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Inhibition of photosynthesis and respiration by substituted 2,6-dinitroaniline herbicides
PESTICIDE
BIOCHEMISTRY
Inhibition
AND
PHYSIOLOGY
2,
354-363
of Photosynthesis
and
2, &Dinitroaniline
Respiration
by
Substituted
Herbicides
II. Effects on Responses in Excised Plant Tissues and Treated D. E. MORELAND, Plant
Science Research Division, Crop Science Department,
F. S. FARMER,
Agricultural Research North Carolina State
Seedlings’
AND G. G. HUSSEY
Service, United States Department University, Raleigh, North Carolina
of Agricu :&we, R7607
Received May 22, 1972; accepted August 11, 1972 Effects of 12 substituted 2,6-dinitroaniline herbicides were measured on photosynthesis and respiration in excised tissue, and tissue excised from seedlings treated with oc,cu,cu-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine (trifluralin). The compounds partially inhibited photosynthesis (measured as oxygen evolution) of strips cut from spinach (Spinacia oleracea L.) leaf discs. Only 3,5-dinitro-N4,N4-dipropylsulfanilamide (oryaalin) strongly inhibited respiration (measured as oxygen uptake) of excised mung bean (Phaseolus aureus Roxb.) root tips and hypocotyls after a short treatment time. However, root tips excised from corn (Zea wzuys L.) seedlings germinated in contact with one representative 2,6-dinitroaniline (trifluralin) showed suppressed oxygen uptake. In addition, phosphorylation was completely uncoupled from oxidation in mitochondria isolated from primary roots of corn seedlings after germination of the seed in contact with trifluralin for 96 hr. Interference with photosynthesis and respiration both in vivo and in vitro by the 2,6-dinitroanilines has been demonstrated. Deviations in growth and metabolism produced by these herbicides can be explained partially by interference with the oxidative and photoproduction of ATP, if recognition is extended to the role played by ATP energy in maintaining cellular and nuclear activities. However, the importance of interference with ATP production to the phytotoxic action of the 2,6dinitroanilines remains to be established. INTRODUCTION
Inhibition of electron transport and phosphorylation in isolated spinach (Spinaciu olerucea L.) chloroplasts and mung bean (Phaseolus aureus Roxb. var. Jumbo) mitochondria by 12 substituted 2,6-dinitro1 Cooperative investigations of the North Carolina Agricultural Experiment Station and the Plant Science Research Division, Agricultural Research Service, United States Department of Agriculture, Raleigh, NC. Paper number 3773 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, NC. Investigations supported in part by Public Health Service Grant ES 90644. 354 Copyright All righta
0 1972 by Academic Press, of reproduction in any form
Inc. reserved.
aniline herbicides was reported in the first paper of t’his series(1). The compounds were identified structurally and literature was reviewed also in the aforementioned paper. The responseswith isolated organelles suggested that if the 2,6-dinitroanilines reach the chloroplasts and mitochondria, inhibition of photosynthesis and respiration in viva could be expected. Therefore, the objectives of the studies reported herein were to measure, correlate, and evaluate effects of the 2,6-dinitroanilines on photosynthesis and respiration in excised tissue, and tissue excised from seedlings treated with trifluralin.
DINITROANILINE
HERBICIDES
ON
PLANT
TISSUE
:i.iT,
Photosynthetic activity (oxygen evolution) of excised foliar tissue was measured polarographically, with a Clark electrode, in the thermostated glass cell described previously for studies with isolated mitochondria (1). Two leaf discs (9 mm diam, approx 40 mg fr wt), taken from growth chamber-grown spinach plants, were sliced into 1 mm-widr strips, placed in 2.0 ml of reaction medium, and deaerated under partial vacuum for 5 min. The reaction medium contained equal parts of CO2 buffer and modified Avron’s medium (2). hfter deaeration, the tissue was transferred to fresh reaction medium to which was added the test chrmical. After incubation for 30 min at 25”C, the tissue and reaction mixture were transferred to the thermostated glass cell. After purging of the reaction mixture with nitrogrn t’o lower the oxygen content, the sample chamber was irradiated (60,000 lx) with a Unitron LKR microscope illuminator.? Oxygen uptake by root tips and hypocotyl spctions, excised from 3-day-old, darkgrown mung bean seedlings, was also measurc>d polarographically with the apparatus used in studies mit,h isolated mitochondria (1). Hypocotyl sections (64 discs, 0.6 mm thick, 0.15 g fr wt) obtained from the region of elongation and root tips (30 tips 5 mm long, 0.04 g fr w-t) mere deaerated under partial vacuum in 2.0 ml reaction medium [0.03 M potassium phosphate buffer (pH 5.5)) 1% glucose, and the chemical treatment]. After deaeration, air was bubbled through thtl reaction medium during the lhr incubation period. The tissue was then transferred t.o fresh reaction medium in the
thermostated glass cell, and oxygen uplake measured. Effects of trifluralin on t,he respiration ot treated seedlings were determined in root tips excised, and mitochondria isolated, frc )m primary roots of corn (Zea mays L. var. Pioneer 3369A) seedlings. Seed had !XWI germinated between filter paper moistened with 25 ppm trifluralin in 0.57 Dh1803 (v/v) in the dark at 25°C. Aft’er 48 and 72 hr, four sequential a-mm sections Ivere excised from the primary roots starting at the tip. Sections from 20 roots were pooled, suspended in buffer [0.03 M potassium phosphate (pH 5.5) and 1% glucose], dcaerated for 5 min, and transferred to the thermostated cell in fresh buffer for oxygen uptake measurements. Mitochondria were also isolated from the root tissue of (*orn seedlings after germination of the seed in contact. with trifluralin by procedures described previously (1). Effects of the treat,ment on mitochondrial oxidative behavior and ATPase a&iv&y mere also measured with previously described procedures (1). The substituted 2,6-dinitroanilines used in this investigation were identified previously (I, Table 1). Because of the relative insolubility of the compounds in water, solutions of the desired concentrations were prepared in absolute ethanol for the foliar tissue study; and in DIMS0 for t’he excised hypocotyl, excised root t’ip, and seedgermination studies. The final concentration of ethanol in all assays including the controls was I % (v/v) and for the studies in which DMSO was the solvent, t#hefinal conrc:ntratjion was 0.5 %I (v/v). Data for the exci& tissue studies are arithmetic averages of al, least duplicate determinations.
2 Mention is made for uot imply Government. Experiment.
3 The following abbreviations text: DMSO, dimethylsulfoxide; dichlorophenyl) -l,l-dimethylurea; heptyl-4-hydroxyquinoline-N-oxide; dinitrophenol; It/C, respiratory
MATERIALS
AND
METHODS
of specific instruments or trade names ident,ification purposes only and does endorsement by the United States or the North Carolina Agricultural Station.
are used in the diuron, :3-(3,4HO&NC 1: 2DNP, 2,4control r&o.
356
MORELAND,
TABLE Effect
FARMER,
1
of
substituted d,6-dinitroanilines evolution of oxygen by illuminated leaf tissue
on
Inhibition”
A-820 AN 56477 BAS 392-H Benefin CGA-10832 GS-38946 GS-39985 Isopropalin Nitralin Oryealin Trifluralin USB 3584
12 17 47 10 9 46 24 2 31 57 0 25
(To)
a Compounds were tested at 0.6 mM, a saturating concentration. RESULTS
Photosynthesis
Most of the substituted 2,6-dinitroanilines, at a saturating concentration of 0.6 mM, inhibited photosynthesis as measured
300
‘OO-
HUSSEP
by suppression of oxygen evolution by illuminated spinach leaf tissue. All compounds were tested at this high concentration in an attempt to maintain a saturated solution external to the tissue so that a relative measure of their maximum inhibitory effectiveness could be obtained. Shown in Fig. 1 are traces of oxygen evolution obtained with untreated control tissue and three 2,6dinitroanilines (AN 56477, nitralin, and oryzalin). The compounds differed only by the amount of inhibition that was expressed. Evolution of oxygen, under the conditions described in Methods, was light-dependent and linear for the initial 2-3 min. In untreated control tissue, oxygen evolution averaged 326 nmoles/hr/dg fr wt. Diuron (3 MM), which is a potent inhibitor of noncyclic electron transport in isolated chloroplasts, was included as a reference and completely inhibited oxygen evolution. Inhibitory percentages for all compounds, calculated by relating the rate of oxygen evolution measured in the presence of a test compound to the rate expressed by un-
the
spinach
Compound
AND
-
liphl on
Time
FIG. 1. Representative polarographic traces depicting oxygen evolution by illuminated spinach leaf tissue. Compared are traces obtained with no-treatment control tissue, and suppression of oxygen evolution by AN 66.&V (0.6 mM), nitralin (0.6 mM), oryzalin (0.6 mM), and diuron (3.0 PM).
DINITTROANILINE
1
HERBICIDES
ON
PLANT
TISSUE
2 min 1 I
1
Time
FIG. 2. Polarographic traces depicting oxygen uptake by mung bean hypocotyl sections. traces obtained with. no-treatment control tissue, oryzalin (0.1, 0.4, and f .O mM), and sodium Rates of oxygen utilization (nmoles 02 per min per I ml) are indicated above the traces.
treated control &sue, are presented in Table 1. Rates were obtained from the initial linear segment of the traces and adjusted for differences in fresh weight of the tissue. Trifluralin and isopropalin had essentially no effect on oxygen evolution. The remainder of the compounds produced some inhibition, with BAS 392-H, GS-38946, and oryzalin being the strongest inhibitors. None of the compounds were as active as diuron (Fig. 1) in inhibiting photosynthesis. With the stronger inhibitors, inhibition of oxygen evolution could be detected after only a few minutes of incubation time and at molar concentrations lower than 0.6 mM. However, for comparative purposes, the concentration tested and the incubation time were held constant as identified in Jlethods. Differences in inhibitory potency may be related to relative penet,rabilit.ies of the compounds.
Oxygen uptake by excised mung bean hypocotyl sections and root tips, handled as described in Methods, was linear (Fig. 2) until anaerobiosis occurred. Oxygen utiliza-
Compared ore azidr (1 m&f).
tion by both tissues was also inhibited by 1 mM standard respiratory inhibitors: sodium azide (Fig. 2), 50 PM antimycin A, and 50 PM HOQNO. Of the 12 substituted 2,6-dinitroanilines tested, only oryzalin, which is the most water soluble, inhibited oxygen uptake. The amount of inhibition obtained was a function of the concentration of the inhibitor (Fig. 2) and at the highest concentration tested (1 mM) reached 60 %. Similar responses with oryzalin were obtained also with root tips, but only the data for hypocotyls are presented herein. Results presented previously (1) indicated that the 2,6-dinitroanilines interfered with the oxidation of malat,c, NADH, and succinate by isolated mitochondria. However, in studies with root tip and hypocotyl sections, that had been incubated with the compounds for 1 hr, only oryzalin inhibited respiration (Fig. 2). Therefore, the studies were extended to permit absorption and penetration to occur over a longer interval. For these evaluations, corn was used as the experimental speciesinstead of mung beans. Only the effects of trifluralin were studied, but
358
MORELAND,
FARMER,
because of similarities in behavior in studies reported previously (1)) comparable results might be expected with the other 2,6-dinitroanilines. Corn seed were germinated in the presence and absence of 25 ppm trifluralin. In comparison to control seedlings, the primary roots of the corn seedlings that had germinated in contact with trifluralin exhibited retarded elongation, inhibited lateral root development, and had a swollen tuberous-shape. These characteristic morphological changes have been observed by other investigators (1). Sections were excised at 2-mm intervals starting from the tip as described in LXIethods. Oxygen uptake (nanomoles per minute per decigram fresh weight) by trifluralin-treated and control tissue are compared in Fig. 3. In Graph A are shown the oxygen uptake patterns of primary root tissue from seed which had been germinated in continuous contact with 25 ppm trifluralin for 72 hr. Data in Graph B were obtained from roots of seed that were germinated initially in water, but after 48 hr were treated with 25 ppm trifluralin for 24 hr prior to excision. Under both conditions,
O-2
2-4
4-6 Dirtonce
6-8 from
O-2 roof
tip
2-4
4-6
6-6
(Inn;)
FIG. 3. Oxygen uptake by excised sections of the primary root of corn germinated in the presence and absence of 26 ppm tri$uralin. Graph A: Sections obtained from seeds germinated between filter paper moistened with water containing 0.6% DMSO (v/v) or 86 ppm Irifluralin for 7.2 hr. Graph B: Sections obtained from seed germinated in contact with water-saturated jilter paper for 48 hr. After 48 hr some of the seed were placed, and remained, in contact with 26 ppm trijluralin for 94 hr. (Legend: open bars, control tissue; slashed bars, treated tissue.)
AND
HUSSEY
trifluralin-treated root tissue showed a depression in respiration, as measured by oxygen uptake, relative to the untreated control tissue. In the root sections from the untreated controls, oxygen uptake was highest in the terminal 2 mm, decreased curvilinearly for one or two sections, and approached a constant value between 4 and 6 mm from the tip. However, the decrease in rate of oxygen uptake with distance from the tip was not observed in the trifluralintreated tissue. This behavior is shown in Graph A where all sections from treated roots utilized oxygen at approximately the same rate. In Graph B, the treated terminal section did utilize oxygen at a rate t’hat was slightly greater than the subsequent sections. In the untreated control tissue, the decrease in respiration with distance from the root tip correlates with mitotic activity. However, in the trifluralin-treated tissue, mitosis has probably been arrested (1) and the respiration is contributed primarily by nondividing cells. Respiratory and ATPase activities of mitochondria isolated from the corn seedlings also were examined 48, 72, and 96 hr after initiation of germination. Data are presented in Table 2 for only the 72- and 96-hr treatments. Trends obtained for the 48-hr treatment paralleled those of the 72-hr treatment and, hence, are not presented. As shown in Table 2, mitochondria isolated from primary roots of corn seedlings germinat.ed in contact with 25 ppm trifluralin for 72 hr generally had higher endogenous, state 3, and state 4 rates than mitochondria isolated from roots of untreated control seedlings. In addition, the R/C (respiratory control) and ADP/O ratios tended to be lower in mitochondria isolated from the treated tissue. There were no major differences in the level of endogenous ATPase activity or the response of the endogenous ATPase to Mg2+, but there was a decrease in the ATPase activity induced by DNP.
DINITROANILINE
HERBICIDES
TABLE 2 and ATPase activities of mitochondria
ON
PLANT
TISSUE
359
respiratory rates (endogenous, state 3, and state 4) that were slightly lower than those from roots of corn seedlings 72 and 96 hr measured after 72 hr (Table 2). The R/C the initiation of germination. Seed ratios also tended to be somewhat lower, wwrre germinated in the presence and but the ADP/O ratios were about the same. absence of 26 ppm tri$uralin However, mitochondria isolated from the Response measured Duration of treatment 96-hr treat.ed tissue generally had lower 72 hr 96 hr endogenous and state 3 respiratory rates -.-~ Control Treated Control Treated than those from untreated tissue. Strikingly, all control normally exerted by ADI’ in Malate oxidation 43 41 Endogmm?’ 45 65 tightly coupled mitochondria was lost for 111 82 state 3” 152 168 h the oxidation of malate, NADH, and succ38 St&e 4" 36 65 h 4.2 2.6 2.9 R/C nate (Table 2). The mit’ochondria IWVW h ADP/O 1.8 1.8 2.2 exhausted ADP and did not enter the cxNADH midtkim Endqq=nous 75 83 57 51 pectcd state 4 rate of respiration. Cc)nsc:i1Q state 3 205 236 137 h quently, R/C and ADP/O ratios could not 56 state 4 il 105 h 2.5 R/C 2.Y 2.3 be calculated. The loss in ADI’ control is h I.1 1.1 ADP/O 1.0 also presented in Fig. 4. In Trace A is shown Succinate oxidation 104 119 66 72 Endoaenous the oxidation of malate by mitochondria state 3 181 170 120 loo * from untreated 96hr corn tissue and the stat 4 104 108 72 6 1.7 1.6 1.7 R/C control exerted by ADP. In comparison, 6 1.3 1.3 ADP.IO 1.1 Trace B was obtained with mitochondria ATPase wtivity’ Endqgmms - Mg* 0.73 0.83 0.59 1.01 from trifluralin-treated tissue. After cwtabEndogenous + Mgz+ 2.42 2.26 1.93 1.88 lishment of the trend measured for endoge4.93 4.16 3.68 1.60 DNP (80& now respiratory activity, a limit,ing amount, of ADP was added. Respiration increased ?JRespirntifm nirt under ADP control, hence, values could and would have continued without an apnr,t he calculated. preciable change in respiratory rate until ’ Dsta presented as rmoles P, relessed per mg protein in 20 min. anaerobiosis occurred. R-o control was imposed by ADP and electron transport apIn intact mitochondria, Mg”+ appears to he peared to be complet’ely uncoupled from an imegral part, of the ATP-generating phosphorylation. Under t~hese conditions, complex and no requirement for exogenous the mitochondria were still capable of oxi11gz+ is shown. However, in structurally dizing substrates, but no ATI’ was being damaged mitochondria, the &I&+ is dissoci- generated. The mitochondrial preparation ated from the complex and a requirement for from the 96-hr treat’ed tissue also possessed Mgz+ is manifested (3). Endogenous mito- considerably higher cndogenous ATPase ehondrial ATPase activity is characteristicactivity t.han the controls and no longer ally stimulated by the addition of Mg2+. strongly responded to DW in the induction After treatment for 72 hr, the mitochon- of ATPase activit.y (Table 2). dria manifested small deviations in behavior In the treated tissue, between 72 and 96 which suggested that alteration in various hr, not only did the mitochondria become functions had been initiated by the treat uncoupled, but the respiration also shifted ment. However, 24 hr later, after 96 hr, toward a type which was resistant to trimore drastic effects were apparent. The root fluralin and other electron transport inCssue of the 96-hr control seedlings was hibitors. For example, with malate as subquite fiberous and the mitochondria had strate, the st#atc 3 respiration of mitochonRespiratory isolated after
MORELAND,
FARMER,
AND
HUSSEY
400-
0” ” i JOOE
200.
1
FIG. 4. Polarographic traces depicting oxygen utilization by mitochondria isolated from primary roots of corn seedlings that had been germinated for 96 hr. Trace A: Oxidation of malate by mitochondria from untreated seedlings showing control exercised by ADP. Trace B: Oxidation of malate by mitochondria from seedlings that had been treated with 26 ppm trifluralin showing the absence of respiratory control. Rates of oxygen utilization (nmoles0s per min per 2 ml) are indicated above the traces. Mitochondria (Mit), malate (0.2 mmoles), and A DP (0 S rmoles) were added at the points indicated. The mitochondrial aliquot used to obtain Trace A contained 0.30 and Trace B 0.3’7 mg protein.
dria obtained from both the 72-hr control and treated tissues, and 96-hr control tissue responded similarly to 40 PM trifluralin, and approx 60% inhibition was obtained. However, the state 3 respiration of mitochondria from 96-hr treated tissue was inhibited only by approx 30%. In addition, malate oxidation by mitochondria from 96-hr control seedlings was inhibited 60-75 % by electron transport inhibitors [sodium azide (1.0 mM), antimycin A (0.1 PM), and rotenone (1.0 mM)], whereas, malate oxidation by mitochondria from 96-hr trifluralin-treated tissue was inhibited only by approx 35 %. DISCUSSION
Studies with isolated chloroplasts and mitochondria showed that the 2,6-dinitroanilmes inhibited both electron transport and phosphorylation (1). Consequently, if the compounds successfully penetrate cells and partition into the organelles in viva, either by translocation or in vapor form from
the atmosphere, inhibition of photosynthesis and respiration could be anticipated. Results of the studies with excised foliar tissue suggested that photosynthesis (measured as oxygen evolution) was inhibited, at least partially, by all of the 2,6-dinit~anilines except trifluralin (Table 1, Fig. 1). Under the conditions of the experiments, oxygen evolution measurements were made after allowing 30 min for penetration of the compounds into the tissue. Respiration (measured as oxygen uptake) of excised root tips and hypocotyls was inhibited only by oryzalin, the most water soluble of the 2,6dinitroanilines. In these experiments, measurements were made after the sections had been treated with the compounds for only 1 hr. Stronger effects on photosynthesis and respiration could be anticipated if penetration occurred over longer periods of time because of the limited water solubilities of the compounds, and the restricted uptake and mobility of the compounds in viva (1).
DINITROANILINE
HERBICIDES
Thcl treatment period was extended by germinating corn seed in contact with 25 ppm trifluralin. Oxygen utilization of root tips excised from the seedlings was measured 48 and 72 hr after the initiation of germinaCon. In a sec.ond approach, the seed were germinat’ed in contact. with water for 48 hr and then treated with 2.5 ppm trifluralin for 24 hr prior to excision of the root tips. Under both conditions, oxygen uptake of the treated tissue was reduced drastically in the terminal O-6 mm (Fig. 3). However, the level of respiration in all sections of the trifluralin-treated roots was only slightly lower t,han that of control tissue locat’ed 6-8 mm behind the tip. Hence, the reduction in oxygen uptake appeared to correlat’e with the previously established suppression of cell division by the 2 ,&dinitroanilines (4-9, 11) and trifluralin did not inhibit strongly rrspiration of nonmeristematic tissue. Mitochondria isolated from corn seedlings germinated in contact with 25 ppm t,rifluralin appeared to be functionally int,act after 48 and 7’1 hr of growth. Nevertheless, some indications of slightly alt’ered functions \\-crc’ clvidcnt. However, between 72 and 96 Iv thrrcx was a complete and apparently irrevcrsiblt shift in the oxidative behavior of the-mitochondria. This alteration was manifested in the uncoupling of oxidation from phosphorylation, the increase in endogenous ATPase activity, the failure of DNP to induct ATPase activity (Table 2, Fig. 4), and Dhe loss in sensitivity of oxidation to electron transport iuhibitors. The latter observation suggests that development of an alternate oxidation pathway has been promoted. The alterations in mitochondrial behavior resemble those associat’ed with developmental changes of tissue. However, these changes w(‘rtt not. observed in mitochondria from control tissue of the same age. The developmental changes could be associated with the effect of t’rifluralin on cell division or to the cffcct, of trifluralin on mitochondrial act,ivit#ies. In addkion, trifluralin might also in
ON
PLANT
TISSUE
:3ti1
some way hasten the maturation of the tisxuc. The capacity of the mitochondria to gcnerate ATP definitely was impaired. However, the mechanism through which impairment, occurred remains to be determined. If it were possible to isolate mitochondria only from cells in which mitosis had been suppressed instead of from all cells of the t.issu(:, a more critical evaluation of effects expressed by trifluralin on the mitochondria could have been made. The mitochondrial preparations consisted of a pooled sample obtained from all cells, only some of which may have been penetrated by trifluralin. .\ strong effect on mitochondrial functions conceivably would not have been observed unless the chemical uniformly penetrated the majority of the cells. The relation between interference with ATP generation by the 2,Cdinitroanilines and inhibition of cell division remains to be elucidated. The 2,6dinitroanilines do suppress cell division. The mechanism through which cell division is supprenscd is not known. Int’erferencc with ATP generation could be involved, but there is as yet no conclusive evidence t,o support, this speculstion. Unfortunately, the compounds are not uniformly distributed within root tissue and are not t’ranslocated readily. Even mitotica abnormalities are produced randomly (4). The enlargement of cells in the root region tiuggests t’hat the selective permeability of cullular membranes has been altered. Altrrations to the membranes could ocrur in several ways including the partitioning into and binding of the 2,6-dinitroanilincs to the membrane and the unavailability of XTP energy needed to maintain selectivity. Any hypothesis proposed to explain t,he biochemical mechanism of action of the 2,6dinitroanilines must account, for the &kt, expressed on meristematic cells. If active uptake of trifluralin is involved, meristematic cells with their higher respiratory activit’y would absorb more of the chemical than nondividing cells. Penetration and
362
MORELAND,
FARMER,
partitioning of trifluralin into nuclei and mitochondria could result in interference with the generation of ATP. Energy derived either directly or indirectly from ATP is required both for the synthetic processes (DNA, RNA, and protein) in interphase and to drive chromosome movement during mitosis. The ATP energy is needed for the orientation and contraction of spindle fibers, and movement of chromosomes toward the poles. The orientation of spindle fibers is a prerequisite for cell plate formation. If the orientation does not occur, the cell plate does not form but the nucleus divides. Formation of multinucleate cells after treatment with the 2,6-dinitroanilines has been reported (4-9, 11) . The ATP may have a mitochondrial origin, but there is some evidence, at least in certain types of animal cells, for the aerobic production of ATP in the nucleus (10, 12). Unfortunately, there is little direct information available at this time on the occurrence of nuclear phosphorylation in plant cells. Nuclear phosphorylation like oxidative phosphorylation is inhibited by cyanide, azide, and DNP (12). Because of the sensitivity of nuclear phosphorylation to inhibitors of oxidative phosphorylation, it is conceivable that the nuclear process, would also be inhibited by the 2,6-dinitroanilines. Respiratory inhibitors and uncouplers do not usually produce multinucleate cells or promote cellular enlargement like the 2,6dinitroanilines. However, in general, most inhibitors of cell division do not have the limited water solubility, high lipophilicity, and limited mobility of the 2,6-dinitroanilines. In addition, the action of the 2,6dinitroanilines on mitochondrial activity appears to be considerably different and more complex than compounds such as DNP, sodium azide, HO&NO, and antimycin A even though the end result (interference with ATP generation) is the same. Unfortunately, it has not been possible from the studies reported herein, to relate clearly the
AND
HUSSEY
importance of interference with oxidative phosphorylation to the in vivo mechanism of action of the 2,6-dinitroanilines. Investigators should be cognizant of the potential of the 2,6-dinitroanilines for interfering with the oxidative and photosynthetic production of ATP. Hence, if the effects of the compound8 are being examined on energy requiring metabolic reactions, an attempt should be made to separate effect’s exerted directly on the response or reaction being studied from ways in which a reduced energy supply might influence the response and the interpretation of results obtained. Only in this way will it be possible to elucidate the actual biochemical mechanisms involved in the phytotoxic action of 2,6-dinitroaniline herbicides. ACKNOWLEDGMENTS
Samples of the 2,6-dinitroanilines were provided generously by Amchem Products, Incorporated, Ambler, PA; The Ansul Company, Weslaco, TX; BASF Corporation, Parsippany, NJ; Eli Lilly and Company, Greenfield, IN; Geigy Agricultural Chemicals, Ardsley, NY; Shell Development Company, Modesto, CA; and U.S. Borax Research Corporation, Anaheim, CA. REFERENCES
1. D.
2.
3.
4.
5.
6.
E. Moreland, F. S. Farmer, and G. G. Hussey, Inhibition of photosynthesis and respiration by substituted 2,6-dinitroaniline herbicides. I. Effects on chloroplast and mitochondrial activities, PCS&. B&hem. Physiol. 2, 342 (1972). J. De Greef, W. L. Butler, and T. F. Roth, Greening of etiolated bean leaves in far red light, Plant Physiol. 47, 457 (1971). W. J. Blackman and D. E. Moreland, Adenosine triphosphatase activity associated with mung bean mitochondria, Plant Physiol. 47, 532 (1971). D. E. Bayer, C. L. Foy, T. E. Mallory, and E. G. Cutter, Morphological and histological effects of trifluralin on root development, Amer. J. Bot. 54,945 (1967). W. A. Gentner and L. G. Burk, Gross morphological and cytological effects of nitralin on corn roots, Weed Sci. 16, 259 (196@. J. Hacskaylo and V. A. Amato, Effect of tri-
DINITROANILINE
HERBICIDES
fluralin on roots of (wrn and cotton, Weed Sci. 16, 513 (1968). 7. C. A. Kust and B. E. Struckmeyer, Effects of trifluralin on growth, nodulation, and anat,omy of soybeans, Weed Sci. 19, 147 (1971). 8. 8. Sawamura and W. T. Jackson, Cytological studies in viva of picloram, pyriclor, triflrtraliu, 2,3,6-TBA, 2,3,5,6-TBA, and nitralin, Cyfologia 33, 545 (1968). 9. U. P. Schultz, H. H. Funderburk, Jr., and N. S. Negi, Effect of t,rifluralin on growth, morphology, and nucleic arid synthesis, Phnf
Ph ysiol.
43, 265 (1968).
ON
PLAKT
TISSSCE
::ti::
10. B. A. Kihlman, “Artions of Chemicals IIII Dividing Cells,” Prentice-Hall, lhgle\l;~~~~l Cliffs, NJ. 1966. 11. B. P. Lantican, P. RI. Zamora, R. P. Roblr~n. and 11. L. Talatala,, Morphological antI physiological responses of rice to triflttralin, Philippp. Agr. 32, 553 (1969). 12. T. E. Conover, Respiration and adenosine triphosphate synthesis in nuclei. in ” Crtrrent~ Topics in Hioenergetics” (R. I r. Sanatli, Ii:d.), Vol. 2. p. 235. Academic, Press. Nt~w York, 1967.
BIOCHEMISTRY
Inhibition
AND
PHYSIOLOGY
2,
354-363
of Photosynthesis
and
2, &Dinitroaniline
Respiration
by
Substituted
Herbicides
II. Effects on Responses in Excised Plant Tissues and Treated D. E. MORELAND, Plant
Science Research Division, Crop Science Department,
F. S. FARMER,
Agricultural Research North Carolina State
Seedlings’
AND G. G. HUSSEY
Service, United States Department University, Raleigh, North Carolina
of Agricu :&we, R7607
Received May 22, 1972; accepted August 11, 1972 Effects of 12 substituted 2,6-dinitroaniline herbicides were measured on photosynthesis and respiration in excised tissue, and tissue excised from seedlings treated with oc,cu,cu-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine (trifluralin). The compounds partially inhibited photosynthesis (measured as oxygen evolution) of strips cut from spinach (Spinacia oleracea L.) leaf discs. Only 3,5-dinitro-N4,N4-dipropylsulfanilamide (oryaalin) strongly inhibited respiration (measured as oxygen uptake) of excised mung bean (Phaseolus aureus Roxb.) root tips and hypocotyls after a short treatment time. However, root tips excised from corn (Zea wzuys L.) seedlings germinated in contact with one representative 2,6-dinitroaniline (trifluralin) showed suppressed oxygen uptake. In addition, phosphorylation was completely uncoupled from oxidation in mitochondria isolated from primary roots of corn seedlings after germination of the seed in contact with trifluralin for 96 hr. Interference with photosynthesis and respiration both in vivo and in vitro by the 2,6-dinitroanilines has been demonstrated. Deviations in growth and metabolism produced by these herbicides can be explained partially by interference with the oxidative and photoproduction of ATP, if recognition is extended to the role played by ATP energy in maintaining cellular and nuclear activities. However, the importance of interference with ATP production to the phytotoxic action of the 2,6dinitroanilines remains to be established. INTRODUCTION
Inhibition of electron transport and phosphorylation in isolated spinach (Spinaciu olerucea L.) chloroplasts and mung bean (Phaseolus aureus Roxb. var. Jumbo) mitochondria by 12 substituted 2,6-dinitro1 Cooperative investigations of the North Carolina Agricultural Experiment Station and the Plant Science Research Division, Agricultural Research Service, United States Department of Agriculture, Raleigh, NC. Paper number 3773 of the Journal Series of the North Carolina State University Agricultural Experiment Station, Raleigh, NC. Investigations supported in part by Public Health Service Grant ES 90644. 354 Copyright All righta
0 1972 by Academic Press, of reproduction in any form
Inc. reserved.
aniline herbicides was reported in the first paper of t’his series(1). The compounds were identified structurally and literature was reviewed also in the aforementioned paper. The responseswith isolated organelles suggested that if the 2,6-dinitroanilines reach the chloroplasts and mitochondria, inhibition of photosynthesis and respiration in viva could be expected. Therefore, the objectives of the studies reported herein were to measure, correlate, and evaluate effects of the 2,6-dinitroanilines on photosynthesis and respiration in excised tissue, and tissue excised from seedlings treated with trifluralin.
DINITROANILINE
HERBICIDES
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:i.iT,
Photosynthetic activity (oxygen evolution) of excised foliar tissue was measured polarographically, with a Clark electrode, in the thermostated glass cell described previously for studies with isolated mitochondria (1). Two leaf discs (9 mm diam, approx 40 mg fr wt), taken from growth chamber-grown spinach plants, were sliced into 1 mm-widr strips, placed in 2.0 ml of reaction medium, and deaerated under partial vacuum for 5 min. The reaction medium contained equal parts of CO2 buffer and modified Avron’s medium (2). hfter deaeration, the tissue was transferred to fresh reaction medium to which was added the test chrmical. After incubation for 30 min at 25”C, the tissue and reaction mixture were transferred to the thermostated glass cell. After purging of the reaction mixture with nitrogrn t’o lower the oxygen content, the sample chamber was irradiated (60,000 lx) with a Unitron LKR microscope illuminator.? Oxygen uptake by root tips and hypocotyl spctions, excised from 3-day-old, darkgrown mung bean seedlings, was also measurc>d polarographically with the apparatus used in studies mit,h isolated mitochondria (1). Hypocotyl sections (64 discs, 0.6 mm thick, 0.15 g fr wt) obtained from the region of elongation and root tips (30 tips 5 mm long, 0.04 g fr w-t) mere deaerated under partial vacuum in 2.0 ml reaction medium [0.03 M potassium phosphate buffer (pH 5.5)) 1% glucose, and the chemical treatment]. After deaeration, air was bubbled through thtl reaction medium during the lhr incubation period. The tissue was then transferred t.o fresh reaction medium in the
thermostated glass cell, and oxygen uplake measured. Effects of trifluralin on t,he respiration ot treated seedlings were determined in root tips excised, and mitochondria isolated, frc )m primary roots of corn (Zea mays L. var. Pioneer 3369A) seedlings. Seed had !XWI germinated between filter paper moistened with 25 ppm trifluralin in 0.57 Dh1803 (v/v) in the dark at 25°C. Aft’er 48 and 72 hr, four sequential a-mm sections Ivere excised from the primary roots starting at the tip. Sections from 20 roots were pooled, suspended in buffer [0.03 M potassium phosphate (pH 5.5) and 1% glucose], dcaerated for 5 min, and transferred to the thermostated cell in fresh buffer for oxygen uptake measurements. Mitochondria were also isolated from the root tissue of (*orn seedlings after germination of the seed in contact. with trifluralin by procedures described previously (1). Effects of the treat,ment on mitochondrial oxidative behavior and ATPase a&iv&y mere also measured with previously described procedures (1). The substituted 2,6-dinitroanilines used in this investigation were identified previously (I, Table 1). Because of the relative insolubility of the compounds in water, solutions of the desired concentrations were prepared in absolute ethanol for the foliar tissue study; and in DIMS0 for t’he excised hypocotyl, excised root t’ip, and seedgermination studies. The final concentration of ethanol in all assays including the controls was I % (v/v) and for the studies in which DMSO was the solvent, t#hefinal conrc:ntratjion was 0.5 %I (v/v). Data for the exci& tissue studies are arithmetic averages of al, least duplicate determinations.
2 Mention is made for uot imply Government. Experiment.
3 The following abbreviations text: DMSO, dimethylsulfoxide; dichlorophenyl) -l,l-dimethylurea; heptyl-4-hydroxyquinoline-N-oxide; dinitrophenol; It/C, respiratory
MATERIALS
AND
METHODS
of specific instruments or trade names ident,ification purposes only and does endorsement by the United States or the North Carolina Agricultural Station.
are used in the diuron, :3-(3,4HO&NC 1: 2DNP, 2,4control r&o.
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TABLE Effect
FARMER,
1
of
substituted d,6-dinitroanilines evolution of oxygen by illuminated leaf tissue
on
Inhibition”
A-820 AN 56477 BAS 392-H Benefin CGA-10832 GS-38946 GS-39985 Isopropalin Nitralin Oryealin Trifluralin USB 3584
12 17 47 10 9 46 24 2 31 57 0 25
(To)
a Compounds were tested at 0.6 mM, a saturating concentration. RESULTS
Photosynthesis
Most of the substituted 2,6-dinitroanilines, at a saturating concentration of 0.6 mM, inhibited photosynthesis as measured
300
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HUSSEP
by suppression of oxygen evolution by illuminated spinach leaf tissue. All compounds were tested at this high concentration in an attempt to maintain a saturated solution external to the tissue so that a relative measure of their maximum inhibitory effectiveness could be obtained. Shown in Fig. 1 are traces of oxygen evolution obtained with untreated control tissue and three 2,6dinitroanilines (AN 56477, nitralin, and oryzalin). The compounds differed only by the amount of inhibition that was expressed. Evolution of oxygen, under the conditions described in Methods, was light-dependent and linear for the initial 2-3 min. In untreated control tissue, oxygen evolution averaged 326 nmoles/hr/dg fr wt. Diuron (3 MM), which is a potent inhibitor of noncyclic electron transport in isolated chloroplasts, was included as a reference and completely inhibited oxygen evolution. Inhibitory percentages for all compounds, calculated by relating the rate of oxygen evolution measured in the presence of a test compound to the rate expressed by un-
the
spinach
Compound
AND
-
liphl on
Time
FIG. 1. Representative polarographic traces depicting oxygen evolution by illuminated spinach leaf tissue. Compared are traces obtained with no-treatment control tissue, and suppression of oxygen evolution by AN 66.&V (0.6 mM), nitralin (0.6 mM), oryzalin (0.6 mM), and diuron (3.0 PM).
DINITTROANILINE
1
HERBICIDES
ON
PLANT
TISSUE
2 min 1 I
1
Time
FIG. 2. Polarographic traces depicting oxygen uptake by mung bean hypocotyl sections. traces obtained with. no-treatment control tissue, oryzalin (0.1, 0.4, and f .O mM), and sodium Rates of oxygen utilization (nmoles 02 per min per I ml) are indicated above the traces.
treated control &sue, are presented in Table 1. Rates were obtained from the initial linear segment of the traces and adjusted for differences in fresh weight of the tissue. Trifluralin and isopropalin had essentially no effect on oxygen evolution. The remainder of the compounds produced some inhibition, with BAS 392-H, GS-38946, and oryzalin being the strongest inhibitors. None of the compounds were as active as diuron (Fig. 1) in inhibiting photosynthesis. With the stronger inhibitors, inhibition of oxygen evolution could be detected after only a few minutes of incubation time and at molar concentrations lower than 0.6 mM. However, for comparative purposes, the concentration tested and the incubation time were held constant as identified in Jlethods. Differences in inhibitory potency may be related to relative penet,rabilit.ies of the compounds.
Oxygen uptake by excised mung bean hypocotyl sections and root tips, handled as described in Methods, was linear (Fig. 2) until anaerobiosis occurred. Oxygen utiliza-
Compared ore azidr (1 m&f).
tion by both tissues was also inhibited by 1 mM standard respiratory inhibitors: sodium azide (Fig. 2), 50 PM antimycin A, and 50 PM HOQNO. Of the 12 substituted 2,6-dinitroanilines tested, only oryzalin, which is the most water soluble, inhibited oxygen uptake. The amount of inhibition obtained was a function of the concentration of the inhibitor (Fig. 2) and at the highest concentration tested (1 mM) reached 60 %. Similar responses with oryzalin were obtained also with root tips, but only the data for hypocotyls are presented herein. Results presented previously (1) indicated that the 2,6-dinitroanilines interfered with the oxidation of malat,c, NADH, and succinate by isolated mitochondria. However, in studies with root tip and hypocotyl sections, that had been incubated with the compounds for 1 hr, only oryzalin inhibited respiration (Fig. 2). Therefore, the studies were extended to permit absorption and penetration to occur over a longer interval. For these evaluations, corn was used as the experimental speciesinstead of mung beans. Only the effects of trifluralin were studied, but
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because of similarities in behavior in studies reported previously (1)) comparable results might be expected with the other 2,6-dinitroanilines. Corn seed were germinated in the presence and absence of 25 ppm trifluralin. In comparison to control seedlings, the primary roots of the corn seedlings that had germinated in contact with trifluralin exhibited retarded elongation, inhibited lateral root development, and had a swollen tuberous-shape. These characteristic morphological changes have been observed by other investigators (1). Sections were excised at 2-mm intervals starting from the tip as described in LXIethods. Oxygen uptake (nanomoles per minute per decigram fresh weight) by trifluralin-treated and control tissue are compared in Fig. 3. In Graph A are shown the oxygen uptake patterns of primary root tissue from seed which had been germinated in continuous contact with 25 ppm trifluralin for 72 hr. Data in Graph B were obtained from roots of seed that were germinated initially in water, but after 48 hr were treated with 25 ppm trifluralin for 24 hr prior to excision. Under both conditions,
O-2
2-4
4-6 Dirtonce
6-8 from
O-2 roof
tip
2-4
4-6
6-6
(Inn;)
FIG. 3. Oxygen uptake by excised sections of the primary root of corn germinated in the presence and absence of 26 ppm tri$uralin. Graph A: Sections obtained from seeds germinated between filter paper moistened with water containing 0.6% DMSO (v/v) or 86 ppm Irifluralin for 7.2 hr. Graph B: Sections obtained from seed germinated in contact with water-saturated jilter paper for 48 hr. After 48 hr some of the seed were placed, and remained, in contact with 26 ppm trijluralin for 94 hr. (Legend: open bars, control tissue; slashed bars, treated tissue.)
AND
HUSSEY
trifluralin-treated root tissue showed a depression in respiration, as measured by oxygen uptake, relative to the untreated control tissue. In the root sections from the untreated controls, oxygen uptake was highest in the terminal 2 mm, decreased curvilinearly for one or two sections, and approached a constant value between 4 and 6 mm from the tip. However, the decrease in rate of oxygen uptake with distance from the tip was not observed in the trifluralintreated tissue. This behavior is shown in Graph A where all sections from treated roots utilized oxygen at approximately the same rate. In Graph B, the treated terminal section did utilize oxygen at a rate t’hat was slightly greater than the subsequent sections. In the untreated control tissue, the decrease in respiration with distance from the root tip correlates with mitotic activity. However, in the trifluralin-treated tissue, mitosis has probably been arrested (1) and the respiration is contributed primarily by nondividing cells. Respiratory and ATPase activities of mitochondria isolated from the corn seedlings also were examined 48, 72, and 96 hr after initiation of germination. Data are presented in Table 2 for only the 72- and 96-hr treatments. Trends obtained for the 48-hr treatment paralleled those of the 72-hr treatment and, hence, are not presented. As shown in Table 2, mitochondria isolated from primary roots of corn seedlings germinat.ed in contact with 25 ppm trifluralin for 72 hr generally had higher endogenous, state 3, and state 4 rates than mitochondria isolated from roots of untreated control seedlings. In addition, the R/C (respiratory control) and ADP/O ratios tended to be lower in mitochondria isolated from the treated tissue. There were no major differences in the level of endogenous ATPase activity or the response of the endogenous ATPase to Mg2+, but there was a decrease in the ATPase activity induced by DNP.
DINITROANILINE
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TABLE 2 and ATPase activities of mitochondria
ON
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respiratory rates (endogenous, state 3, and state 4) that were slightly lower than those from roots of corn seedlings 72 and 96 hr measured after 72 hr (Table 2). The R/C the initiation of germination. Seed ratios also tended to be somewhat lower, wwrre germinated in the presence and but the ADP/O ratios were about the same. absence of 26 ppm tri$uralin However, mitochondria isolated from the Response measured Duration of treatment 96-hr treat.ed tissue generally had lower 72 hr 96 hr endogenous and state 3 respiratory rates -.-~ Control Treated Control Treated than those from untreated tissue. Strikingly, all control normally exerted by ADI’ in Malate oxidation 43 41 Endogmm?’ 45 65 tightly coupled mitochondria was lost for 111 82 state 3” 152 168 h the oxidation of malate, NADH, and succ38 St&e 4" 36 65 h 4.2 2.6 2.9 R/C nate (Table 2). The mit’ochondria IWVW h ADP/O 1.8 1.8 2.2 exhausted ADP and did not enter the cxNADH midtkim Endqq=nous 75 83 57 51 pectcd state 4 rate of respiration. Cc)nsc:i1Q state 3 205 236 137 h quently, R/C and ADP/O ratios could not 56 state 4 il 105 h 2.5 R/C 2.Y 2.3 be calculated. The loss in ADI’ control is h I.1 1.1 ADP/O 1.0 also presented in Fig. 4. In Trace A is shown Succinate oxidation 104 119 66 72 Endoaenous the oxidation of malate by mitochondria state 3 181 170 120 loo * from untreated 96hr corn tissue and the stat 4 104 108 72 6 1.7 1.6 1.7 R/C control exerted by ADP. In comparison, 6 1.3 1.3 ADP.IO 1.1 Trace B was obtained with mitochondria ATPase wtivity’ Endqgmms - Mg* 0.73 0.83 0.59 1.01 from trifluralin-treated tissue. After cwtabEndogenous + Mgz+ 2.42 2.26 1.93 1.88 lishment of the trend measured for endoge4.93 4.16 3.68 1.60 DNP (80& now respiratory activity, a limit,ing amount, of ADP was added. Respiration increased ?JRespirntifm nirt under ADP control, hence, values could and would have continued without an apnr,t he calculated. preciable change in respiratory rate until ’ Dsta presented as rmoles P, relessed per mg protein in 20 min. anaerobiosis occurred. R-o control was imposed by ADP and electron transport apIn intact mitochondria, Mg”+ appears to he peared to be complet’ely uncoupled from an imegral part, of the ATP-generating phosphorylation. Under t~hese conditions, complex and no requirement for exogenous the mitochondria were still capable of oxi11gz+ is shown. However, in structurally dizing substrates, but no ATI’ was being damaged mitochondria, the &I&+ is dissoci- generated. The mitochondrial preparation ated from the complex and a requirement for from the 96-hr treat’ed tissue also possessed Mgz+ is manifested (3). Endogenous mito- considerably higher cndogenous ATPase ehondrial ATPase activity is characteristicactivity t.han the controls and no longer ally stimulated by the addition of Mg2+. strongly responded to DW in the induction After treatment for 72 hr, the mitochon- of ATPase activit.y (Table 2). dria manifested small deviations in behavior In the treated tissue, between 72 and 96 which suggested that alteration in various hr, not only did the mitochondria become functions had been initiated by the treat uncoupled, but the respiration also shifted ment. However, 24 hr later, after 96 hr, toward a type which was resistant to trimore drastic effects were apparent. The root fluralin and other electron transport inCssue of the 96-hr control seedlings was hibitors. For example, with malate as subquite fiberous and the mitochondria had strate, the st#atc 3 respiration of mitochonRespiratory isolated after
MORELAND,
FARMER,
AND
HUSSEY
400-
0” ” i JOOE
200.
1
FIG. 4. Polarographic traces depicting oxygen utilization by mitochondria isolated from primary roots of corn seedlings that had been germinated for 96 hr. Trace A: Oxidation of malate by mitochondria from untreated seedlings showing control exercised by ADP. Trace B: Oxidation of malate by mitochondria from seedlings that had been treated with 26 ppm trifluralin showing the absence of respiratory control. Rates of oxygen utilization (nmoles0s per min per 2 ml) are indicated above the traces. Mitochondria (Mit), malate (0.2 mmoles), and A DP (0 S rmoles) were added at the points indicated. The mitochondrial aliquot used to obtain Trace A contained 0.30 and Trace B 0.3’7 mg protein.
dria obtained from both the 72-hr control and treated tissues, and 96-hr control tissue responded similarly to 40 PM trifluralin, and approx 60% inhibition was obtained. However, the state 3 respiration of mitochondria from 96-hr treated tissue was inhibited only by approx 30%. In addition, malate oxidation by mitochondria from 96-hr control seedlings was inhibited 60-75 % by electron transport inhibitors [sodium azide (1.0 mM), antimycin A (0.1 PM), and rotenone (1.0 mM)], whereas, malate oxidation by mitochondria from 96-hr trifluralin-treated tissue was inhibited only by approx 35 %. DISCUSSION
Studies with isolated chloroplasts and mitochondria showed that the 2,6-dinitroanilmes inhibited both electron transport and phosphorylation (1). Consequently, if the compounds successfully penetrate cells and partition into the organelles in viva, either by translocation or in vapor form from
the atmosphere, inhibition of photosynthesis and respiration could be anticipated. Results of the studies with excised foliar tissue suggested that photosynthesis (measured as oxygen evolution) was inhibited, at least partially, by all of the 2,6-dinit~anilines except trifluralin (Table 1, Fig. 1). Under the conditions of the experiments, oxygen evolution measurements were made after allowing 30 min for penetration of the compounds into the tissue. Respiration (measured as oxygen uptake) of excised root tips and hypocotyls was inhibited only by oryzalin, the most water soluble of the 2,6dinitroanilines. In these experiments, measurements were made after the sections had been treated with the compounds for only 1 hr. Stronger effects on photosynthesis and respiration could be anticipated if penetration occurred over longer periods of time because of the limited water solubilities of the compounds, and the restricted uptake and mobility of the compounds in viva (1).
DINITROANILINE
HERBICIDES
Thcl treatment period was extended by germinating corn seed in contact with 25 ppm trifluralin. Oxygen utilization of root tips excised from the seedlings was measured 48 and 72 hr after the initiation of germinaCon. In a sec.ond approach, the seed were germinat’ed in contact. with water for 48 hr and then treated with 2.5 ppm trifluralin for 24 hr prior to excision of the root tips. Under both conditions, oxygen uptake of the treated tissue was reduced drastically in the terminal O-6 mm (Fig. 3). However, the level of respiration in all sections of the trifluralin-treated roots was only slightly lower t,han that of control tissue locat’ed 6-8 mm behind the tip. Hence, the reduction in oxygen uptake appeared to correlat’e with the previously established suppression of cell division by the 2 ,&dinitroanilines (4-9, 11) and trifluralin did not inhibit strongly rrspiration of nonmeristematic tissue. Mitochondria isolated from corn seedlings germinated in contact with 25 ppm t,rifluralin appeared to be functionally int,act after 48 and 7’1 hr of growth. Nevertheless, some indications of slightly alt’ered functions \\-crc’ clvidcnt. However, between 72 and 96 Iv thrrcx was a complete and apparently irrevcrsiblt shift in the oxidative behavior of the-mitochondria. This alteration was manifested in the uncoupling of oxidation from phosphorylation, the increase in endogenous ATPase activity, the failure of DNP to induct ATPase activity (Table 2, Fig. 4), and Dhe loss in sensitivity of oxidation to electron transport iuhibitors. The latter observation suggests that development of an alternate oxidation pathway has been promoted. The alterations in mitochondrial behavior resemble those associat’ed with developmental changes of tissue. However, these changes w(‘rtt not. observed in mitochondria from control tissue of the same age. The developmental changes could be associated with the effect of t’rifluralin on cell division or to the cffcct, of trifluralin on mitochondrial act,ivit#ies. In addkion, trifluralin might also in
ON
PLANT
TISSUE
:3ti1
some way hasten the maturation of the tisxuc. The capacity of the mitochondria to gcnerate ATP definitely was impaired. However, the mechanism through which impairment, occurred remains to be determined. If it were possible to isolate mitochondria only from cells in which mitosis had been suppressed instead of from all cells of the t.issu(:, a more critical evaluation of effects expressed by trifluralin on the mitochondria could have been made. The mitochondrial preparations consisted of a pooled sample obtained from all cells, only some of which may have been penetrated by trifluralin. .\ strong effect on mitochondrial functions conceivably would not have been observed unless the chemical uniformly penetrated the majority of the cells. The relation between interference with ATP generation by the 2,Cdinitroanilines and inhibition of cell division remains to be elucidated. The 2,6dinitroanilines do suppress cell division. The mechanism through which cell division is supprenscd is not known. Int’erferencc with ATP generation could be involved, but there is as yet no conclusive evidence t,o support, this speculstion. Unfortunately, the compounds are not uniformly distributed within root tissue and are not t’ranslocated readily. Even mitotica abnormalities are produced randomly (4). The enlargement of cells in the root region tiuggests t’hat the selective permeability of cullular membranes has been altered. Altrrations to the membranes could ocrur in several ways including the partitioning into and binding of the 2,6-dinitroanilincs to the membrane and the unavailability of XTP energy needed to maintain selectivity. Any hypothesis proposed to explain t,he biochemical mechanism of action of the 2,6dinitroanilines must account, for the &kt, expressed on meristematic cells. If active uptake of trifluralin is involved, meristematic cells with their higher respiratory activit’y would absorb more of the chemical than nondividing cells. Penetration and
362
MORELAND,
FARMER,
partitioning of trifluralin into nuclei and mitochondria could result in interference with the generation of ATP. Energy derived either directly or indirectly from ATP is required both for the synthetic processes (DNA, RNA, and protein) in interphase and to drive chromosome movement during mitosis. The ATP energy is needed for the orientation and contraction of spindle fibers, and movement of chromosomes toward the poles. The orientation of spindle fibers is a prerequisite for cell plate formation. If the orientation does not occur, the cell plate does not form but the nucleus divides. Formation of multinucleate cells after treatment with the 2,6-dinitroanilines has been reported (4-9, 11) . The ATP may have a mitochondrial origin, but there is some evidence, at least in certain types of animal cells, for the aerobic production of ATP in the nucleus (10, 12). Unfortunately, there is little direct information available at this time on the occurrence of nuclear phosphorylation in plant cells. Nuclear phosphorylation like oxidative phosphorylation is inhibited by cyanide, azide, and DNP (12). Because of the sensitivity of nuclear phosphorylation to inhibitors of oxidative phosphorylation, it is conceivable that the nuclear process, would also be inhibited by the 2,6-dinitroanilines. Respiratory inhibitors and uncouplers do not usually produce multinucleate cells or promote cellular enlargement like the 2,6dinitroanilines. However, in general, most inhibitors of cell division do not have the limited water solubility, high lipophilicity, and limited mobility of the 2,6-dinitroanilines. In addition, the action of the 2,6dinitroanilines on mitochondrial activity appears to be considerably different and more complex than compounds such as DNP, sodium azide, HO&NO, and antimycin A even though the end result (interference with ATP generation) is the same. Unfortunately, it has not been possible from the studies reported herein, to relate clearly the
AND
HUSSEY
importance of interference with oxidative phosphorylation to the in vivo mechanism of action of the 2,6-dinitroanilines. Investigators should be cognizant of the potential of the 2,6-dinitroanilines for interfering with the oxidative and photosynthetic production of ATP. Hence, if the effects of the compound8 are being examined on energy requiring metabolic reactions, an attempt should be made to separate effect’s exerted directly on the response or reaction being studied from ways in which a reduced energy supply might influence the response and the interpretation of results obtained. Only in this way will it be possible to elucidate the actual biochemical mechanisms involved in the phytotoxic action of 2,6-dinitroaniline herbicides. ACKNOWLEDGMENTS
Samples of the 2,6-dinitroanilines were provided generously by Amchem Products, Incorporated, Ambler, PA; The Ansul Company, Weslaco, TX; BASF Corporation, Parsippany, NJ; Eli Lilly and Company, Greenfield, IN; Geigy Agricultural Chemicals, Ardsley, NY; Shell Development Company, Modesto, CA; and U.S. Borax Research Corporation, Anaheim, CA. REFERENCES
1. D.
2.
3.
4.
5.
6.
E. Moreland, F. S. Farmer, and G. G. Hussey, Inhibition of photosynthesis and respiration by substituted 2,6-dinitroaniline herbicides. I. Effects on chloroplast and mitochondrial activities, PCS&. B&hem. Physiol. 2, 342 (1972). J. De Greef, W. L. Butler, and T. F. Roth, Greening of etiolated bean leaves in far red light, Plant Physiol. 47, 457 (1971). W. J. Blackman and D. E. Moreland, Adenosine triphosphatase activity associated with mung bean mitochondria, Plant Physiol. 47, 532 (1971). D. E. Bayer, C. L. Foy, T. E. Mallory, and E. G. Cutter, Morphological and histological effects of trifluralin on root development, Amer. J. Bot. 54,945 (1967). W. A. Gentner and L. G. Burk, Gross morphological and cytological effects of nitralin on corn roots, Weed Sci. 16, 259 (196@. J. Hacskaylo and V. A. Amato, Effect of tri-
DINITROANILINE
HERBICIDES
fluralin on roots of (wrn and cotton, Weed Sci. 16, 513 (1968). 7. C. A. Kust and B. E. Struckmeyer, Effects of trifluralin on growth, nodulation, and anat,omy of soybeans, Weed Sci. 19, 147 (1971). 8. 8. Sawamura and W. T. Jackson, Cytological studies in viva of picloram, pyriclor, triflrtraliu, 2,3,6-TBA, 2,3,5,6-TBA, and nitralin, Cyfologia 33, 545 (1968). 9. U. P. Schultz, H. H. Funderburk, Jr., and N. S. Negi, Effect of t,rifluralin on growth, morphology, and nucleic arid synthesis, Phnf
Ph ysiol.
43, 265 (1968).
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PLAKT
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10. B. A. Kihlman, “Artions of Chemicals IIII Dividing Cells,” Prentice-Hall, lhgle\l;~~~~l Cliffs, NJ. 1966. 11. B. P. Lantican, P. RI. Zamora, R. P. Roblr~n. and 11. L. Talatala,, Morphological antI physiological responses of rice to triflttralin, Philippp. Agr. 32, 553 (1969). 12. T. E. Conover, Respiration and adenosine triphosphate synthesis in nuclei. in ” Crtrrent~ Topics in Hioenergetics” (R. I r. Sanatli, Ii:d.), Vol. 2. p. 235. Academic, Press. Nt~w York, 1967.