
of malonyl-CoAwith variousacylCoA fattyacidprimers(1, 4, 83, 84). Shownin Figure10 is
the inhibition of elongation of stearoyl-CoAby carbamothioates.
Evidence suggesting that carbamothioatesinhibit one or
more acyl-CoA elongases is largely indirect. Treatmentwith
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WEED SCIENCE
carbamothioatesreduces the amountof surface lipid on plants
(7, 53, 75, 141, 145, 147). Furthermore,in vivo studies with
[14C]acetatehave indicated that elongation of C:16 and C:18
fatty acids is more sensitive to these herbicides than is their
de novo synthesis (7, 75). Recently, in vitro studies indicated
that diallate and triallate inhibitedthe synthesis of VLCFA in
a microsomal fraction from germinating pea seed (54).
However, relatively high concentrations (100 il) of the
herbicides were requiredand total fatty acid biosynthesis was
also strongly inhibited at these concentrations. It has been
proposed that the sulfoxide derivatives of carbamothioates,
which are known to be formed in vivo (19, 79), or CoA
conjugates of these derivatives, which have been hypothesized to be formed in vivo (43), may inhibit lipid synthesis. It
is possible that one of these carbamothioatemetabolites may
selectively inhibit acyl-CoA elongases in vivo. Further
research is needed to address this question.
The effects of carbamothioateson surfacelipids are one of
the best documentedeffects of this herbicide class. However,
this effect probably makes only a minor contributionto the
herbicidal activity of carbamothioates. It is unlikely that
impaired synthesis of cutin, wax, and suberinwould be toxic
in itself.
Chloroacetamides. There is not a clear picture regardingthe
effects of the chloroacetamides on lipid biosynthesis.
Chloroacetamides,like carbamothioates,inhibit the synthesis
of epicuticularwax (33, 34). However, reportsconceming the
effects of this herbicide class on de novo fatty acid
biosynthesis are contradictory. Recently, Weisshaar et al.
(140) suggested that this herbicide class may inhibit plastidic
desaturases. Chloroacetamides (like carbamothioates)have
been proposed to interfere with lipid metabolism by
alkylating CoA or key enzymes involved in lipid metabolism
(43, 68, 69).
Substituted pyridazinones. Certain representatives of this
chemistry appearto inhibit one or more fatty acid desaturases
associated with the chloroplast envelope (29, 54, 60). Based
on the in vivo effects of BASF 13-338 on fatty acid
composition, this herbicide appears to be a rather selective
inhibitor of the A-15 desaturaseresponsible for desaturation
of 18:2 bound to MGDG (29, 54, 60) (Figure 10). Recently, it
was demonstrated in vitro that BASF 13-338 inhibits
desaturationof 18:2 bound to MGDG (102). Although not as
selective in its mechanism of action as is BASF
13-338, SAN 6706 apparentlyinhibits the desaturaseconverting 16:0 to 16:1(t) in the sn-2 position of phosphatidylglycerol (25, 29, 54, 60, 71). BASF 13-338 and other
substituted pyridazinones should prove to be useful tools to
learn more about fatty acid desaturasesin plants and their role
in plant adaptation to various stresses.
While the substituted pyridazinones inhibit fatty acid
desaturationto varying degrees, it does not appear that this
effect makes a significant contribution to their herbicidal
activity. BASF 13-338, the substituted pyridazinone that
selectively inhibits fatty acid desaturationwith little or no
effect on photosynthesis or carotenoid biosynthesis, has
minimal phytotoxicity to plants. The most effective herbicides of the substituted pyridazinones are those that also
inhibit photosynthesis and/or carotenoid biosynthesis (123).
Aryloxyphenoxypropionic acids/cyclohexanediones. There
is strong evidence that aryloxyphenoxypropionicacids and
cyclohexanediones inhibit ACCase, a key enzyme that
catalyzes the first committed step in de novo biosynthesis of
fatty acids (14, 15, 42, 72, 106, 107, 113) (Figure 10). Of the
two partialreactionscatalyzed by ACCase, the carboxyltransferase reaction is most susceptible to inhibition by these
chemistries(16, 107, 108). Both chemistries appearto bind at
a common domain on ACCase (16, 107, 108). The
aryloxyphenoxypropionic acids and cyclohexanediones
should prove to be useful tools to furtherour understanding
about this important enzyme and to provide information
about the intriguing difference between grass and dicot
ACCase which governs the selectivity of these herbicide
classes.
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