Light-dependent herbicides: an overview

Author(s): F. Dan Hess

Source: Weed Science, 48(2):160-170. 2000.
Published By: Weed Science Society of America
DOI: http://dx.doi.org/10.1614/0043-1745(2000)048[0160:LDHAO]2.0.CO;2
URL: http://www.bioone.org/doi/full/10.1614/0043-1745%282000%29048%5B0160%3ALDHAO%5D2.0.CO
%3B2

BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and
environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published by
nonprofit societies, associations, museums, institutions, and presses.
Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of BioOne’s
Terms of Use, available at www.bioone.org/page/terms_of_use.
Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries or
rights and permissions requests should be directed to the individual publisher as copyright holder.

BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research libraries,
and research funders in the common goal of maximizing access to critical research.
Weed Science, 48:160–170. 2000

Review

Light-dependent herbicides: an overview

F. Dan Hess
AffyAgro Unit of Affymax Research Institute,
3410 Central Expressway, Santa Clara, CA 95051;
dan[email protected]

Activities of a surprisingly large number of herbicide fam- from a Mn cluster in the water-splitting complex. The elec-
ilies are directly or indirectly influenced by light. Herbicide tron in pheophytin is then passed to a protein-bound plas-
mechanisms that are dependent on light or are enhanced by toquinone molecule called QA, which in turn passes the
light fall into several different categories. These herbicides electron to a protein-bound plastoquinone called QB via a
inhibit electron flow in photosystem II in the photosyn- nonheme iron (Figure 1). When a second electron is passed
thetic light reaction (e.g., triazines, phenyl ureas, and ura- to QB from QA, the fully reduced quinone then becomes
cils), capture electrons in photosystem I in the photosyn- protonated (two hydrogen ions are added) to form a bound
thetic light reaction (e.g., bipyridiniums such as paraquat), plastohydroquinone (PQH2). The binding affinity of PQH2
inhibit glutamine synthetase in the nitrogen assimilation to its binding site is low; thus, another plastoquinone mol-
pathway (e.g., glufosinate), inhibit protoporphyrinogen ox- ecule from the plastoquinone pool in the membrane can
idase during chlorophyll biosynthesis (e.g., diphenyl ethers easily displace it (Figure 1). The function of the reduced
[DPEs] and N-phenyl heterocycles), directly inhibit carot- PQH2 is to transfer electrons between PS II and a cyto-
enoid biosynthesis by inhibiting one of the desaturase en- chrome b6f complex, which in turn transfers electrons to
zymes (e.g., norflurazon and fluridone), or indirectly inhibit photosystem I (PS I) via plastocyanin. Hankamer et al.
carotenoid biosynthesis by inhibiting a quinone cofactor in- (1997), Tsiotis et al. (1996), and Vermaas (1993) have pub-
volved in the desaturase reaction (e.g., triketones and isox- lished detailed reviews of the PS II reaction center.
azoles). Surprisingly, in all instances the involvement of light Herbicides that inhibit photosynthesis in PS II do so by
in herbicide action is not a direct interaction of light and binding to a protein at the lipophilic binding niche for the
herbicide at the primary site of action. In addition, the plastoquinone QB. Work published from Arntzen’s labora-
events that cause tissue damage (necrosis) are not associated tory (Pfister et al. 1981; Steinback et al. 1981) using azido-
with the primary target and are always due to membrane atrazine to achieve covalent bonding (photoaffinity labeling)
damage caused by lipid peroxidation of polyunsaturated fat- of the herbicide at its binding site was a key milestone in
ty acids. identifying the location of herbicide binding in PS II. This
protein was initially termed the 32-kDa protein, but it is
now known to be the D1 protein in PS II. Specificity of
Photosystem II the site was demonstrated by showing that binding of her-
Herbicide types such as triazines (e.g., atrazine) and phe- bicides to this protein did not occur in a triazine-resistant
nyl ureas (e.g., diuron) have their site of action in photo- mutant. Two years earlier, Arntzen et al. (1979) concluded
system II (PS II) in the photosynthesis light reaction. Symp- that because of small molecular weight differences identified
toms from herbicides that interfere with PS II slowly evolve between the resistant and susceptible biotypes of two pro-
over several days. First, treated plants develop a marked teins (a ⬃22 and ⬃24 kDa), one of these two proteins
chlorosis (yellowing), which is then followed by necrosis (tis- would turn out to contain the triazine binding site. In a
sue death). The chlorosis is due to chlorophyll destruction recent conversation, Charlie Arntzen stated ‘‘. . . the data in
through photooxidation reactions in the chloroplast, and the the 1979 publication made me sure we had a lead on the
necrosis is due to membrane destruction through lipid per- identity of the triazine receptor. But when Klaus Pfister and
oxidation. The association of light with the mechanism of Kit Steinback came to me only a year later with the first
action of these herbicides is indirect and due to the light autoradiogram of proteins separated by gel electrophoresis
energy absorbed by chlorophyll not being dissipated in PS after photoaffinity labeling of thylakoid proteins with azido
II. There is no direct involvement of light at the herbicide atrazine, I immediately changed my mind. It clearly showed
target in PS II. a tag on a 32-kilodalton protein. It was the cleanest first
In PS II, light energy captured by light-harvesting pig- experiment I’ve ever seen.’’ After repeating the experiment,
ments (chlorophylls and carotenoids) is transferred to a spe- the photoaffinity data were used as the basis for the 1981
cial reaction center (P680; a chlorophyll a dimer) providing publications.
the necessary energy for electron transfer from P680 to pheo- Herbicides that inhibit electron flow in PS II show com-
phytin (Figure 1). An electron obtained from a tyrosine res- petitive binding kinetics with QB and, therefore, compete
idue (YZ) in the D1 protein (Figure 1) replaces the lost for this binding niche in the D1 protein. This competition
electron in P680. The tyrosine residue obtains its electron leads to herbicide displacement of the QB from its binding

160 • Weed Science 48, March–April 2000

 FIGURE 2. Initiation of lipid peroxidation by blocking electron flow in pho-
FIGURE 1. Electron transport in the photosystem II (PS II) complex em- tosystem II (PS II). When herbicide (HERB) replaces plastoquinone at the
bedded in thylakoid membranes of chloroplasts. D1 and D2 are proteins exchangeable quinone binding site (QB), energy transfer from singlet chlo-
and arrows within these proteins represent electron flow. Electrons move rophyll (1CHL) to PS II is inhibited. In the light, singlet chlorophyll ac-
from the water-splitting complex to a tyrosine residue in the D1 protein cumulates and undergoes intersystem crossing (i.s.c.) to generate a longer
(YZ), then to a chlorophyll a dimer (P680) bound in the PS II complex. lived triplet chlorophyll (3CHL). Triplet chlorophyll may be capable of
Electrons are then transferred to pheophytin (PHEO), to a bound plasto- directly initiating lipid peroxidation in polyunsaturated fatty acids in the
quinone at the QA domain in the D2 protein, and finally to an exchange- chloroplast membrane. Alternatively, triplet chlorophyll interacts with ox-
able plastoquinone at the QB domain in the D1 protein. When the ex- ygen to form singlet oxygen (1O2), which can then serve as the initiating
changeable plastoquinone receives two electrons from the bound plasto- factor for lipid peroxidation. Carotenoids quench triplet chlorophyll and
quinone at the QA binding domain and two hydrogen ions from the stro- singlet oxygen; however, the abundance of these reactive substances formed
ma, the plastohydroquinone (PQH2) releases into the membrane and is when electron flow in PS II is blocked by a herbicide are substantially above
replaced by another plastoquinone (PQ) molecule. Photosynthesis inhibitor the capacity of the carotenoids.
herbicides (HERB) compete with the exchangeable plastoquinone within
the QB binding domain. Electron flow stops when herbicide molecules
occupy the QB binding domain. phyll is formed during normal photosynthesis, but it is dis-
sipated by carotenoids. Because of what must be a massive
site, which stops electron flow through PS II (Figure 1). A number of triplet chlorophyll molecules produced by block-
great deal is now known about the structure of the D1 ing electron flow through PS II, the carotenoid quenching
protein (e.g., Trebst and Draber 1986). The availability of system is completely overloaded. The excess triplet chloro-
high-resolution X-ray crystal structures of the reaction center phyll causes initiation of lipid peroxidation by two mecha-
from photosynthetic bacteria has yielded several computer nisms. Although somewhat equivocal, one is the direct for-
models of the QB binding niche in the D1 protein (e.g., mation of a lipid radical in unsaturated fatty acids. In a
Tietjen et al. 1991; Xiong et al. 1996). There are five hy- second mechanism, triplet chlorophyll reacts with oxygen to
drophobic amino acid helices in the D1 protein that span produce singlet oxygen (Figure 2). Some singlet oxygen is
the thylakoid membrane. The QB binding pocket is located produced during normal photosynthesis and is dissipated by
near the surface of the membrane on the stroma side be- carotenoids, but as in the case of carotenoid quenching of
tween the fourth and fifth span of the D1 peptide through triplet chlorophyll, the ability of the carotenoid system to
the membrane and a connecting parallel helix between these quench singlet oxygen becomes completely overloaded in
two membrane-crossing helices. the presence of photosynthesis inhibitors. Research has
Early reports suggested weeds treated with PS II inhibi- shown measurable singlet oxygen levels occur in chloroplasts
tors died by ‘‘starving to death’’ as a result of the inhibition exposed to light and diuron (Chauhan et al. 1992). The
of the light reaction of photosynthesis, causing an indirect excess singlet oxygen then initiates lipid destruction through
inhibition of sugar production in the Calvin cycle through the formation of lipid radicals in polyunsaturated fatty acids
the depletion of ATP and NADPH supplied from the light (see last section).
reaction. However, plants die faster if treated with PS II
inhibitors and placed in the light than if treated and placed
in the dark. This shows that something other than inhibi-
Photosystem I
tion of carbohydrate synthesis is responsible for the observed In contrast to development of chlorosis and necrosis over
herbicidal effect. The leaf necrosis that develops after her- several days by inhibitors of electron flow in PS II, herbi-
bicide treatment is due to membrane damage. When chlo- cides that interfere with PS I, such as paraquat and diquat
rophyll accepts light energy, it changes from a ground energy (the bipyridiniums), develop symptoms within a few hours
state to a singlet energy state. This singlet energy is normally after treatment. Initial symptoms are the occurrence of dark
transferred to the P680 reaction center, and the chlorophyll green areas on treated leaves, which is often termed ‘‘water
molecule returns to the ground state (Figure 2). When elec- soaking.’’ These dark green areas are the result of substantial
tron flow is blocked by herbicide binding in the QB pocket plasma membrane disruption that allows cell contents to
of the D1 protein, the singlet chlorophyll energy cannot be leak into the intercellular spaces, changing the refractive in-
transferred to the PS II reaction centers. The short-lived dex of these areas. Within 1 or 2 d after this membrane
singlet energy state of chlorophyll molecules accumulate and disruption, affected areas become necrotic due to tissue des-
some are transformed to a longer lived triplet energy state iccation. As in the case of PS II inhibitors, the involvement
through intersystem crossing (i.s.c.). Some triplet chloro- of light in the mechanism of action of bipyridylium herbi-

Hess: Light-dependent herbicides • 161

 FIGURE 4. Paraquat, a di-cation bipyridylium herbicide, captures electrons
from PS I during electron flow in photosynthesis and becomes a free radical
(mono-cation). The paraquat free radical is unstable and rapidly undergoes
autooxidation back to the parent ion. During the autooxidation process,
FIGURE 3. (A) Electron transport in the photosystem I (PS I) complex superoxide radicals (O2·⫺) are produced from molecular oxygen. Superoxide
embedded in thylakoid membranes of chloroplasts. Arrows within the PS can undergo enzymatic dismutation (superoxide dismutase [SOD]) to form
I complex represent electron flow. P700, a dimer of chlorophyll a, accepts hydrogen peroxide (H2O2). As hydrogen peroxide and superoxide accu-
light energy from the chlorophylls and carotenoids associated with PS I. mulate in the cell after paraquat treatment, they react to produce hydroxyl
Other electron carriers are: A0, a chlorophyll a molecule; A1, a phylloqui- radicals (OH·) (Haber–Weiss reaction). The reaction is catalyzed by tran-
none; and FX, FA, and FB, 4Fe–4S iron–sulfur clusters (B). Plastocyanin sition metals (iron and/or copper) (Fenton reaction). Hydroxyl radicals ef-
and ferredoxin are soluble electron carrier proteins that dock to PS I. Plas- ficiently initiate lipid peroxidation in polyunsaturated fatty acids in mem-
tocyanin gives up an electron to PS I and ferredoxin accepts an electron branes.
from PS I. The structural proteins of PS I are termed Psa. PsaA and PsaB
make up the core of PS I and are embedded in the thylakoid membrane,
whereas PsaC is a peripheral protein housing the iron–sulfur clusters FA iron–sulfur clusters (FA and FB) (Figure 3B), which serve as
and FB. PsaD and PsaE assist with the docking of ferredoxin, whereas PsaF electron carriers to ferredoxin (a photoreduction reaction).
assists with the docking of plastocyanin. The bipyridylium herbicides com-
pete with ferredoxin for a binding site at or near PsaC. Ferredoxin is a small, water soluble iron–sulfur protein that
shuttles electrons from PS I to NADP⫹. Proper docking of
ferredoxin to PS I for electron transfer requires the assistance
cides is associated with the functioning of the photosyn- of two proteins (PsaD and PsaE). The site in the PS I light
thetic electron transport system rather than a direct effect reaction where paraquat and diquat dock to obtain electrons
of light and herbicide at the target site. is also at or near the PsaC protein. The role, if any, of PsaD
Within the photosynthesis light reaction, these di-cation and PsaE in docking paraquat at its binding site near PsaC
herbicides do not stop electron flow in PS I, but rather is unknown. This is not the only location where these her-
become free radicals (mono-cations) by capturing electrons bicides can accept electrons because toxicity also occurs to
from PS I during routine electron flow. Throughout the past a small extent in the dark. In this instance, the electron
decade, substantial progress, including obtaining high-reso- transport chain of respiration (mitochondria) probably sup-
lution X-ray crystal structures, has been made in under- plies electrons for free radical formation of paraquat.
standing the PS I structure and its electron transfer se- The paraquat and diquat free radicals (mono-cations) are
quence. For reviews of PS I see Brettel (1997) and Chitnis not the agents causing tissue damage. These free radicals are
(1996). The PS I complex is an assembly of more than 10 unstable and rapidly undergo autooxidation back to the par-
proteins, which are termed Psa proteins. PsaA and PsaB pro- ent ion (Figure 4). This rapid cycling between the paraquat
teins make up the core of the PS I complex (Figure 3A) and ion and radical, coupled with the high flow rate of electrons
bind the four redox factors (P700, A0, A1, and FX) necessary in PS I, probably explains the rapid development of symp-
to move electrons from docked plastocyanin (photooxida- toms. During the autooxidation process, superoxide radicals
tion reaction) to PsaC. Embedded within PsaC are two are produced from molecular oxygen. Superoxide undergoes

162 • Weed Science 48, March–April 2000

enzymatic dismutation (superoxide dismutase [SOD]) to
form hydrogen peroxide. Hydrogen peroxide, which is pro-
duced to some extent in nontreated plants, is enzymatically
dissipated, with the ascorbate–glutathione cycle being the
most commonly cited pathway (for review see Noctor and
Foyer [1998]). The abundance of superoxide and hydrogen
peroxide formed when paraquat cycles between the parent
ion and the radical are substantially above what the enzymes
are able to dissipate. The accumulated superoxide and hy-
drogen peroxide react with each other to produce hydroxyl
radicals (Haber–Weiss reaction). The reaction is catalyzed
by transition metals (iron and/or copper) (Fenton reaction).
For a review of these reactions, see the reviews by Hippeli
et al. (1999) and Saran et al. (1998). Hydroxyl radicals are
the most potent biological oxidants known and quickly and
effectively initiate membrane degradation through lipid per-
oxidation of polyunsaturated fatty acids, as described in the
last section of this overview.

Glutamine Synthetase
The only herbicide that inhibits the glutamine synthetase
enzyme in the nitrogen assimilation pathway is glufosinate.
The primary symptom is chlorosis, which is then followed
by necrosis. These symptoms usually begin to develop with-
in 3 to 5 d after treatment. The symptoms resemble PS II
inhibitors, but the primary target is not photosynthesis. If
treated plants are placed in the dark immediately after treat-
FIGURE 5. The herbicide glufosinate competes at the glutamate binding site
ment, symptoms develop, but at a greatly reduced rate; thus, on the glutamine synthetase (GS) enzyme, which inhibits the formation of
light is somehow involved in the full expression of herbicidal glutamine. In the absence of glutamine, glutamate cannot be synthesized
activity. due to the lack of substrate for the enzyme. Glutamine and glutamate are
After glufosinate application, the ammonia level in leaves, required for many nitrogen assimilation reactions in the cell; thus, inhibi-
tion of their synthesis yields substantial disruption of metabolism in plant
which is usually low, increases dramatically. Within a few cells. When glufosinate inhibits glutamine synthetase there is a substantial
hours after treatment, the ammonia level can be 10 or more increase in ammonia concentration because it is not incorporated into glu-
times higher than in nontreated leaves. In the dark, the tamine. Although ammonia accumulation may directly inhibit the light
ammonia accumulation caused by glufosinate treatment is reaction in photosynthesis, it is more likely that photosynthesis is indirectly
substantially less. This is attributed to two important am- inhibited by an accumulation of glyoxylate in photorespiration. Regardless
of the mechanism, inhibition of the photosynthesis light reaction leads to
monia-producing reactions in plants (nitrite reduction and initiation of lipid peroxidation in membranes.
the photorespiratory conversion of glycine to serine) that are
dependent on light. The accumulation of ammonia in glu-
fosinate-treated plants is known to be due to a direct inhi- development after glufosinate treatment is somehow due to
bition of the glutamine synthetase (GS) enzyme, which is the ammonia accumulation. Evidence shows ammonia is not
responsible for converting glutamate plus ammonia to glu- directly responsible for the toxic effects of glufosinate. For
tamine (Figure 5). Both the chloroplast and cytoplasm forms example, Seelye et al. (1995) reported that when glutamine
of GS are inhibited (Manderscheid and Wild 1986). Glu- was added to Asparagus officinalis L. (asparagus) tissue cul-
fosinate inhibition of GS results in decreased levels of glu- tures (callus) treated with glufosinate, phytotoxicity was
tamine. In the absence of glutamine, glutamate is not syn- eliminated, even though ammonia accumulated to high con-
thesized due to lack of substrate for the enzyme. Glufosinate centrations.
is competitive with the substrate glutamate for the GS en- Photosynthesis is inhibited after glufosinate treatment,
zyme. At this point, binding of glufosinate to GS is revers- which may cause the primary symptom development, in-
ible. Then ATP phosphorylates glufosinate bound to GS. cluding slow but significant membrane disruption. Whereas
The phosphorylated from of glufosinate becomes irreversibly inhibition of photosynthesis can be associated with an ac-
bound to GS (Manderscheid and Wild 1986). Glutamine cumulation of ammonia (for an overview of the effect of
and glutamate are required for many metabolic reactions in ammonia on photosynthesis, see Lea and Ridley [1989]),
the cell involving nitrogen (nitrogen assimilation); thus, there is experimental evidence that inhibition of photosyn-
their absence may well cause substantial disruption of plant thesis by glufosinate is due to something other than am-
cell function. monia accumulation. For example, adding high concentra-
It is always tempting to conclude that the high level of tions of ammonia to non-glufosinate-treated plants had little
ammonia accumulation must somehow cause the herbicidal to no effect on photosynthesis (Sauer et al. 1987). Adding
symptoms. However, the effect of ammonia accumulation glutamine or glutamate to glufosinate-treated plants reduced
on phytotoxicity caused by glufosinate remains controver- the inhibition of photosynthesis even though ammonia ac-
sial, and caution is advised in concluding that symptom cumulation was increased more than eightfold (Wendler et

Hess: Light-dependent herbicides • 163

al. 1990). Alternatively, evidence shows inhibition of pho- ticed that his first sample of ␣-tocopherol, a compound
tosynthesis occurs through a circuitous route associated with known to be somewhat susceptible to degradation, was quite
the inhibition of the GS enzyme. After glufosinate treat- brown, so another sample was ordered from a different sup-
ment, the loss of amino donors (i.e., glutamate) for the gly- plier. When this sample was used in the experiments, injury
colate pathway (glycolate → glyoxylate → glycine) leads to was consistently reduced. Treating thylakoids isolated from
a breakdown of the transamination reaction of glyoxylate to plant chloroplasts with DPE herbicides caused a large in-
glycine in the photorespiration cycle. This causes phosphog- crease in the formation of singlet oxygen (sometimes abbre-
lycolate, glycolate, and glyoxylate to accumulate (Wild and viated 1O2) (Haworth and Hess 1988). In these experi-
Wendler 1993). González-Moro et al. (1997), Wendler et ments, singlet oxygen was measured by monitoring the
al. (1992), and Wild and Wendler (1993) have shown that bleaching of N,N-dimethyl p-nitrosoaniline. In the presence
the accumulation of glyoxylate inhibits ribulose-1,5 bis- of oxyfluorfen, generation of singlet oxygen required light,
phosphate carboxylase/oxygenase (rubisco), which in turn which provided the first evidence of the direct connection
leads to an inhibition of the light reaction in photosynthesis between light and herbicide action. Singlet oxygen is known
(Figure 5). Membrane disruption then occurs by the process to be an efficient initiating factor of lipid peroxidation, but
described for PS II inhibitors. As would be expected, pho- determining the event that was causing the accumulation of
tosynthesis inhibition by glufosinate occurs more slowly in singlet oxygen was elusive.
C4 plants, where photorespiration is reduced, than in C3 Early studies used other herbicides and pigment mutants
plants (Wendler et al. 1990). of plants as tools to pinpoint where and how these herbi-
cides were interacting with light. Using inhibitors of pho-
Protoporphyrinogen Oxidase tosynthesis (diuron) or yellow (nonphotosynthesizing) mu-
tants of plants, in most cases, did not reduce the damage
Symptoms associated with herbicides that inhibit the pro- caused by treatment with DPE herbicides. These results sug-
toporphyrinogen oxidase (Protox) enzyme are similar to the gest the light requirement for herbicide activity does not
bipyridylium herbicides (membrane disruption) but develop involve photosynthesis. However, research from Peter Bög-
somewhat slower. As with paraquat, the first visible symp- er’s laboratory using the alga Scenedesmus repeatedly showed
tom is a dark green appearance of treated leaf tissue (water that photosynthesis was somehow involved because adding
soaking), which is followed by desiccation of the affected photosynthesis inhibitors to their system reduced the phy-
tissue. Initial research clearly showed that light was an ob- totoxicity caused by DPE herbicides (e.g., Kunert and Böger
ligate requirement for the herbicidal activity of these her- 1981). Bowyer et al. (1989) showed that in their Scenedes-
bicides. Whereas many herbicide types are known to have a mus cultures, the decrease in DPE herbicide activity after
mechanism at Protox (Anderson et al. 1994), most of the blocking photosynthesis was due to a depletion of the ox-
mechanism of action research has focused on DPEs, partic- ygen necessary for tetrapyrrole formation (oxidation of pro-
ularly oxyfluorfen and the methyl ester of acifluorfen. How- toporphyrinogen to protoporphyrin). Under conditions of
ever, other chemical classes of Protox inhibitors (e.g., N- high aeration, diuron did not protect Scenedesmus from DPE
phenyl heterocyclic compounds) have been commercialized herbicide activity in their test system. However, Böger’s lab-
(e.g., azafenidin, oxadiazon, carfentrazone, and sulfentra- oratory (Nicolaus et al. 1989) provided evidence that the
zone).
involvement of photosynthesis in DPE action was not re-
Researchers agree that membrane damage induced by
lated to oxygen evolution. Their proposal was that because
DPE herbicides is the result of lipid peroxidation of poly-
synthesis of the precursor to tetrapyrrole formation, ␦-ami-
unsaturated fatty acids in membranes. Two lines of evidence
have been known for many years. First, short-chain hydro- nolevulinic acid, obtains the needed carbon skeletons, ATP
carbon gases are produced after herbicide treatment. For ex- and NADPH from photosynthesis, inhibiting photosynthe-
ample, ethane evolution was detected within 3 h after ex- sis could reduce the activity of DPEs in some systems.
posing Scenedesmus cells to oxyfluorfen (Kunert and Böger Initial research using mutant plants [yellow and white
1981). Second, various products of lipid peroxide decom- mutants of Oryza sative L. (rice) (Matsunaka 1969) as well
position that are precursors of malondialdehyde (sometimes as a yellow mutant of Glycine max (L.) Merr. (soybean) and
abbreviated MDA) are detected within a few hours after a white mutant of Zea mays L. (corn) (Fadayomi and Warren
treatment. In one study, malondialdehyde precursors were 1976)] strongly implicated carotenoids as the target site of
detected within 1 to 2 h after light exposure of Cucumis DPE herbicides. In all instances, the yellow, achlorophyllous
sativus L. (cucumber) cotyledons pretreated with acifluorfen- mutants were sensitive and the white mutants were tolerant
methyl for 4 h in the dark (Orr and Hess 1982). to these herbicides. However, these early studies did not
Determining the substance responsible for initiating lipid evaluate chlorophyll biosynthesis prior to the final steps of
peroxidation and determining which plant pigments were forming functional chlorophyll. In the yellow mutants, the
involved in producing the initiating factor took several years. chlorophyll biosynthesis pathway must have been blocked
First, it was shown that some type of free radical reaction at a late step or one or more of the chlorophyll intermediates
was involved during lipid peroxidation of polyunsaturated must have been unstable. In the white mutants, the block
fatty acids in plant membranes because adding ␣-tocoph- in chlorophyll biosynthesis must have been early in the por-
erol, an antioxidant known to scavenge lipophilic free rad- phyrin pathway. These and other early studies strongly im-
icals, protected against acifluorfen-methyl damage (Orr and plicated a site of action for DPE herbicides in carotenoid
Hess 1982). The first experiments with ␣-tocopherol almost biosynthesis, but a target in this pathway was never identi-
missed this important observation because no reduction in fied. In addition, several researchers reported that DPE her-
lipid peroxidation was detected. Fortunately, Greg Orr no- bicide toxicity occurs in red light that is outside the absorp-

164 • Weed Science 48, March–April 2000

 a review), which then initiates lipid peroxidation and mem-
brane damage. Based on the accumulation of protoporphy-
rin IX, the first enzyme implicated was magnesium chela-
tase; however, the activity of DPE herbicides on this enzyme
was, at best, weak. Two groups (Matringe et al. 1989; Wit-
kowski and Halling 1989) surprisingly reported the inhib-
ited enzyme to be protoporphyrinogen oxidase (abbreviated
Protox or PPO). Protox is a membrane-bound enzyme lo-
cated in the chloroplast envelope, where it is involved in
chlorophyll biosynthesis (Figure 6). This enzyme converts
protoporphyrinogen IX to protoporphyrin IX. DPE herbi-
cides compete for the substrate (protoporphyrinogen IX)
binding site on the Protox enzyme (e.g., Nicolaus et al.
1993).
Considering that Protox is the enzyme prior to proto-
porphyrin IX, how can its inhibition lead to an accumula-
tion of the enzyme product protoporphyrin IX? The para-
dox was solved by a series of interactions between the lab-
oratories of Steve Duke and Kevin Vaughn. At this time,
Larry Lehnen, a postdoctoral student in Vaughn’s laboratory,
was conducting a series of experiments utilizing fluorescence
microscopy. At the request of Duke, Lehnen examined
achlorophyllous C. sativus tissue treated with acifluorfen and
reported the fluorescence from porphyrins accumulated out-
side the plastid, particularly at the plasma membrane. After
FIGURE 6. Diphenyl ethers and several other herbicide types inhibit the additional experiments, this key observation was published
enzyme protoporphyrinogen oxidase (Protox) in the chlorophyll biosynthe- (Lehnen et al. 1990). Duke presented these interesting find-
sis pathway. Inhibiting this enzyme, which is located in the chloroplast
envelope, results in an accumulation of protoporphyrinogen IX, which then ings, along with the kinetics of accumulation, at a seminar
leaks into the cytoplasm. Enzymatic oxidation of protoporphyrinogen IX at Dartmouth Medical School. After hearing the seminar,
in the cytoplasm yields a significant accumulation of protoporphyrin IX Nick Jacobs hypothesized that when the Protox enzyme in
away from the location of the chlorophyll biosynthesis sequence in chlo- the chloroplast membrane is inhibited, the accumulated pro-
roplasts. The accumulated protoporphyrin IX reacts with oxygen and light toporphyrinogen IX leaks from the plastid and then is con-
to produce singlet oxygen (1O2). Singlet oxygen disrupts membrane integ-
rity by initiating lipid peroxidation in polyunsaturated fatty acids. verted to protoporphyrin IX by herbicide-insensitive en-
zymes in the cytoplasm. This hypothesis was confirmed dur-
ing a series of subsequent experiments (e.g., Jacobs et al.
tion range of carotenoids (see Scalla and Matringe [1994] 1991; Jacobs and Jacobs 1993; Lee et al. 1993). Protopor-
for details]). phyrin IX now being away from the reaction center for for-
Early models of herbicide action proposed that the NO2 mation of Mg protoporphyrin IX by Mg chelatase, reacts
group on the DPE molecule accepted an electron from a with oxygen and light to form singlet oxygen, which initi-
plant pigment to become a free radical (e.g., Kunert and ates lipid peroxidation (Figure 6). These protoporphyrino-
Böger 1981). It was proposed that this herbicide free radical gen IX ‘‘oxidizer’’ enzymes are much less sensitive to DPE
directly induced lipid peroxidation. It is now known free herbicides than is the Protox enzyme. The exact identity of
radical formation on the herbicide is not involved (Ensmin- these enzymes is unknown; however, they appear to be some
ger et al. 1985). Replacing the NO2 group with a Cl or a type of peroxidase associated with the plasma membrane,
H did not change the mechanism of action of these herbi- endoplasmic reticulum, or microsomes (Lee and Duke
cides, even though Cl and H derivatives are not able to 1994; Retzlaff and Böger 1996; Yamato et al. 1994).
accept electrons to become free radicals in a physiological
environment.
A flurry of publications during 1988 and 1989 substan- Carotenoid Biosynthesis
tially increased the understanding of DPE action. Matringe
and Scalla (1987) authored the first publication implicating Although carotenoid biosynthesis is an excellent target for
the porphyrin biosynthesis pathway. Then a series of five herbicide action, few commercial products exist. The prin-
publications by four groups appeared in the literature (Ly- ciple reason is that most derivatives evaluated to date show
don and Duke 1988; Matringe and Scalla 1988a, 1988b; inadequate crop selectivity. The most striking symptom re-
Sandmann and Böger 1988; Witkowski and Halling 1988). sulting from treating plants with herbicides having a site of
Data in all these publications implicated accumulation of action in carotenoid biosynthesis is the white foliage pro-
the tetrapyrrole protoporphyrin IX as being central to the duced following treatment, which is sometimes termed ‘‘al-
mechanism of action of DPE herbicides (Figure 6). Proto- bino growth.’’ The white foliage is the result of a primary
porphyrin IX is a precursor in chlorophyll biosynthesis (for inhibition of carotenoid biosynthesis coupled to a secondary
a review of chlorophyll biosynthesis, see Reinbothe and inhibition of chlorophyll biosynthesis and a destruction of
Reinbothe [1996] and von Wettstein et al. [1995]). Oxygen existing chlorophyll. Because of the coupling of these pro-
and light are known to interact with protoporphyrin IX to cesses, herbicides of this type are often termed ‘‘bleaching
produce singlet oxygen (see Scalla and Matringe [1994] for herbicides’’ or ‘‘bleachers.’’ Growth does continue for a time,

Hess: Light-dependent herbicides • 165

 rotenoid biosynthesis. Then, through a series of linear ad-
ditions, three molecules of IPP are sequentially added to
dimethylallyl pyrophosphate (an allylic isomer of IPP
formed by IPP isomerase) to form the 20-carbon chain ger-
anylgeranyl pyrophosphate. Two molecules of geranylgeranyl
pyrophosphate condense to form the colorless 40-carbon ca-
rotenoid intermediate phytoene. Then a series of desatura-
tion reactions occur to form additional double bonds. This
lengthens the series of conjugated double bonds and pro-
vides the chromophore color to carotenoids. Cyclization re-
actions catalyzed by one or two cyclase enzymes occur at
both ends of the lycopene chain to yield ␣- and ␤-carotene.
Elegant molecular biology research in carotenoid biosyn-
thesis during the 1990s by Gerhard Sandmann and co-
workers yielded significant advances in understanding the
desaturation enzymes in several different organisms. Using
the sequence of phytoene desaturase genes obtained from
different organisms, a structural heterogeneity was evident,
indicating the existence of two groups of enzymes: one in-
cluding the phytoene desaturases from plants and cyano-
bacteria and the other from all remaining bacteria and fungi.
In a recent conversation, Sandmann summarized that ‘‘het-
erologous expression of phytoene desaturase genes from both
groups was the experimental breakthrough for purification
and biochemical characterization, including enzyme kinetic
studies with herbicidal inhibitors (see Fraser et al. 1992;
Schneider et al. 1997). Not only was the reaction product
of the bacterial enzyme different from the plant-type enzyme
(lycopene with four newly introduced double bonds was the
end product instead of ␨-carotene with two additional dou-
FIGURE 7. Carotenoid biosynthesis begins with the formation of isopentenyl
pyrophosphate (IPP). Then, three molecules of IPP are sequentially added ble bonds), it was also completely unaffected by the typical
to dimethylallyl pyrophosphate, an isomer of IPP, to form geranylgeranyl bleaching herbicides (see Sandmann and Fraser 1993). It
pyrophosphate. Two molecules of geranylgeranyl pyrophosphate condense was obvious to us that the bacterial phytoene desaturase
to form phytoene, then a series of desaturation reactions form four addi- must lack the inhibition site of the plant enzyme. This find-
tional double bonds. Cyclization occurs at both ends of the lycopene chain
to yield ␣- and ␤-carotene. Several sites of action for herbicides exist in the
ing led us to generate transgenic tobacco plants by inserting
carotenoid biosynthesis pathway (circled enzymes). The most frequently the naturally resistant phytoene desaturase gene from the
identified target is phytoene desaturase. Although not commercial, inhibi- enterobacterium Erwinia uredovora. The outcome was a
tors of ␨-carotene desaturase, lycopene cyclase, and prenyl transferase have plant in which the foreign bacterial phytoene desaturase
also been identified. takes over the catalysis of phytoene desaturation to lycopene
when a phytoene or ␨-carotene desaturase inhibitor inacti-
vates the endogenous enzyme.’’ The details of this research
but without production of green photosynthetic tissue, are reviewed in a publication by Sandmann et al. (1996).
growth of affected plants cannot be maintained indefinitely. A combination of inhibition of carotenoid biosynthesis
Growth ceases and then necrosis begins to occur. Those her- coupled with the destruction of chlorophyll by light (pho-
bicides that inhibit carotenoid biosynthesis do not affect tooxidation) and an inhibition of chlorophyll biosynthesis
preexisting carotenoids. Thus, plant tissues formed before results in completely white new growth in treated plants.
treatment do not show typical albino symptoms. There is One important role of carotenoids is to protect chlorophyll
turnover of carotenoid pigments; thus, tissue formed prior from photooxidation. After chlorophyll is synthesized and
to treatment will eventually show chlorosis and then necro- becomes functional, some of the chlorophyll that has been
sis. electronically excited by absorbing light photons is trans-
The carotenoid biosynthesis pathway (Figure 7) is well formed from the short-lived singlet form to the longer lived
characterized (see Cunningham and Gantt [1998] for a triplet form. With their multiple conjugated double bonds
complete review). The starting material for carotenoid bio- (11 in ␤-carotene), carotenoids are able to quench the en-
synthesis is a five-carbon building block, isopentenyl pyro- ergy of triplet chlorophyll, as well as singlet oxygen formed
phosphate (IPP). For many years, it was assumed all IPP from triplet chlorophyll, when generated under high light
was derived from acetate. However, Lichtenthaler has shown intensity (Figure 2). When carotenoids are not present, sin-
that there are two pathways for IPP generation: one in the glet oxygen, and perhaps triplet chlorophyll, initiate degrad-
cytoplasm utilizing acetate as the starting material and one ing reactions, including chlorophyll and membrane destruc-
in chloroplasts starting with glyceraldehyde-3-phosphate and tion. Thus without carotenoids, plant cells cannot survive
pyruvate (see Lichtenthaler [1999] for review). This series in high light. However, if plants are treated with carotenoid
of reactions, named the 1-deoxy-D-xylulose-5-phosphate synthesis inhibitor herbicides and then grown in very low
(DOXP) pathway, provides the IPP building blocks for ca- light intensities, new growth can contain up to 70% of the

166 • Weed Science 48, March–April 2000

 steps in carotenoid biosynthesis, a second site of action for
carotenoid synthesis inhibitors is ␨-carotene desaturase,
which further desaturates ␨-carotene and neurosporene to
form lycopene. As expected, when ␨-carotene desaturase is
inhibited, there is an accumulation of ␨-carotene and, to
some extent, neurosporene. A third identified site of action
for carotenoid biosynthesis inhibitors is at the cyclization
step following lycopene synthesis, which yields an accumu-
lation of lycopene. Böger (1996) published a review of in-
hibitors for these latter two sites of action.
FIGURE 8. Several herbicide types inhibit the phytoene desaturase enzyme A new class of potential herbicides, the bisphosphonates,
responsible for double bond formation (desaturation) in phytoene and phy- has a site of action early in the carotenoid biosynthesis path-
tofluene in the carotenoid biosynthesis pathway. These herbicides bind di- way. Although new growth exhibits chlorosis rather than
rectly to the phytoene desaturase enzyme and inhibit its function. The
phytoene desaturase enzyme contains a dinucleotide binding domain (e.g., albinism, these derivatives inhibit prenyl transferase in the
FAD), which accepts electrons and hydrogen ions (protons) from phytoene isoprenoid portion of the carotenoid biosynthesis pathway
during desaturation and transfers them to plastoquinone (PQ), which then (Cromartie et al. 1999; Oberhauser et al. 1998).
shuttles the electrons and protons to the photosynthesis light reaction. Clomazone, a commercial herbicide with symptoms sim-
ilar to the desaturase inhibitors described above, has a
unique site of action. Duke and Kenyon (1986) reported
that this herbicide did not cause an accumulation of phy-
chlorophyll present in nontreated plants (Bartels and Hyde toene. Because clomazone leads to an accumulation of the
1970). This newly formed chlorophyll will not be destroyed sesquiterpenoids gossypol and hemigossypol, Duke et al.
by photooxidation if plants are maintained in low light, but (1991) concluded clomazone inhibited an enzyme in carot-
80% is destroyed if plants are returned to high light. In enoid biosynthesis prior to geranylgeranyl pyrophosphate.
addition to chlorophyll destruction by photooxidation, the Sandmann and Böger (1986, 1987) provided evidence in-
absence of carotenoids disrupts formation of pigment–pro- dicating the enzyme inhibited was IPP isomerase. However,
tein complexes required for the assembly of PS II in the Lützow et al. (1990) and others could not find an effect of
thylakoids of chloroplasts and causes some inhibition of clomazone on isolated enzymes involved in the synthesis of
chlorophyll biosynthesis (for review see Bramley and Pallett phytoene (IPP isomerase, a prenyl transferase, or phytoene
[1993] and Moskalenko and Karapetyan [1996]). This in- synthase). There has been some speculation that clomazone
hibition of chlorophyll biosynthesis, coupled with destruc- is a ‘‘proherbicide’’ and thus would not ‘‘directly’’ inhibit
tion of chlorophyll and perhaps precursors to chlorophyll, any of the enzymes in the carotenoid biosynthesis pathway
may explain why pretreating plants with carotenoid synthe- in in vitro test systems.
sis inhibitors reduced the activity of DPE herbicides (Protox
inhibitors) in laboratory test systems.
The best-studied site of herbicide action in the carotenoid Inhibition of 4-Hydroxyphenylpyruvate
biosynthesis pathway is inhibition of phytoene desaturation, Dioxygenase (HPPD)
which is responsible for the formation of two additional
double bonds in the carbon backbone of phytoene. Phyto- During 1993, a new site of action was defined for carot-
ene desaturase enzymes contain a dinucleotide-binding do- enoid synthesis inhibitors (for review of chemical types see
main for FAD or NAD⫹/NADP⫹, which accepts electrons Lee et al. [1997]). The herbicide sulcotrione (ICIA-0051;
and hydrogen ions (protons) from phytoene during the de- also published as SC-0051), a triketone herbicide used com-
saturation reaction. Plastoquinones are then thought to act mercially in Europe, and isoxaflutole (RPA-201772), an is-
as electron carriers between the reduced FAD or NAD⫹/ oxazole herbicide now used in the United States, produce
NADP⫹ bound to the carotenoid desaturase enzymes and carotenoid synthesis inhibition symptoms (new growth is
the photosynthetic electron transport chain (Norris et al. white). Initial research on the mechanism of action of these
1995) (Figure 8). Phytoene desaturase is a membrane-bound herbicides reported an accumulation of phytoene, suggesting
enzyme in the chloroplast thylakoids. Because inhibition oc- a classical inhibition of phytoene desaturase. Surprisingly,
curs in an in vitro system (e.g., isolated Narcissus pseudonar- the site of action was shown not to be the phytoene desa-
cissus L. [daffodil] chromoplasts), the mechanism of action turase enzyme (Sandmann et al. 1990). Pallett et al. (1998),
is directly on the functioning enzyme (Mayer et al. 1989). Schulz et al. (1993), and Secor (1994) showed the site of
Norflurazon binding to phytoene desaturase is reversible and action is at the enzyme p-hydroxyphenylpyruvate dioxygen-
noncompetitive with the substrate phytoene (Mayer et al. ase (HPPD), which converts p-hydroxyphenylpyruvate to
1989; Sandmann et al. 1989). Inhibition of this enzyme homogentisate (Figure 9). Secor (1994) partially purified
causes a large accumulation of phytoene in treated plants. HPPD from Echinochloa crus-galli (L.) Beauv. (barn-
The same enzyme (phytoene desaturase) is involved in two yardgrass) and found sulcotrione was a competitive inhibitor
desaturation reactions (two double bonds formed), with the with the substrate (p-hydroxyphenylpyruvate). Similar re-
end product being ␨-carotene. Therefore, phytofluene also sults were reported by Viviani et al. (1998) for HPPD iso-
accumulates to some extent if phytoene desaturase is inhib- lated from Daucus carota L. (carrot) when treated with the
ited. Although not infallible, the accumulation of phytoene diketonitrile derivative of isoxaflutole, which is known to be
is the most commonly reported proof for herbicide action the active form of the herbicide. Feeding homogentisate to
at the phytoene desaturase site. plants eliminates the carotenoid synthesis inhibition. The
Although there are no commercial herbicides for later inhibition of carotenoid synthesis at the phytoene desaturase

Hess: Light-dependent herbicides • 167

 FIGURE 10. Lipid peroxidation of polyunsaturated fatty acids in plant mem-
branes. An initiating factor (R·) such as singlet oxygen, hydroxyl radical,
and perhaps triplet chlorophyll removes a hydrogen from a polyunsaturated
fatty acid (LH) in the membrane. This hydrogen abstraction process gen-
erates a lipid radical (L·). Oxygen reacts with the lipid radical to form a
peroxidized lipid radical (LOO·). This peroxidized lipid radical reacts with
another polyunsaturated lipid, which propagates the reaction within a lo-
calized region of the membrane. The lipid peroxides (LOOH) formed dur-
ing propagation are unstable and degrade to compounds such as short chain
hydrocarbon gases (e.g., ethane [C2H6]).

erated from bipyridylium herbicide interaction with PS I

FIGURE 9. Triketone and isoxazole herbicides indirectly inhibit the forma-
and perhaps triplet chlorophyll formation from inhibiting
tion of carotenoids. These herbicides inhibit the desaturation of phytoene PS II. These initiating factors are able to remove (abstract)
and phytofluene by inhibiting the generation of the plastoquinones (PQ) a hydrogen from a methylene group positioned near unsat-
necessary for shuttling electrons and protons to photosynthesis from the urated sites on a polyunsaturated fatty acid. The lipid radical
reduced dinucleotide (e.g., FADH2) associated with phytoene desaturase. generated reacts with molecular oxygen to form a peroxi-
As would be expected, phytoene and phytofluene accumulate and carot-
enoid synthesis stops. The inhibition site is at the enzyme p-hydroxyphen- dized lipid radical. These reactions are called initiation. The
ylpyruvate dioxygenase which is responsible for generating homogentisate peroxidized lipid radicals are reduced to lipid peroxides
from p-hydroxyphenylpyruvate. when they extract hydrogen from other polyunsaturated fat-
ty acids in the membrane (propagation reaction). The lipid
peroxides formed are not stable and undergo degradation to
site is indirect and due to depletion of plastoquinones syn- malondialdehyde precursors that can be measured by reac-
thesized from homogentisate. These plastoquinones are tion with thiobarbituric acid and short-chain hydrocarbon
needed as cofactors for proper functioning of the phytoene gases such as pentane from linoleic acid and ethane from
desaturase enzyme, as proposed by Mayer et al. (1992) and linolenic acid (termination reaction). This degradation of
Norris et al. (1995) (Figure 8). polyunsaturated fatty acids destabilizes the overall integrity
of the membrane. For reviews of lipid peroxidation, see Gi-
rotti (1998) and Saran et al. (1998).
The Ultimate Target—Lipid Peroxidation
For herbicides where light is involved in phytotoxicity, a Acknowledgments
key component of symptom development is disruption of
the integrity of cell membranes, which yields substantial tis- The drawings in this overview are from a Continuing Education
sue damage and, subsequently, plant death. Initially, loss of course on Herbicide Action taught at Purdue University and are
membrane integrity eliminates the electrochemical and hy- public domain. This overview is dedicated to G. Fred Warren who
started the Herbicide Action course at Purdue in 1980.
drogen ion gradient, both of which are essential for normal
cell function. Further membrane degradation provides an
avenue for the liquid within the cell to leak into the inter- Literature Cited
cellular space, which changes the refractive index of the leaf. Anderson, R. J., A. E. Norris, and F. D. Hess. 1994. Synthetic organic
As described earlier in this overview, a change in refractive chemicals that act through the porphyrin pathway. Pages 18–33 in S.
index causes the affected leaf tissue to appear darker green; O. Duke and C. A. Rebeiz, eds. Porphyric Pesticides: Chemistry, Tox-
hence, the term water-soaked leaf is often associated with icology, and Pharmaceutical Applications. Washington, DC: American
herbicides that disrupt membrane integrity. Chemical Society Symposium Series 559.
Arntzen, C. J., C. L. Ditto, and P. E. Brewer. 1979. Chloroplast membrane
The mechanism of membrane disruption caused by a alterations in triazine-resistant Amaranthus retroflexus biotypes. Proc.
wide variety of herbicides is essentially the same (peroxida- Natl. Acad. Sci. USA 76:278–282.
tion of polyunsaturated fatty acids). The pathway for dis- Bartels, P. G. and A. Hyde. 1970. Choroplast development in 4-chloro-5-
ruption of polyunsaturated fatty acids in plant membranes (dimethylamino)-2-( ␣ , ␣ , ␣ -trifluoro-m-tolyl)-3(2H)-pyridazinone
is well characterized and similar after different initiating fac- (Sandoz 6706)-treated wheat seedlings. Plant Physiol. 45:807–810.
Böger, P. 1996. Mode of action of herbicides affecting carotenogensis. J.
tors start the process. Lipid peroxidation in polyunsaturated Pestic. Sci. 21:473–478.
fatty acids such as linoleic acid (18:2; 18-carbon chain with Bowyer, J. R., B. J. Hallahan, P. Camilleri, and J. Howard. 1989. Mode of
two double bonds) and linolenic acid (18:3) involves three action studies on nitrodiphenyl ether herbicides. II. The role of pho-
steps: initiation, propagation, and termination (Figure 10). tosynthetic electron transport in Scenedesmus obliquus. Plant Physiol.
89:674–680.
Herbicides interfering with various plant reactions yield sev- Bramley, P. M. and K. E. Pallett. 1993. Phytoene desaturase: a biochemical
eral different initiating factors. Examples are singlet oxygen target of many bleaching herbicides. Proc. Brighton Crop Prot. Conf.
from inhibition of PS II or Protox, hydroxyl radicals gen- Weeds 2:713–722.

168 • Weed Science 48, March–April 2000

Brettel, K. 1997. Electron transfer and arrangement of the redox cofactors not inhibit the prenyl diphosphate-forming enzymes in plastids. Z.
in photosystem I. Biochim. Biophy. Acta Bioenergetics 1318:322–373. Naturforsch. 45c:856–858.
Chauhan, N. P., T. Fatma, and R. K. Mishra. 1992. Protection of wheat Lydon, J. and S. O. Duke. 1988. Porphyrin synthesis is required for pho-
chloroplasts from lipid peroxidation and loss of photosynthetic pig- tobleaching activity of the p-nitrosubstituted diphenyl ether herbicides.
ments by quercetin under strong illumination. J. Plant Physiol. 140: Pestic. Biochem. Physiol. 31:74–83.
409–413. Manderscheid, R. and A. Wild. 1986. Studies on the mechanism of inhi-
Chitnis, P. R. 1996. Update on photosynthetic electron transport: photo- bition by phosphinothricin of glutamine synthetase isolated from Trit-
system I. Plant Physiol. 111:661–669. icum aestivum L. J. Plant Physiol. 123:135–142.
Cromartie, T. H., K. J. Fisher, and J. N. Grossman. 1999. The discovery Matringe, M., J. M. Camadro, P. Labbe, and R. Scalla. 1989. Protopor-
of a novel site of action for herbicidal bisphosphonates. Pestic. Bio- phyrinogen oxidase as a molecular target for diphenyl ether herbicides.
chem. Physiol. 63:114–126. Biochem. J. 260:231–235.
Cunningham, F. X. Jr., and E. Gantt. 1998. Genes and enzymes of carot- Matringe, M. and R. Scalla. 1987. Induction of tetrapyrrole accumulation
enoid biosynthesis in plants. Ann. Rev. Plant Physiol. Plant Mol. Biol. by diphenyl ether-type herbicides. Proc. Br. Crop Prot. Conf. Weeds
49:557–583. 3:981–988.
Duke, S. O. and W. H. Kenyon. 1986. Effects of dimethazone (FMC Matringe, M. and R. Scalla. 1988a. Effects of acifluorfen-methyl on cu-
57020) on chloroplast development. II. Pigment synthesis and pho- cumber cotyledons: porphyrin accumulation. Pestic. Biochem. Physiol.
tosynthetic function in cowpea (Vigna unguiculata L.) primary leaves. 32:164–172.
Pestic. Biochem. Physiol. 25:11–18. Matringe, M. and R. Scalla. 1988b. Studies on the mode of action of
Duke, S. O., R. N. Paul, J. M. Becerril, and J. H. Schmidt. 1991. Clom- acifluorfen-methyl in nonchlorophyllous soybean cells. Plant Physiol.
azone causes accumulation of sesquiterpenoids in cotton (Gossypium 86:619–622.
hirsutum L.). Weed Sci. 39:339–346. Matsunaka, S. 1969. Acceptor of light energy in photoactivation of diphe-
Ensminger, M. P., F. D. Hess, and J. T. Bahr. 1985. Nitro free radical nyl ether herbicides. J. Agric. Food Chem. 17:171–175.
formation of diphenyl ether herbicides is not necessary for their toxic Mayer, M. P., D. L. Bartlett, P. Beyer, and H. Kleinig. 1989. The in vitro
action. Pestic. Biochem. Physiol. 23:163–170. mode of action of bleaching herbicides on the desaturation of 15-cis-
Fadayomi, O. and G. F. Warren. 1976. The light requirement for herbicidal phytoene and cis-␨-carotene in isolated daffodil chromoplasts. Pestic.
activity of diphenyl ethers. Weed Sci. 24:598–600. Biochem. Physiol. 34:111–117.
Fraser, P. D., N. Misawa, H. Linden, S. Yamano, K. Kobayashi, and G. Mayer, M. P., V. Nievelstein, and P. Beyer. 1992. Purification and charac-
Sandmann. 1992. Expression in Escherichia coli, purification and re- terization of a NADPH dependent oxidoreductase from chromoplasts
of Narcissus pseudonarcissus: a redox-mediator possibly involved in ca-
activation of the recombinant Erwinia uredovora phytoene desaturase.
rotenoid desaturation. Plant Physiol. Biochem. 30:389–398.
J. Biol. Chem. 267:19891–19895.
Moskalenko, A. A. and N. V. Karapetyan. 1996. Structural role of carot-
Girotti, A. W. 1998. Lipid hydroperoxide generation, turnover, and effector
enoids in photosynthetic membranes. Z. Naturforsch. 51c:763–771.
action in biological systems. J. Lipid Res. 39:1529–1542.
Nicolaus, B., G. Sandmann, and P. Böger. 1993. Molecular aspects of her-
Gonzáles-Moro, M. B., M. Lacuesta, N. Iriberri, A. Muñoz-Rueda, and C.
bicide action on protoporphyrinogen oxidase. Z. Naturforsch. 48c:
Gonzáles-Murua. 1997. Comparative effects of PPT and AOA on
326–333.
photosynthesis and fluorescence chlorophyll parameters in Zea mays.
Nicolaus, B., G. Sandmann, H. Watanabe, K. Wakabayashi, and P. Böger.
J. Plant Physiol. 151:641–648. 1989. Herbicide-induced peroxidation: influence of light and diuron
Hankamer, B., J. Barber, and E. J. Boekema. 1997. Structure and mem- on protoporphyrin IX formation. Pestic. Biochem. Physiol. 35:192–
brane organization of photosystem II in green plants. Ann. Rev. Plant 201.
Physiol. Plant Mol. Biol. 48:641–671. Noctor, G. and C. H. Foyer. 1998. Ascorbate and glutatione: keeping active
Haworth, P. and F. D. Hess. 1988. The generation of singlet oxygen (1O2) oxygen under control. Ann. Rev. Plant Physiol. Plant Mol. Biol. 49:
by the nitrodiphenyl ether herbicide oxyfluorfen is independent of 249–279.
photosynthesis. Plant Physiol. 86:672–676. Norris, S. R., T. R. Barrette, and D. DellaPenna. 1995. Genetic dissection
Hippeli, S., I. Heiser, and E. F. Elstner. 1999. Activated oxygen and free of carotenoid synthesis in Arabidopsis defines plastoquinone as an es-
radicals in pathology: new insights and analogies between animals and sential component of phytoene desaturation. Plant Cell 7:2139–2149.
plants. Plant Physiol. Biochem. 37:167–178. Oberhauser, V., J. Gaudin, R. Fonné-Pfister, and H.-P. Schär. 1998. New
Jacobs, J. M. and N. J. Jacobs. 1993. Porphyrin accumulation and export target enzyme(s) for bisphosphonates: inhibition of geranylgeranyl di-
by isolated barley (Hordeum vulgare) plastids. Plant Physiol. 101: phosphate synthase. Pestic. Biochem. Physiol. 60:111–117.
1181–1187. Orr, G. L. and F. D. Hess. 1982. Mechanism of action of the diphenyl
Jacobs, J. M., N. J. Jacobs, T. D. Sherman, and S. O. Duke. 1991. Effect ether herbicide acifluorfen-methyl in excised cucumber (Cucumis sa-
of diphenyl ether herbicides on oxidation of protoporphyrinogen to tivus L.) cotyledons. Plant Physiol. 69:502–507.
protoporphyrin in organellar and plasma membrane enriched fractions Pallett, K. E., J. P. Little, M. Sheekey, and P. Veerasekaran. 1998. The mode
of barley. Plant Physiol. 97:197–203. of action of isoxaflutole. I. Physiological effects, metabolism, and se-
Kunert, K. J. and P. Böger. 1981. The bleaching effect of the diphenyl lectivity. Pestic. Biochem. Physiol. 62:113–124.
ether oxyfluorfen. Weed Sci. 29:169–173. Pfister, K., K. E. Steinback, G. Gardner, and C. J. Arntzen. 1981. Photo-
Lea, P. J. and S. M. Ridley. 1989. Glutamine synthetase and its inhibition. affinity labeling of an herbicide receptor protein in chloroplast mem-
Pages 137–170 in A. D. Dodge, ed. Herbicides and Plant Metabolism. branes. Proc. Natl. Acad. Sci. USA 78:981–985.
Cambridge, UK: Cambridge University Press, Society of Experimental Reinbothe, S. and C. Reinbothe. 1996. Regulation of chlorophyll biosyn-
Biology Seminar Series 38. thesis in angiosperms. Plant Physiol. 111:1–7.
Lee, D. L., M. P. Prisbylla, T. H. Cromartie, D. P. Dagarin, S. W. Howard, Retzlaff, K. and P. Böger. 1996. An endoplasmic reticulum plant enzyme
W. McLean-Provan, M. K. Ellis, T. Fraser, and L. C. Mutter. 1997. has protoporphyrinogen IX oxidase activity. Pestic. Biochem. Physiol.
The discovery and structural requirements of inhibitors of p-hydrox- 54:105–114.
yphenylpyruvate dioxygenase. Weed Sci. 45:601–609. Sandmann, G. and P. Böger. 1986. Interference of dimethazone with for-
Lee, H. J., M. V. Duke, and S. O. Duke. 1993. Cellular localization of mation of terpenoid compounds. Z. Naturforsch. 41c:729–732.
protoporphyrinogen-oxidizing activities of etiolated barley (Hordeum Sandmann, G. and P. Böger. 1987. Interconversion of prenyl pyrophos-
vulgare L.) leaves. Plant Physiol. 102:881–889. phates and subsequent reactions in the presence of FMC 57020. Z.
Lee, H. J. and S. O. Duke. 1994. Protoporphyrinogen IX-oxidizing activ- Naturforsch. 42c:803–807.
ities involved in the mode of action of peroxidizing herbicides. J. Sandmann, G. and P. Böger. 1988. Accumulation of protoporphyrin IX in
Agric. Food Chem. 42:2610–2618. the presence of peroxiding herbicides. Z. Naturforsch. 43c:699–704.
Lehnen, L. P. Jr., T. D. Sherman, J. M. Becerril, and S. O. Duke. 1990. Sandmann, G., P. Böger, and I. Kumita. 1990. Atypical inhibition of phy-
Tissue and cellular localization of acifluorfen-induced porphyrins in toene desaturation by 2-(4-chloro-2-nitrobenzoyl)-5,5-dimethylcyclo-
cucumber cotyledons. Pestic. Biochem. Physiol. 37:239–248. hexane-1,3-dione. Pestic. Sci. 30:353–355.
Lichtenthaler, H. K. 1999. The 1-deoxy-D-xylulose-5-phosphate pathway Sandmann, G. and P. D. Fraser. 1993. Differential inhibition of phytoene
of isoprenoid biosynthesis in plants. Ann. Rev. Plant Physiol. Plant desaturase from diverse origins and analysis of resistant cyanobacterial
Mol. Biol. 50:47–65. mutants. Z. Naturforsch. 48c:307–311.
Lützow, M., P. Beyer, and H. Kleinig. 1990. The herbicide Command does Sandmann, G., H. Linden, and P. Böger. 1989. Enzyme-kinetic studies on

Hess: Light-dependent herbicides • 169

 the interaction of norflurazon with phytoene desaturase. Z. Natur- topology of the herbicide and QB binding polypeptide in the thyla-
forsch. 44c:787–790. koid membrane. Photosyn. Res. 10:381–392.
Sandmann, G., N. Misawa, and P. Böger. 1996. Steps towards genetic en- Tsiotis, G., G. McDermott, and D. Ghanotakis. 1996. Progress towards
gineering of crops resistant against bleaching herbicides. Pages 189– structural elucidation of photosystem II. Photosyn. Res. 50:93–101.
200 in S. O. Duke, ed. Herbicide-Resistant Crops: Agricultural, Eco- Vermaas, W. 1993. Molecular-biological approaches to analyze photosystem
nomic, Environmental, Regulatory, and Technological Aspects. Boca II structure and function. Annu. Rev. Plant Physiol. Plant Mol. Biol.
Raton, FL: Lewis Publishers. 44:457–481.
Saran, M., C. Michel, and W. Bors. 1998. Radical functions in vivo: a Viviani, F., J. P. Little, and K. E. Pallett. 1998. The mode of action of
critical review of current concepts and hypotheses. Z. Naturforsch. isoxaflutole. II. Characterization of the inhibition of carrot 4-hydrox-
53c:210–227. yphenylpyruvate dioxygenase by the diketonitrile derivative of isoxaf-
lutole. Pestic. Biochem. Physiol. 62:125–134.
Sauer, H., A. Wild, and W. Rühle. 1987. The effect of phosphinothricin
von Wettstein, D., S. Gough, and C. G. Kannangara. 1995. Chlorophyll
(glufosinate) on photosynthesis. II. The causes of inhibition of pho- biosynthesis. Plant Cell 7:1039–1057.
tosynthesis. Z. Naturforsch. 42c:270–278. Wendler, C., M. Barniske, and A. Wild. 1990. Effect if phosphinothricin
Scalla, R. and M. Matringe. 1994. Inhibitors of protoporphyrinogen oxi- (glufosinate) on photosynthesis and photorespiration in C3 and C4
dase as herbicides: diphenyl ethers and related photobleaching mole- plants. Photosyn. Res. 24:55–61.
cules. Rev. Weed Sci. 6:103–132. Wendler, C., A. Putzer, and A. Wild. 1992. Effect of glufosinate (phos-
Schneider, C., P. Böger, and G. Sandmann. 1997. Phytoene desaturase: phinothricin) and inhibitors of photorespiration on photosynthesis
heterologous expression in an active state, purification, and biochem- and ribulose-1,5-bisphosphate carboxylase activity. J. Plant Physiol.
ical properties. Protein Expr. Purif. 10:175–179. 139:666–671.
Schulz, A., O. Ort, P. Beyer, and H. Kleinig. 1993. SC-0051, a 2-benzoyl- Wild, A. and C. Wendler. 1993. Inhibitory action of glufosinate on pho-
cyclohexane-1,3-dione bleaching herbicide, is a potent inhibitor of the tosynthesis. Z. Naturforsch. 48c:369–373.
enzyme p-hydroxyphenylpyruvate dioxygenase. FEBS Lett. 318:162– Witkowski, D. A. and B. P. Halling. 1988. Accumulation of photodynamic
166. tetrapyrroles induced by acifluorfen-methyl. Plant Physiol. 87:632–
Secor, J. 1994. Inhibition of barnyardgrass 4-hydroxyphenylpyruvate diox- 637.
ygenase by sulcotrione. Plant Physiol. 106:1429–1433. Witkowski, D. A. and B. P. Halling. 1989. Inhibition of plant protopor-
Seelye, J. F., W. M. Borst, G. A. King, P. J. Hannan, and D. Maddocks. phyrinogen oxidase by the herbicide acifluorfen-methyl. Plant Physiol.
1995. Glutamine synthetase activity, ammonium accumulation and 90:1239–1242.
Xiong, J., S. Subramaniam, and Govindjee. 1996. Modeling of the D1/D2
growth of callus cultures of Asparagus officinalis L. exposed to high
proteins and cofactors of the photosystem II reaction center: impli-
ammonium or phosphinothricin. J. Plant Physiol. 146:686–692. cations for herbicide and bicarbonate binding. Protein Sci. 5:2054–
Steinback, K. E., L. McIntosh, L. Bogorad, and C. J. Arntzen. 1981. Iden- 2073.
tification of the triazine receptor protein as a chloroplast gene product. Yamato, S., M. Katagiri, and H. Ohkawa. 1994. Purification and charac-
Proc. Natl. Acad. Sci. USA 78:7463–7467. terization of a protoporphyrinogen-oxidizing enzyme with peroxidase
Tietjen, K. G., J. F. Kluth, R. Andree, M. Haug, M. Lindig, K. H. Müller, activity and light-dependent herbicide resistance in tobacco cultured
H. J. Wroblowsky, and A. Trebst. 1991. The herbicide binding niche cells. Pestic. Biochem. Physiol. 50:72–82.
of photosystem II—a model. Pestic. Sci. 31:65–72.
Trebst, A. and W. Draber. 1986. Inhibitors of photosystem II and the Received August 3, 1999, and approved January 21, 2000.

170 • Weed Science 48, March–April 2000