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`Mechanisms of herbicide absorption across plant membranes and
`accumulation in plant cells
`
`Research Gate
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`Article  in  Weed Science · January 1994
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`

`Weed Science, 1994. Volume 42:263-276
`
`Mechanisms of Herbicide Absorption Across Plant Membranes and Accumulation in Plant Cells 1
`
`TRACY M. STERLING2
`
`Abstract. In most cases, a herbicide must traverse the cell
`wall, the plasma membrane, and organellar membranes of a
`plant cell to reach its site of action where accumulation causes
`phytotoxicity. The physicochemical characteristics of the
`herbicide molecule including lipophilicity and acidity, the
`plant cell membranes, and the electrochemical potential in
`the plant cell control herbicide absorption and accumulation.
`Most herbicides move across plant membranes via nonfacili(cid:173)
`tated diffusion because the membrane's lipid bilayer is per(cid:173)
`meable to neutral, lipophilic xenobiotics. Passive absorption
`of lipophilic, ionic herbicides or weak acids can be mediated
`by an ion-trapping mechanism where the less lipophilic,
`anionic form accumulates in alkaline compartments of the
`plant cell. A model that includes the pH and electrical gradi(cid:173)
`ents across plant cell membranes better predicts accumula(cid:173)
`tion concentrations in plant cells of weak acid herbicides
`compared to a model that uses pH only. Herbicides also may
`accumulate in plant cells by conversion to nonphytotoxic
`metabolites, binding to cellular constituents, or partitioning
`into lipids. Evidence exists for herbicide transport across cell
`membranes via carrier-mediated processes where herbicide
`accumulation is energy dependent; absorption is saturable
`and slowed by metabolic inhibitors and compounds of siJni(cid:173)
`lar structure.
`Additional index words: Active transport, carrier-mediated,
`herbicide transport, herbicide uptake, ion trapping, passive
`diffusion, passive absorption, weak acids, #3 ABUTH,
`AEGCY, BROTE, CASOB, CYNDA, HELTU, IPOHG,
`LEMNI, LOLMU, SETFA, SETVI, SETVP.
`
`INTRODUCTION
`
`Prior to phytotoxic action, a herbicide must be absorbed by
`the plant and reach its site of action, usually located within an
`organelle in the plant cell. Once at the active site, the herbicide
`may accumulate to concentrations that will elicit a phytotoxic
`response. Several factors can influence the final herbicide con(cid:173)
`centration at sites of action, including root or shoot absorption,
`translocation from sites of absorption to sites of action, or meta(cid:173)
`bolism of the herbicide to nonphytotoxic compounds (38, 40).
`Once the herbicide reaches the plant cell, it must traverse the cell
`wall and the plasma membrane to enter the cytoplasm, and in
`
`many cases traverse organellar membranes to reach sites of
`action. This review will focus on movement of herbicides into
`plant cells.
`Research examining mechanisms of herbicide absorption
`across plant membranes is limited; however, over the past dec(cid:173)
`ade, numerous studies have explained the mechanisms of herbi(cid:173)
`cide absorption at the cellular level. To obtain relatively accurate
`measurements of herbicide concentration in plant cells, re(cid:173)
`searchers have used either excised tissues such as roots (24, 26,
`49, 73), tuber slices (4, 58, 68, 97), cotyledon (33, 44) or leaf
`tissues (27, 55, 77, 96), suspension-cultured plant cells (15, 21,
`34, 81), protoplasts (6, 19, 22, 54), plasma membrane vesicles
`(80), or algal cells (62, 100). The use of excised tissues reduces
`the confounding of translocation which continually reduces her(cid:173)
`bicide concentrations at the site of absorption (73). Suspension(cid:173)
`cultured cells are more simply organized compared to tissue
`sections allowing for accurate estimates of cellular herbicide
`concentrations. Use of protoplasts (plant cells lacking cell walls)
`or plasma membrane vesicles allows for direct exposure of the
`plasma membrane to the external solution (19, 80). Generally,
`radio labeled herbicid~ are used to detect final herbicide concen(cid:173)
`trations and patterns of herbicide absorption in these tissues. This
`review is limited to research conducted on such tissue, where
`tissue was bathed in 1.1edium containing nontoxic concentrations
`of herbicide and no metabolism of the herbicide was detected
`unless stated otherwise.
`An understanding of the principles of solute transport in
`plants is essential for describing mechanisms of herbicide ab(cid:173)
`sorption by plant cells; these principles are discussed by Briskin
`(10) in this review series. Physicochemical characteristics of the
`herbicide molecule, the plant cell membrane, and the electro(cid:173)
`chemical potential across the plant cell membrane control herbi(cid:173)
`cide absorption across plant membranes and subsequent
`accumulation in the cell. In general, mechanistic studies have
`demonstrated that herbicides are absorbed across plant mem(cid:173)
`branes by either passive or active processes. This review will
`discuss the various mechanisms by which specific herbicides are
`absorbed across plant membranes, and in certain cases how
`herbicides are accumulated within plant cells. The chemical
`names of herbicides discussed in the text are listed in Table l.
`
`PASSIVE ABSORPTION
`
`1Received for publication April 8, 1993, and in revised form July 10, 1993.
`Research supported by the New Mexico Agric. Exp. Stn., New Mexico State
`Univ.
`2Asst. Prof., Dep. Entomol., Plant Pathol. and Weed Sci .. New Mexico State
`Univ., Las Cruces, NM 88003.
`3Leners following this symbol are a WSSA-approved computer code from
`Composite List of Weeds, Revised 1989. Available from WSSA. 1508 West
`University Ave., Champaign, IL 61821-3133.
`
`Most herbicides are thought to move across plant membranes
`via nonfacilitated diffusion (40). Nonfacilitated diffusion is sol(cid:173)
`ute movement down an electrochemical gradient (63). Herbi(cid:173)
`cides absorbed via passive diffusion can be separated into two
`classes depending on the physicochemical characteristics of the
`herbicide molecule: a lipophilic, neutral molecule or a lipophilic
`molecule with a functional group sensitive to pH which can
`
`263
`
`

`

`STERLING: HERBICIDE ABSORPTION AND ACCUMULATION
`
`Table 1. Chemical names of herbicides and herbicide metabolites mentioned in the text.
`
`Ametryn
`Amitrole
`Atratooe
`Atrazine
`
`Bensulfuroo
`Bentazon
`Bromoxynil
`
`Chloramben
`Chlorbromuron
`Chlorimuron
`Chlorpropharn
`Chlorsulfuron
`Chlortoluron
`Clopyralid
`Cyanazine
`
`Dalapon
`Diclofop
`Dinoseb
`Diuron
`
`EPrC
`
`Fenuron
`Fluorodifen
`Fluometuron
`
`Glyphosate
`
`N-ethyI-N'-( l-methylethyI)-6-(methylthio )-1,3,5-triazine-2,4-diarnine
`IH-1,2,4-triazoI-3-arnine
`2-methoxy-4-(ethylamino)-6-(isopropylamino)-s-triazine
`6-chloro-N-ethyl-N'-( 1-methylethyl )-1,3,5-triazi ne-2,4-diamine
`
`2-[[[[[(4,6-dimethoxy-2-pyrimidinyl)amino]carbonyl)amino]sulfonyl]methyl]benzoic acid
`3-( 1-methylethyl)-( IH)-2, 1,3-benzothiadiazi n-4(3H)-one 2,2-dioxide
`3,5-dibromo-4-hydrox ybenzonitri le
`
`3-arnino-2,5-dichlorobenzoic acid
`3-( 4-bromo-3-chlorophenyl)- l-methox y-1-methyI-urea
`2-[[[[(4-chloro-6-methoxy-2-pyrimidinyl)amino]carbonyl)amino]sulfonyl]benzoic acid
`1-methylethyl 3-chlorophenylcarbamate
`2-chloro-N-[ [ ( 4-methox y-6-meth yl- J ,3,5-triazi n-2-yl )amino ]carbony I ]benzenesul f onamide
`N'-(3-chloro-4-methylphenyl)-N,N-d'.methylurea
`3,6-dichloro-2-pyridinecarboxylic acid
`2-[[ 4-chloro-6-( ethylami no )-1,3,5-triazi n-2-yl ]amino J-2-methy lpropaneni tri le
`
`2,2-dichloropropanoic acid
`(±)-2-14-(2,4-dichlorophenox y)phenoxy ]propanoic acid
`2-( l-methylpropyl)-4,6-dini trophenol
`N'-(3,4-dichlorophenyl)-N,N-dimethylurea
`
`S-ethyl dipropyl carbarnothioate
`
`N,N-dimethyl-N'-phenylurea
`p--nitrophen yl-a,a,a-tri fluoro-2-ni tro-p--tolyI
`N,N-dimethyl-N'-[3-(trifluoromethyl)phenyl]urea
`
`N-(phosphonomethyl)glycine
`
`Haloxyfop
`Hydroxyatrazine
`
`2-(4-[[3-chloro-5-(trifluoromethyl)-2-pyridinyl]oxy]phenoxy]propanoic acid
`2-hydroxy-4-(ethylamino)-6-(isopropylamino)-s-triazine
`
`lmazapyr
`lmazaquin
`lmazethapyr
`Ioxynil
`
`Linuron
`
`MCPA
`Metribuzin
`Metsul furon
`MH
`Monuron
`
`Napropamide
`Norllurawn
`
`Oryzalin
`
`Paraquat
`Picloram
`Propazine
`
`Sethoxydim
`Simazine
`Sulfometuron
`
`Thi fensul furon
`Trifluralin
`2.4-D
`2.4.5-T
`
`264
`
`(±)-2-(4,5-dihydro-4-( I-methylethyl)-5-oxo-lH-imidazol-2-yl]-3-pyridinecarboxylic acid
`2-[ 4,5-dihydro-4-methyl-4-( l -methylethyl)-5-oxo- I H-i midazol-2-yI ]-3-quinol i necarboxylic acid
`2-( 4 ,5-dihydro-4-meth yl-4-( 1-rnethylethy 1)-5-oxo- LH-imidazol-2-yl ]-5-ethyl-3-pyridinecarboxyiic acid
`4-hydroxy-3,5-diiodobenzonitrile
`
`N'-(3,4-dichlorophenyl)-N-methoxy-N-methylurea
`
`(4-chloro-2-methylphenoxy)acetic acid
`4-amino-6-( I, l-dimethylethyl)-3-(methylthio)- J,2,4-triazin-5(4H)-one
`2-(([[ ( 4-methox y-6-methyl-1,3,5-triazin-2-yl)amino Jcarbonyl]arnino]sulfooyl]benzoic acid
`I ,2-dihydro-3,6-pyridazinedione
`N'-( 4-chlorophen yl )-N,N-dirnethyl urea
`
`N,N-diethyl-2-( 1-naphthalenyloxy)propanarnide
`4-chloro-5-(methylamino)-2-(3-(trifluoromethyI)phenyl)-3(2H)-pyridazinone
`
`4-(dipropylarnino)-3,5-dinitrobenzenesulfonarnide
`
`I. l'-dimethyl-4.4'-bipyridinium ion
`4-amino-3.5.6-trichloro-2-pyridinecarboxylic acid
`6-chloro-N.N'-bis( 1-methylethyl)-1 ,3,5-triazine-2,4-diamine
`
`2-[ l-(ethoxyimino )butyl J-5-(2-( ethylthio )propyl )-3-hydrox y-2-cyclohexene- J-one
`6-chloro-N.N'-diethyl-1.3,5-triazine-2.4-diamine
`2-[[([(4.6-dimethyl-2-pyrimidinyl)amino]carbonyl]amino]sulfonyl]benzoic acid
`
`3-[ I [ [ ( 4-methox y-6-methyl-1.3.5-triazin-2-yl )amino ]carbonyl]amino[sul fonyl]-2-thiophenecarboxylic acid
`2.6-dinitro-N.N-dipropyl-4-(trifluoromethyl)benzenamine
`(2.4-dichlorophenoxy)al-etic acid
`(2.4.5-trichlorophenoxy)acetic acid
`
`Volume 42. Issue 2 (April-June) 1994
`
`

`

`C.:yloplasm
`(pll 7 .5)
`
`Vac:uole
`(pll 5 .:-,)
`
`. . p ln s rnc1
`111e111 brunc: _. l.ono p lc1 !-i l
`
`e
`
`\()
`
`\" 0
`
`,. -
`_\
`
`~ 9 (I
`
`.... \-
`
`•
`
`\(}
`"
`:.
`'
`\ -
`"
`' ' _\
`~- + \
`
`' I
`t,'
`~ , ·
`'
`,,
`
`I
`
`•
`
`WEED SCIENCE
`
`Apoplasm
`(pH 5.5)
`
`cel l wc1ll ~
`
`a .
`
`\
`
`l; .
`
`,. '
`~ -
`...
`
`\()
`
`~
`
`yo
`- \. .
`
`1,
`' ·,
`
`(cid:141)
`
`dissociate into a less lipophilic ion. The most important physico(cid:173)
`chemical properties of a herbicide molecule in terms of absorp(cid:173)
`tion by plant cells are Iipophilicity and acidity ( 12). Lipophilicity
`or polarity is assessed by the octan-1-ol water1 partition
`coefficient <Kow)4 of the herbicide molecule, either by direct
`measurement or estimation using water solubility values. The
`dissociation constant (pKa)4, a measure of acidity, determines the
`relative proportion of the acid and its conjugate base present at
`a particular pH and can be measured or estimated. The impor(cid:173)
`tance of these physicochemical characteristics for whole plant
`translocation are discussed in many reviews ( 12, 23, 25) and will
`not be addressed here.
`Lipophilic, neutral herbicides. Lipophilic, neutral herbicide
`molecules are thought to passively diffuse into plant cells. The
`lipophilic nature of these herbicides allows them to diffuse
`rapidly across the lipid bilayer of plant membranes down their
`concentration gradient to reach equilibrium concentrations be(cid:173)
`tween external solutions and the cell interior (Figure la). Several
`mechanistic studies strongly support this hypothesis for amitrole
`(49, 88), monuron (26), norflurazon (56), oryzalin (99), and
`triazines (73, 87). In addition, a variety of other studies add
`supplemental support for this hypothesis. Although amitrole is
`amphoteric5 and the triazines are weak bases, they will be in(cid:173)
`cluded in this section because they behave, in terms of cellular
`absorption at physiological pH, as lipophilic, neutral herbicides.
`Experimental criteria supporting passive diffusion of herbicides
`include saturation of uptake over time to equilibrium concentra(cid:173)
`tions, herbicide absorption rates related linearly to external her(cid:173)
`bicide concentrations, rapid herbicide efflux, temperature
`coefficients (Qw) of absorption which are less than 2, and insen(cid:173)
`sitivity of absorption to metabolic inhibitors.
`In general, passive diffusion of herbicides into plant tissues
`and cells to reach equilibrium concentrations occurs rapidly.
`Amitrole absorption by bean (Phaseolus vulgaris L.) roots was
`biphasic with an initial rapid phase through 4 h followed by a
`slower absorption phase through 24 h where equilibrium concen(cid:173)
`trations were nearly reached (49). Oryzalin absorption by com
`(Zea mays L.) root apices plateaued after 1 h (99). Another
`dinitroaniline, trifluralin, and a diphenyl ether, fluorodifen, were
`absorbed by isolated Zinnia elegans Jacq. leaf cells or tomato
`(Lycopersicon esculentum Mill.) immature fruit protoplasts
`within 2 h (6). Norflurazon absorption by sicklepod (Cassia
`obtusifolia L. #3 CASOB) and cotton (Gossypium hirsutum L.)
`root segments reached equilibrium concentrations after 10 min
`(56). Simazine absorption by barley (Hordeum vulgare L.) roots
`was complete in 5 min (87). Similarly, diuron reached equilib(cid:173)
`rium concentrations after only 5 min in unicellular microalgae
`(Ankistrodesmus braunii Naegeli) (62), whereas monuron ab-
`
`4Abbreviations: A-, conjugate base or anionic species; DNP, dinitrophenol;
`CCCP, carbonyl cyanide m-chlorophenylhydrazone; DCCD, N,W-dicyclohexyl(cid:173)
`carbodiimide; DCMU, 3-(3,4-dichlorophenyl)-l- l-dirnethylurea; FCCP, car(cid:173)
`bonyl cyanide p-trifluoromethoxyphenylhydrazone; HA, undissociated acid;
`H+-ATPase, proton-ATPase; l<,,w, octan-1-ol water- • partition coefficient; PCIB,
`p-chlorophenoxyisobutyric acid; p~. dissociation constant.
`51. Baron, Rhone-Poulenc Agric. Co., Res. Triangle Park, NC. 1993. Personal
`communication.
`
`Volume 42, Issue 2 (April-June) 1994
`
`Figure 1. Models of herbicide absorption and accumulation by plant cells: a)
`passive diffusion of lipophilic, neutral herbicides, X; b) passive diffusion of
`, nondissociated acid; x-, anionic species) and
`lipophilic, ionic herbicides (X0
`accumulation of anionic species in celJ compartments due to inability to diffuse
`across the lipid bilayer of plant membranes; c) passive diffusion of lipophilic,
`, nondissociated acid; x-, anionic species) and diffusion of
`ionic herbicides (X0
`anionic species across membranes; and d) carrier-mediated transport of herbi(cid:173)
`cide, X. Membrane potentials across the plasma membrane and tonoplast are
`equal to ca. -120 and -90 m V, respectively.
`
`sorption by barley roots was complete after 1 h (26). In contrast,
`other phenylurea herbicides, fluometuron, and chlorbromuron
`were not absorbed by isolated Zinnia leaf cells or tomato imma(cid:173)
`ture fruit protoplasts although these results may be due to the
`extensive rinsing techniques causing efflux of any absorbed
`herbicide (6). Atrazine rapidly diffused into excised roots of
`velvetleaf (Abutilon theophrasti Medic. # ABUlH) (73, 75),
`corn (20, 75), oat (Avena sativa L.) (75), potato tuber slices
`(Solanum tuberosum L.) (68, 75), and microalgae cells (62)
`reaching concentrations equal to external concentrations after 10
`to 30 min. Atrazine reached equilibrium concentrations in oat
`root segments within 10 min (Figure 2a). In contrast, atrazine
`absorption by com root protoplasts (20) and diclofop-methyl
`absorption by oat seedling protoplasts (98) was complete in 10
`and 5 s, respectively, suggesting that the complex organization
`
`265
`
`

`

`STERLING: HERBICIDE ABSORPTION AND ACCUMULATION
`
`ametryn
`
`atrazine
`
`a.
`
`120
`
`E 100
`:I w .i:
`:::,.:::
`.c 80
`<
`E--
`:I
`'1.. C"
`QJ 60
`....
`:::,
`0
`~ 40
`
`20lL----'----'-----L---'---'---....1._-_.J
`0
`5
`10
`15
`20
`25
`30
`TIME (min)
`
`100 i---~-----.._ ______ b_._
`
`size as atrazine (Figure 2a). Log K,,w values measured for
`ametryn, atrazine, and hydroxyatrazine were 2.9, 2.5, and 0.8,
`respectively {I). Diclofop-methyl uptake by bean and maize
`chloroplasts (41) and oat protoplasts (98) was 8 and 10 times
`greater than the less lipophilic molecule diclofop, respectively.
`These results suggest that although the plasma membrane is not
`a barrier to triazine or diclofop-methyl absorption, the membrane
`is a barrier to less lipophilic molecules such as hydroxyatrazine
`and diclofop, respectively. In whole plant studies, root uptake of
`nonionized chemicals was determined primarily by the lipo-
`philicity of the chemicals and was largely independent of plant
`species (8).
`Further evidence that cell membranes do not present an ap(cid:173)
`preciable barrier to herbicide molecules is the ability of these
`molecules to efflux out of cells or tissues that contain the herbi(cid:173)
`cide. Norflurazon efflux from com and sicklepod root segments
`occurred in two phases: a rapid phase where about 70% of the
`radiolabel was lost within 30 min, and a slow phase, through 6
`h, where an additional 10 to 20% diffused out of the tissues.
`Atrazine diffused rapidly out of excised corn (20, 75), foxtail
`(Setaria faberi Hemn. # SETFA) (66), and oat ( 1, 75) roots,
`potato tuber discs (68) and slices (75), and soybean [ Glycine max
`(L.) Merrill] (59) and velvetleaf (73, 74, 75) roots. For all these
`studies, 50 to 90% of the initial atrazine present in tissues
`effluxed within 30 min. When atrazine efflux from oat roots was
`compared to efflux of the less lipophilic molecules, hydroxya(cid:173)
`trazine and the mineral ion K+ (as tested using 86Rb+), atrazine
`efflux was nearly complete after 30 min followed by hydroxy(cid:173)
`atrazine and K+ with 60% and less than I% eluted after 30 min,
`respectively (Figure 2b). The pools from where the herbicide
`eluted could not be identified; these pools, as proposed for
`mineral ions using compartmental analysis of efflux data, include
`the free space, cytoplasm, and vacuole of the tissue (1 , 20). These
`results support further that cell membranes are not barriers to
`either influx or efflux of lipophilic, neutral herbicides.
`Additional evidence supports absorption oflipophilic, neutral
`herbicides by simple diffusion and not by an energy-dependent
`process such as carrier-mediated transport. A linear increase in
`herbicide absorption with increasing external herbicide concen(cid:173)
`trations indicates absorption is not saturating at higher concen(cid:173)
`trations and absorption is not carrier mediated. Absorption of
`amitrole (49, 88), atrazine (54, 62, 73), diclofop-methyl (98),
`metribuzin (13), monuron (26), and simazine (86) was linear
`with external concentrations of each herbicide, suggesting the
`mechanism by which these herbicides enter plant cells is simple
`diffusion. Temperature coefficient (Q 10) values for arnitrole,
`atrazine, chlorpropham, EPTC, linuron, and naproparnide
`ranged from 1.1 to 1.85 (3, 49, 59, 73, 88). These values are
`similar to Q 10 values near l for physical or passive processes
`while coefficients of 2 or greater indicate metabolic processes
`are involved (63). Amitrole (88), oryzaJin (99), and triazine (66)
`absorption by plant tissues was insensitive to metabolic inhibi(cid:173)
`tors, suggesting absorption did not require energy; however,
`evidence for arnitrole (88) and atrazine (73) was less conclusive,
`suggesting some energy requirement.
`
`Volume 42, Issue 2 (April-June) 1994
`
`atrazine
`0 L__..J...__....1._ __ _ .....1.... _ _;_ _
`0
`30
`60
`90
`120 150 180 210 240
`TIME (min)
`
`__..J._ _
`
`___,_ _
`
`___,Jc...J
`
`Figure 2. Time COUJ'SC of: a) ametryn, alrazine, and hydroxyatrazine influx into
`and b) atrazine, hydroxyatrazine. and K+ (86Rb+) efflux from oat rOOI segments.
`From Balke and Price ( I).
`
`of tissue sections compared to individual protoplasts or cells
`results in slower penetration. Also, these results suggest the
`plasma membrane is not a barrier to lipophilic, neutral herbicide
`absorption.
`Absorption of lipophilic, neutral herbicides by plant cells is
`related to their relative lipophilicities. Absorption of nonelectro(cid:173)
`lytes by Chara intemodal cells was positively correlated with
`partition coefficients of the chemicals between olive oil and
`water (16). Initial rates of triazine (atrazine, atratone, hydroxy(cid:173)
`atrazine, simazine) and phenylurea (diuron) herbicide absorption
`correlated positively with their partition coefficients in olive oil
`and water or in n-dodecane and water (87). Diuron and atrazine
`partition coefficients were independent of pH (62, 87), suggest(cid:173)
`ing the neutral, nonpolar form of atrazine predominates at physi(cid:173)
`ological pH. Atrazine and ametryn were rapidly absorbed by
`excised com (20) and oat (I) roots reaching equilibrium concen(cid:173)
`trations within 10 min in contrast to slower absorption of hy(cid:173)
`droxyatrazine. a less lipophilic molecule approximately the same
`
`266
`
`

`

`WEED SCIENCE
`
`a.
`
`d.
`
`O
`II
`
`1·•
`V SOzNHCKH-{ _ /;N
`Cl
`N-'--. CH 3
`
`N ___, OCH3
`-
`._
`
`bentazon
`
`chlorsulfuron
`
`e.
`
`f.
`
`b.
`
`OCH 2COOH
`
`(cid:144)C l
`
`Cl
`2,4-D
`
`net
`
`C~N::::J-. COOH
`
`clopyralid
`
`C.
`
`0
`imazethapyr
`
`o
`
`~ NOC 2 H 5 D c -cH2CH 2CH3
`
`OH
`
`H 5C2S~HCH2
`CH 3
`sethoxydim
`
`Figure 3. Structures of weak acid herbicides accumulated in plant cells by ion
`trapping.
`
`Weak acid herbicides are absorbed by simple diffusion. Ab(cid:173)
`sence of saturation kinetics indicates that weak acid herbicide
`transport into plant ~lls is not carrier mediated and is via
`nonfacilitated diffusion. Absorption of several weak acid herbi(cid:173)
`cides by various plant species and tissues was linearly related to
`external herbicide concentration including bentazon (Figure 4 ),
`bromoxynil (33), chloramben (95), clopyralid (24), chlorsul(cid:173)
`furon (24), 2,4-D (44, 100), imazapyr (77), imazaquin (77),
`imazethapyr (34, 77), ioxynil (62), dinoseb (62), maleic hydraz(cid:173)
`ide (32), MCPA (31 ), picloram (21, 43, 97), sethoxydim (91, 96),
`and the sulfonylureas bensulfuron methyl, chlorimuron ethyl,
`
`~
`:::i::: --120
`I
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`
`•
`
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`
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`
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`
`Thus, uptake of many lipophilic, neutral herbicides is a pas(cid:173)
`sive process where the neutral herbicide is able to freely diffuse
`across cell membranes, reaching equilibrium concentrations
`with the external solution or tissue apoplasm. Accumulation of
`these herbicides to concentrations greater than the external con(cid:173)
`centrations are discussed below.
`Lipophilic, ionic herbicides. Lipophilic, ionic molecules are
`either weak acids or weak bases. In an aqueous solution, neutral
`weak acids are in equilibrium with their conjugate base which is
`charged ( equation I); neutral weak bases are in equilibrium with
`their conjugate acid which is charged (equation 2). The relative
`concentrations of the hydrophobic, undissociated acid molecules
`(HA)4 or the more polar anions (A-)4 depend on solution pH and
`strength of the acid as described by the Henderson-Hasselbach
`equation (equation 3). Ability of these molecules to be either
`neutral or charged greatly affects rates of movement across plant
`membranes as well as accumulation concentrations.
`
`R-COOH =
`
`R-Coo- + w
`
`[A-J
`pH =pl(,.+ log [HA]
`
`(I)
`
`(3)
`
`Mechanistic evidence for passive absorption of lipophilic,
`ionic herbicides and their accumulation by ion trapping as de(cid:173)
`scribed below exists only for weak acid herbicides such as
`bentazon (94), 2,4-D (19, 81 , 87), clopyralid and chlorsulfuron
`(24), imidazolinones (34, 77), and sethoxydim (96) (Figure 3).
`The principles of passive absorption described in the previous
`section for lipophilic, neutral herbicides apply also to weak acid
`herbicides which passively diffuse into plant cells. However, in
`contrast to lipophilic, neutral herbicides, weak acid herbicide
`absorption saturates over time with the herbicide reaching cellu(cid:173)
`lar concentrations greater than external concentrations (19, 24,
`34, 94, 96). Weak acid herbicide accumulation in plant cells to
`concentrations greater than external concentrations can be re(cid:173)
`duced by metabolic inhibitors or anoxia and is enhanced by
`acidic pH external to the cell, suggesting accumulation is energy
`dependent and is mediated by ion trapping. Higher concentra(cid:173)
`tions of the undissociated herbicide exist at acidic rather than
`neutral pH (equation 3). The undissociated acid diffuses across
`the plasma membrane because cell membranes are more perme(cid:173)
`able to undissociated, neutral molecules compared to dissoci(cid:173)
`ated, charged molecules (76). Once in the alkaline compartments
`of the cell, the acid dissociates creating a concentration gradient
`for further influx of the acid which results in herbicide accumu(cid:173)
`lation as the anion (Figure 1 b ). Cytoplasm of the plant cell is
`maintained alkaline by the continual removal of H+ to the vacu(cid:173)
`ole or to the apoplast by the proton-ATPases (H+-ATPases)4
`located on the plasma membrane and tonoplast of the plant cell
`(10); therefore, accumulation of weak acid herbicides requires
`metabolic energy.
`
`Volume 42, Issue 2 (April-June) 1994
`
`< -E--
`0..
`~ ~
`z """
`1-, 80
`N -<
`0
`E-- s 40
`~ -
`z i:::
`
`0D
`
`0
`
`i:o
`
`0
`
`0 10 20 30 40 50 60 70 80 90 100
`CONCENTRATION ( µM)
`
`Figure 4. Concentration dependence ofbentazon uptake by velvetleaf cells. From
`Sterling et al. (94).
`
`267
`
`

`

`STERLING: HERBICIDE ABSORPTION AND ACCUMULATION
`
`chlorsulfuron, thifensulfuron methyl, metsulfuron methyl, sulfo(cid:173)
`meturon methyl (65), and chlorimuron ethyl ester (61).
`Nonfacilitated movement of weak acid herbicides across
`plant cell membranes is also supported by the ability of the
`herbicides to diffuse out of cells into solutions not containing the
`herbicide. Herbicide efflux from tissues loaded with bentazon
`(94), bromoxynil (33), 2,4-D (44, 100), clopyralid (24),
`chlorimuron ethyl ester (61), chlorsulfuron (24, 55), the irnida(cid:173)
`zolinones (77), and picloram (43, 60, 97) was between 75 and
`95% of initial herbicide concentrations and was complete within
`30 to 90 min. Two phases of efflux were identified for imazaquin
`elution, suggesting initial elution was from cell wall free space
`followed by elution from the cytoplasm and vacuole (77). These
`results suggest the accumulation of weak acid herbicides is not
`due to irreversible binding and that neither the plasma membrane
`nor the tonoplast are effective barriers to weak acid herbicide
`transport. In contrast, frozen potato tuber slices released 2,4-D
`freely after transfer to a solution not containing 2,4-D; however,
`nonfrozen tissue released only 3% after 30 min, suggesting the
`cell membranes were barriers to 2,4-D movement (68). Similarly,
`efflux from intact compared to freeze-thawed tissue loaded with
`imazaquin was more rapid, suggesting that imazaquin is retained
`in the tissue by an intact plasma membrane (39, 77). Overall,
`rates of efflux for weak acid herbicides were slower than efflux
`of lipophilic, neutral herbicides discussed above and may be due
`to lower membrane permeability to weak acid herbicides com(cid:173)
`pared to lipophilic, neutral herbicides.
`The feature controlling the transport of weak acid herbicides
`across membranes is their lipid-soluble, weakly acidic nature.
`Uptake of 12 imidazolinone analogs by sunflower (Helianthus
`annuus L.) and com roots was highly correlated to their lipo(cid:173)
`philicity (51, 52). In addition, irnidazolinone analogs required
`their carboxylic acid group for activity although the acidity and
`lipophilicity of each was more important for transport than for
`enzyme inhibition at the site of action (48). Similarly, rates of
`sulfonylurea analog uptake by velvetleaf cells were governed
`primarily by the lipophilicity (Kow) of each analog (65). Chlor(cid:173)
`sulfuron <Kow = l .3) absorption by excised pea roots was faster
`than clopyralid <Kow = 0.0018) absorption (24). Lipophilicity
`<Kow) values are dependent on pH with greater Kow values at
`lower pH or at pH closer to the pK3 of each herbicide as
`demonstrated for2,4-D (87), dinoseb (62), bromoxynil (33), and
`sethoxydim (96). In addition, rates of irnidazolinone absorption
`were higher at low pH compared to high pH and appeared related
`to the larger Kow values at lower pH compared to higher pH
`(Table 2). Membranes are more permeable to the lipophilic,
`undissociated acid molecules, which predominate at low pH
`(equation 3) compared to their more polar anions (9). Raven (76)
`estimated that the permeability of an algal (Hydrodictyon afri(cid:173)
`canum) cell membrane to nonionized indole-3-acetic acid (IAA)
`is approximately 10-3 m s- 1• about a thousand times greater than
`the permeability of its anion. Therefore. the undissociated acid
`diffuses across the plasma membrane because cell membranes
`are more permeable to undissociated, neutral molecules com(cid:173)
`pared to dissociated, charged molecules. Alternatively, lipo-
`
`Table 2. Effect of pH on imidazolinone Kow values and uptake rates. From Reider
`Van Ellis and Shaner (77).
`
`K,,w
`
`Imidazolinone
`uptake
`
`Herbicide
`
`pH4
`
`pH 7
`
`pH4
`
`pH7
`
`nmol · g dw- 1 • h- 1
`
`lmazapyr
`lmazethapyr
`lrnazaquin
`
`0. 1
`1.4
`7.7
`
`0.004
`0.017
`0.038
`
`13
`24
`45
`
`3
`3
`3
`
`philicity may not be the sole factor determining the behavior of
`weak acids (9). Although ion trapping explains accumulation of
`the irnidazolinones, this mechanism does not fully explain why
`rates of uptake of irnidazolinone analogs vary greatly with small
`changes in chemical structure (39). Therefore, other factors
`besides Kow and pK3 also may regulate uptake of weak acid
`herbicides.
`Although experimental evidence supports simple diffusion as
`the mechanism of weak acid herbicide transport across cell
`membranes, cellular metabolism is necessary for their accumu(cid:173)
`lation to concentrations greater than external concentrations. The
`respiratory inhibitor potassium cyanide (KCN) and low oxygen,
`as induced by bubbling nitrogen gas {N2) through the uptake
`medium, reduced bentazon absorption by velvetleaf cells (94)
`and chlorimuron ethyl ester absorption by excised velvetleaf
`roots (61), suggesting that cellular metabolic energy enhanced
`uptake of weak acid herbicides. The addition of ATP increased
`sethoxydim uptake by wheat (Triticum aestivum L.) leaves,
`further supporting the need for metabolic energy (Table 3). The
`proton ionophore, carbonyl cyanide m-chlorophenylhydrazone
`(CCCP)4. reduced absorption ofbentazon (94), bromoxynil (33),
`MCPA (31), maleic hydrazide (32), imidazolinones (77), and
`sethoxydim (Table 3) indicating that proton gradients across
`membranes are involved in weak acid herbicide uptake. The
`proton ionophore, carbonyl cyanide p-trifluoromethoxyphenyl(cid:173)
`hydrazone (FCCP)4. also reduced absorption of irnidazolinones
`(39, 77). The uncoupler, dinitrophenol (DNP)4, reduced absorp(cid:173)
`tion of MCPA (31), maleic hydrazide (32), picloram (43), and
`
`Table 3. Influence of various compounds on setboxydim uptake by wheat leaf
`sections. From Couderchet and Retzlaff (17).
`
`Substance added
`
`ATP (0.5mM)
`ATP ( l mM)
`ATP (2mM)
`ATP (4mM)
`ATP(7.6mM)
`Fusicoccin (0.5 mM)
`CCCP(20µM)
`Vanadate ( I 00 µM)
`DES (IOOµM)
`
`Sethoxydim
`uptake
`
`(% of control)
`
`121.8
`133.3
`234.7
`254.6
`255.0
`117.5
`75.3
`34.7
`52.0
`
`268
`
`Volume 42, Issue 2 (April-June) 1994
`
`

`

`WEED SCIENCE
`
`several imidazolinones (51, 77) probably by disrupting the hy(cid:173)
`drogen ion concentration gradient and reducing membrane in(cid:173)
`tegrity (51 ). Similarly, imidazolinone uptake was inhibited with
`sodium azide, sodium cyanide, and 3-(3,4-dichlorophenyl)-l-l(cid:173)
`dimethylurea (DCMU)4 (77); and picloram uptake was inhibited
`by sodium azide and sodium arsenate (43). In contrast, KCN
`(100) and DNP (44) did not affect 2,4-D uptake nor did sodium
`azide affect picloram absorption (60). Uptake of 2,4-D and
`2,4,5-Twas independent of available energy as tested by addition
`of CCCP, N,N'-dicyclohexylcarbodiimide (DCCD)4, and leci(cid:173)
`thin (89). The potassium ionophore, valinomycin, did not affect
`bentazon absorption (94), suggesting that potassium gradients
`across cell membranes were not necessary for bentazon uptake.
`Energy-dependent absorption is most likely due to the cell
`expending metabolic ene