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Trifluralin effects on carrot callus tissue
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
Trifluralin
201-208
15,
Effects
MARGARET
Received
October
(1981)
on Carrot
E. SLOAN ANDN.
15, 1980; accepted
Callus Tissue D. CAMPER
February
5. 1981
The effect of trifluralin on the growth. oxygen uptake. and adenosine phosphates level of carrot (DNWUS c’urotu L.) callus tissue was determined over a period of I8 days after subculture. The herbicide (IO-’ and IO-” M) reduced fresh weight gain significantly; the reduction was less with lower trifluralin concentrations. Dry weight accumulation was not inhibited until after Day 6 of the test period and thereafter was reduced by all concentrations tested. Oxygen consumption wab inhibited by trifluralin (IO-’ and IO-” M) throughout the test period. Concentrations of IO-“. 10 ;. and IO-” M produced variable effects. The response profile of 0, consumption in the presence of dinitrophenol was different from that of trifluralin. Analysis of adenosine phosphates level gave no clear response trend. Energy charge values of 0.7 to 0.85 were obtained for untreated tissue. Trifluralin had no effect on energy charge until Day 9 (IO-‘M) and after Day I5 (all concentrations).
INTRODUCTION
photophosphorylation (2, 27); however, such effects in intact plants may be of little physiological significance because trifluralin is not readily translocated (2). Oxygen consumption was unaffected in excised oat seedlings (5), inhibited in isolated mitochondria from soybean and sorghum (23), and stimulated in Ipomoeu (18). Oxidative phosphorylation was uncoupled in isolated corn mitochondria (21). The site of trifluralin’s inhibition in the mitochondria is at or near the site of oligomycin inhibition and a major mechanism of dinitroaniline phytotoxicity may be the disruption of ATP formation either by interference with the energy-generating mechanism or by blocking the energy-transferring mechanism (29). However, ATP formation was not impaired in soybean hypocotyls (7). The importance of interference with ATP production by the phytotoxic action of the dinitroaniline herbicides remains to be established (23). The biochemical mechanism of action of the 2,6-dinitroanilines must account for the effect expressed on meristematic cells (20). We, therefore, chose a system in which cell division was a major cellular event. Callus tissue provides such a system. The study was undertaken to examine the effect of trifluralin on oxygen consumption and on
Trifluralin . ’ a substituted 2,6-dinitroaniline herbicide, is a preemergence herbicide used for selective weed control. Trifluralin has been classified as a mitotic poison (28) with many of its morphological and cytological responses similar to those of colchitine (8). Recent studies with microtubules suggested that trifluralin and colchicine affect cellular metabolism by different mechanisms (4, 9, 10). The effect of trifluralin on plant metabolism is dependent upon plant species, concentration, plant organ, and time (2, 24). Effects on nucleic acid metabolism may be a key to trifluralin’s mode of action (1). However, trifluralin effects on RNA and DNA synthesis remain contradictory (19. 27, 30). Evidence suggests that trifluralin may bind to the DNA template, inhibiting synthesis (25). Protein synthesis and amino acid composition may be altered by trifluralin (17, 19, 30). Effects of trifluralin on organelle phosphorylation and oxygen consumption has been examined. The herbicide inhibited I Abbreviations used: trifluralin, 2.6-dinitro-,Y.N-dipropyl-p-toluidine; chlorophenoxy) acetic acid; PEP. vate: DNP. 2.Cdinitrophenol.
n.u,u-trifluoro2.4.D.(2,4-diphosphoenol pyru-
201 004%3575/81/030201-08%02.00/O CopyrIght All rights
C 1981 by Academic Press. Inc. of reproduction in any form reserved.
202
SLOAN
the level of adenosine phosphates energy charge in carrot callus tissue. MATERIALS
Culture
conditions of carrots (Daucus
AND
AND
and
METHODS
and growth. Taproots carota L.) were ob-
tained locally. No distinctions were made between varieties in the subsequent procedures. Carrot callus was isolated by a modified procedure of Komamine et al. (13-15). Carrot roots were surface sterilized in absolute ethanol for 2 min, rinsed, and resterilized in 20% clorox for 30 min. Transverse sections were excised, cut into 2-mm sections, and cultured on modified Murashige and Skoog medium (22) containing 0.9% agar, 0.9% agar supplemented with 2,4-D (1 mg/ml), or 0.9% agar supplemented with 2,4-D (1 mg/ml) and kinetin (0.1 mg/ ml). The pH was adjusted to 6 with 1 N NaOH before autoclaving (20 min, l/O5 kg/ cmz). Subcultures were made at monthly intervals and cultures were maintained in the dark at 23 to 25°C. These cultures were used in the subsequent studies. Stock solutions of technical trifluralin were prepared in pesticide-grade acetone and Millipore filtered. Serial dilutions were prepared so that to each 125ml flask containing 50 ml of medium was added 0.25 ml regardless of the concentration. Each control flask contained 0.25 ml acetone. Trifluralin was added to autoclaved, but cooled, medium prior to solidification. Fresh herbicide solutions were made for each medium preparation and designated Day 0. DNP was prepared and handled in a similar manner. Tissue was harvested every 3 days for a period of 18 days; all experiments had three replicates and were repeated at least once (data represent an average of six determinations). Flasks were incubated in the dark at 23 + 2°C. Dry weight was determined as described by Gamborg and Wetter (6). Respiration. Respiration was determined by standard manometric techniques utilizing a Gibson differential respirometer. Respiratory rate was determined at 30°C for
CAMPER
1.5 hr with readings taken every 10 min. Conditions were those of Komamine et al. (13-15). Extraction
and assay of total adenylates.
Extraction of ATP was conducted by a modified procedure as described by Karl and Holm-Hanson (11). A 0.03- to 0.05-g sample was removed, immersed immediately in 5 ml of boiling Tris buffer (0.02 M, pH 7.75) for 1 min, and immediately homogenized in a tissue grinder. The sample was boiled for 1 min and brought to a final volume of 10 ml with Tris buffer. The suspension was centrifuged for 10 min at 13,000g (Beckman Model J 2-21). The supernatant was decanted and stored frozen (-4°C) for analysis. Enzymatic determination of total adenylates was performed as described by Karl and Holm-Hanson (11) and Kimmich, Randles, and Brand (12) as modified by the authors. Fire fly extract was reconstituted as prescribed by Sigma Chemical Company. For ATP determinations, 0.4 ml of the sample (or ATP standards) was added to a test tube containing 0.1 ml of a 100 mM TrisHCl (pH 7.4), 35 m&I KCl, and 6 n&I MgC12. Enzymatic conversion of ADP+ATP was determined by adding 0.4 ml of the sample to 0.1 ml of a 100 mM Tris-HCl (pH 7.4), 35 mM KCl, and 6 mM MgCl, containing 0.5 mM PEP, and 20 pg pyruvate kinase. Enzymatic determination of AMP-+ATP was conducted in the same manner as ADP with the addition of 25 pg of myokinase to the reaction buffer. All samples were incubated at 30°C for 30 min and placed in a boiling water bath for 2 min to deactivate the enzymes. The samples were diluted to 2 ml with Tris buffer (0.02 M, pH 7.75) to read 5,000 to 150,000 cpm on a Beckman LS1OOC (3H-channel, 0.1 min, 2% error). The amount of ATP was determined from a standard curve conducted daily. Levels of ADP and AMP were calculated from the difference of these measured values. The energy charge was calculated by the following equation as described by Atkinson (3): (ATP + ‘/ ADP)/(ATP + ADP + AMP).
TRIFLURALIN
AND
RESULTS
Data reported in this study (e.g., fresh and dry weight, adenylates levels, etc.) were not measured until Day 3 of the experiments. Preliminary studies showed that up to 3 days from initial culturing were required for stabilized growth due to tissue source, adjustment to media, and possibly other factors. Growth of untreated tissue increased with time (Fig. 1, control). All concentrations of trifluralin tested significantly inhibited fresh weight gain by the callus tissue (Fig. 1). Growth at highest levels tested (lo-’ and lo-” M) was not different: similar trends were evident for IO-“,
CALLUS
203
TISSUE
10-7, and lop8 M. Significant differences in growth rates can be seen between the higher trifluralin levels (lo-“ and IO-” M) and the lower ones (lOpci, lo-‘. and IO-” M). Similar results were observed with dry weight gain (Fig. 2). Respiratory activity of the control remained essentially constant during the test period; however, there was a trend toward decreased 0, consumption with incubation time (Fig. 3). Trifluralin at concentrations lo-” and lo-” M significantly reduced oxygen uptake as compared to the control throughout the test period. Initially the respiratory rate of lo-” M-treated tissue was
Trifluralln
0
3
6
9
12
15
18 0
3
6
9
12
15
18
204
SLOAN
AND
CAMPER
LEGEND: 1o-4
M-
10-5
M ----_
10-6
M ---_-
lo-'
M --M ---_
10-e o-
CULTURE FIG.
treuted
2. Effect
of trij7urulin
on dry weight
GROWTH TIME
(DAYS)
(g k SE) uccumulation
of carrot
callus
tissue.
Control
(O),
(0).
less than the control until Day 6, thereafter it was not different from the control. Oxygen consumption by tissue treated with 10m7 and 10eE M trifluralin was lower than the control except at Days 12 and 18 (lOpR A4 only). The effect of DNP on oxygen consump-
01
31
6I
tion was determined for comparison with trifluralin. Oxygen consumption by the control tissue increased from Days 3 to 9 and decreased thereafter (Fig. 4). DNP at 5 x IO-” A4 significantly decreased oxygen consumption through Day 9 after which oxygen uptake could not be detected. The
I9
1 12
I 15
ia
CULTURE GROWTH TIME (DAYS) FIG.
Control
3. Effect of frifluralin (0). trrated (0).
on oqvgen
uptukr
(~1 OJIO
minlg
fresh
wjt k SE) by carrot
cullus
tissue.
TRIFLURALIN
AND
CALLUS
205
TISSUE
SO-
IOG 0
tissue.
Cnnlrol
of dinitrophenol ( 0). rretrted (0).
3
6 Culture
on oxygen
9 Growth
rrptuke
concentration of 10-j M significantly stimulated oxygen uptake on Days 9, 12, and 15; whereas 10m7 M significantly stimulated oxygen consumption on Days 12 and 15 only. The level of ATP in the control increased from Days 3 to 9 and decreased thereafter (Fig. 5). Concentrations of ADP and AMP remained essentially at a constant level throughout the test period in the untreated tissue. ATP levels were higher in tissue treated with lop4 and 10e5 M trifluralin than in the control except at Day 9. The level of ATP in tissue treated with 10m6, lo-‘, and lo-* M trifluralin did not show a definite trend. Similarly, the levels of ADP and AMP were not significantly affected by treatment with trifluralin except that the level of AMP was higher than the control from 12-18 days. The energy charge of the treated tissue paralleled that of the control from Days 3 to 15; however, it was lower than the control on Day 15 (Table 1). At Day 9 an exception to this statement can be seen for tissue treated with 10e4 M herbicide. DISCUSSION
The growth data show that carrot callus is susceptible to trifluralin. At Day 6 trifluralin concentrations required to reduce
Time
12 (days)
(p.1 O.JlO
minlg
15
,fresh wt I
18
SE)
fresh and dry weight gain by 30% were <10-x and 5.5 x lO-‘j M, respectively. Throughout the test period fresh weight was more sensitive to the herbicide (e.g., I,, for fresh weight at 18 days was 5.5 x lo-” M and for dry weight, 9.5 x lO-‘j M). This response might suggest that the cellular expansion phase of growth is inhibited to a greater extent than is the synthesis of cellular material (dry weight). Effects of trifluralin on mitochondrial activities reported in the literature are extremely variable. Feeny (5) observed no effect on oxygen consumption in excised oat coleoptiles. In isolated mitochondria, oxygen consumption in soybeans, a resistant plant, was inhibited more than a susceptible plant, sorghum (23). In Zpomoeu, a resistant species, oxygen uptake was stimulated (18). Since resistant plants are more susceptible to trifluralin action, the role that mitochondrial responses play in selectivity is unknown. In isolated mitochondria from corn seedlings germinated in the presence of trifluralin, oxidative phosphorylation was uncoupled (21). This stimulatory effect was confounded by the stronger inhibitory effect of the electron transport chain (20). The state of inhibition was not relieved by dinitrophenol as it was by oligomycin (21). Wang et al. (29) observed that trifluralin’s
206
SLOAN
I
II
I
03
coNTm
T
10dM
6
9
12
15
IO
AND
It
16•M
I
IO-‘M
3
9
6
TIME FIG.
fresh (.--.).
CAMPER
5. Effecf
of triflurulin on concentrations WI c SE) of currot callus tissue. ATP (-),
effect on mitochondria was similar to oligomycin, an electron transfer inhibitor. Trifluralin at a concentration of 10V6 M stimulated oxygen consumption by carrot callus on Day 12, whereas the other concentrations remained inhibitory in our study. Dinitrophenol at low concentrations (lop5 and lo-’ M) had some stimulatory effects on oxygen consumption; DNP at lop5 was inhibitory resulting in complete cessation of 0, consumption by Day 12. Triflura-
12
15
I9
3
6
9
12
15
1B
(days)
of ATP, ADP
ADP, AMP, and toiul (---), AMP (---),
udcnylutes (nglmlig total adenylates
lin does not give an inhibition profile similar to DNP. Thus, in this system, the mechanism of inhibition of 0, consumption by trifluralin is unclear. Interpretation of results obtained with ATP, ADP, AMP, and total adenylates is difficult. In the control tissue both total adenylates and ATP decreased with time; however, ADP and AMP levels remained constant. ADP and AMP concentrations also maintained at a steady state at all levels
TRIFLURALIN
Treatment time (days)
Energy 0
AND
charge
(i
CALLUS
SE)
at trifluralin
0.69 0.78
f 0.03 I 0.04
0.68 0.81
2 0.04 k 0.03
0.77 0.83
0.82
-c 0.05
0.68
c 0.03
0.76
+ 0.03 1-.0.03 t 0.03
12 15
0.74 0.73
2 0.02 I 0.03
0.76 0.61
+ 0.02 + 0.03
0.73 20.64
k 0.06 -t 0.08
in duckweed and in low levels compared to ATP levels (16). In the presence of trifluralin no clear trend was observed in its effects on ATP, ADP, and AMP (except with ATP levels at concentrations of 1O-4 and 10-j M herbicide). AMP levels after 9 days were higher than the control which may suggest an enhanced degradative activity and more AMP released into the cellular pool. Total adenylates were generally higher than the control after 12 days. Part of this increase may have been derived from the increase in AMP levels. Similar variation in nucleic acid metabolism in tissue treated with trifluralin has been observed (30). An examination of energy charge data showed that only in the later stages of the growth period followed was there an effect of trifluralin. These values may be indicative of a general decline in cellular activities which may have been enhanced by trifluralin. Carrot callus tissue was used as a model system in this study to examine various effects of trifluralin. Considerable variation in responses at the molecular level was encountered which may be related to resistance of carrots observed in the field. Oxygen consumption was impaired in treated tissue: however, ATP levels were not drastically affected. Trifluralin has been classed as a multisite inhibition affecting both electron transfer and phosphorylation of ATP. Our data tend to support this concept.
0.68 0.85 0.78 0.7.5 0.59
..~~
IO i
2 0.02 ? 0.02 + 0.01 t 0.05 2 0.08
IO ”
0.74
k 0.04
0.07
f 0.06
0.84
r 0.05
0.81
0.76 0.74 0.59
r 0.06 rt 0.03 t 0.05
0.70 0.69 0.61
-t 0.03 2 0.04 i 0.03 ? 0.04
REFERENCES 1. F. M. Ashton and A. S. Crafts, “The Mode of Action of Herbicide5.” Academic Press, New York, 1973. 2.
3.
F. M. Ashton. W. B. Duke, herbicides.
and 4.
0.
T. de Villiers. R. K. Gleen. and Localization of sites of action of Pt,.\tic,. Bioc~11em. Ph~\io/. 7. 1112
(1977). D. E. Atkinson. Its
“Cellular
5.
F. W. oat
Kelleber.
6.
7.
Culture Methods,” tory. Saskatoon. R. E. Gruenhagan
8.
hypocotyls. D. He55 and
Northetrst and
herbicides
on
IO.
12,
in
171 ( 197.5).
C‘otrttd
Wetter,
Cot$
“Plant
of oat
20.
Ti5\ue
Prairie Regional LaboraSaskatchewan, 1975. and D. E. Moreland. Effects of ATP
levels
in
excised
of dividing plant of cotton (Go.ts.vpiu~~
L. ‘Acala 4-42’), J. Cdl. Sci. D. Hess and D. Bayer. Binding trifluralin to Ch/~rrn~lk,mo,iu.\ .I. (‘rli. Sci. 24, 351 (1977). D. Hess. The influence of the on flagella E.rp. Ceil
against
on the growth of exci5ed
Wed
1.. R.
Margrulis.
soybean
Wred St,;. 19. 3 19 (I971 ). D. Bayer. The effect of trifluralin
the ultrastructure root meristems 9.
C~lohim
Effect of trifluralin and respiration
roots, Prw. 595 t 1966). 0. L. Gamborg
L.
New
is active morphogenesi\
oral
c~o/rru/u.v,
Press.
and
trifluralin
Feeny. seedlings
Metabolism
Academic
The herbicide microtubular-based
Serltor
Energy
Regulation.”
York. 1977. S. Banerjee. J. K.
on
cells
of the
/icr.sitiuni
IS, 429 (1974). of the
herbicide
flagella herbicide
tubulin. trifluralin
regeneration in Ch/o,n~c/,,nfo,fir.\. Rrs. 119. 99 (1979).
1 I.
D. M. Karl and 0. Holm-Hanson. ATP. ADP and AMP determination5 in water samples and algal cultures. it7 “Phycological Methods.” pp. 197 -206. Cambridge Univ. Press. London/New
12.
G. A.
York. ACKNOWLEDGMENTS
(M)
10~~6
3 6 9
Trifluralin was generously ucts Co. Contribution No. tural Experiment Station.
concentration
, o-.Y
IO-’
207
TISSUE
Assay AMP
provided by Elanco Prod1850 of the S. C. Agricul13.
1978. Kimmich.
J.
Randles.
and
J. S.
Brand.
of picomole amounts of ATP, ADP and using the luciferase enzyme system. Atfcri. Bioc.hrm. 69. I87 (297.5). A. Komamine. Y. Morohashi. and M. Shimokori-
208
SLOAN AND CAMPER
yama, Changes in respiratory metabolism in tissue cultures of carrot root. Planf Cell. Physiol. 12, 73 (1969). 14. A. Komamine, T. Shimizu, H. Ashihara, and M. Shimokoriyama. The mechanism of changes in respiratory activity during callus formation in carrot root slices culture, Plutnt Cell. Physiol. 13, 821 (1972). 15. A. Komamine and T. Shimizu. Changes in some enzyme activities and respiration in the early stages of callus formation in a carrot root tissue culture, Physiol. Plufll. 33, 47 (1975). 16. T. Kondo and H. Nakashima. Content of adenosine phosphate compounds in a long day plant duckweed, f.ir?rniu gibha L. 63, under different light and nutritional conditions, Phvsiol.
Piant.
33, 47 (1979).
17. B. E. Lapade and B. L. Mercado, Morphological and physical responses of crops and weeds to trifluralin in VI. Free amino acid content of the rice seedlings. Philipp. Agr. 55, 239 (1971). 18. B. L. Mercado and J. N. Sierra, Respiration in resistant and susceptible seedlings treated with trifluralin. Philipp. Agr. 54, 115 (1970). 19. D. E. Moreland, S. S. Malhotia. R. D. Gruenhagen, and E. H. Shokrah. Effects of herbicides on RNA and protein synthesis, Weed Sci. 17, 556 (1969). 20. D. E. Moreland. F. S. Farmer, and G. G. Hussey. Inhibition of photosynthesis and respiration by substituted 2,6-dinitroaniline herbicides. I. Effects on chloroplasts and mitochondrial activities. Pesfic. Biochem. Physiol. 2,342 (1972). 21. D. E. Moreland. F. S. Farmer, and G. G. Hussey. Inhibition of photosynthesis and respiration by
22. 23.
24. 25.
substituted 2,6-dinitroaniline herbicides. II. Effects on responses in excised plant tissues and treated seedlings, Pesfic. Biochrm. Physiol. 2. 354 (1972). T. Murashige and F. Skoog. A revised media for rapid growth and bioassays with tobacco callus tissue, Physiol. Plum. 15, 473 (1962). M. S. Negi, H. H. Funderburk, Jr., D. P. Schultz. and D. E. Davies, The effect of trifluralin and nitralin on mitochondria activities, Weed Sci. 16, 83 (1968). S. J. Parka and Q. F. Sober, The physiology and mode of action of the dinitroaniline herbicide, Weed Sri. 25, 79 (1977). D. Penner and R. W. Early, Action of trifluralin on chromatin activity in corn and soybeans, Weed Sri.
20. 364 (1972).
26. S. J. Robinson. C. F. Yocum, H. Ikuma, and F. Hayoski, Inhibition of chloroplasts electron transport reactions by trifluralin and diallate, P/UII~ Physiol.
60, 840 (1977).
27. D. D. Schultz. H. H. Funderburk, Jr.. and N. S. Negi, The effect of trifluralin on growth, morphology. and nucleic acid synthesis, Plunt Physiol.
43, 265 (1968).
28. R. E. Talbert, Effect of trifluralin on soybean root development, Proc. Saurhern Weed Conf 18, 652 (1965). 29. B. Wang, S. Grooms, and R. E. Frans, Response of soybean mitochondria to substituted dinitroaniline herbicides, Weed Sci. 22, 64 (1974). 30. L. W. Young and N. D. Camper, Trifluralin effects on tobacco callus tissue: Mitosis and selected metabolic effects, Pesfic. Biochrm. Ph.vsioi.
12, 117 ( 1979).
BIOCHEMISTRY
AND
PHYSIOLOGY
Trifluralin
201-208
15,
Effects
MARGARET
Received
October
(1981)
on Carrot
E. SLOAN ANDN.
15, 1980; accepted
Callus Tissue D. CAMPER
February
5. 1981
The effect of trifluralin on the growth. oxygen uptake. and adenosine phosphates level of carrot (DNWUS c’urotu L.) callus tissue was determined over a period of I8 days after subculture. The herbicide (IO-’ and IO-” M) reduced fresh weight gain significantly; the reduction was less with lower trifluralin concentrations. Dry weight accumulation was not inhibited until after Day 6 of the test period and thereafter was reduced by all concentrations tested. Oxygen consumption wab inhibited by trifluralin (IO-’ and IO-” M) throughout the test period. Concentrations of IO-“. 10 ;. and IO-” M produced variable effects. The response profile of 0, consumption in the presence of dinitrophenol was different from that of trifluralin. Analysis of adenosine phosphates level gave no clear response trend. Energy charge values of 0.7 to 0.85 were obtained for untreated tissue. Trifluralin had no effect on energy charge until Day 9 (IO-‘M) and after Day I5 (all concentrations).
INTRODUCTION
photophosphorylation (2, 27); however, such effects in intact plants may be of little physiological significance because trifluralin is not readily translocated (2). Oxygen consumption was unaffected in excised oat seedlings (5), inhibited in isolated mitochondria from soybean and sorghum (23), and stimulated in Ipomoeu (18). Oxidative phosphorylation was uncoupled in isolated corn mitochondria (21). The site of trifluralin’s inhibition in the mitochondria is at or near the site of oligomycin inhibition and a major mechanism of dinitroaniline phytotoxicity may be the disruption of ATP formation either by interference with the energy-generating mechanism or by blocking the energy-transferring mechanism (29). However, ATP formation was not impaired in soybean hypocotyls (7). The importance of interference with ATP production by the phytotoxic action of the dinitroaniline herbicides remains to be established (23). The biochemical mechanism of action of the 2,6-dinitroanilines must account for the effect expressed on meristematic cells (20). We, therefore, chose a system in which cell division was a major cellular event. Callus tissue provides such a system. The study was undertaken to examine the effect of trifluralin on oxygen consumption and on
Trifluralin . ’ a substituted 2,6-dinitroaniline herbicide, is a preemergence herbicide used for selective weed control. Trifluralin has been classified as a mitotic poison (28) with many of its morphological and cytological responses similar to those of colchitine (8). Recent studies with microtubules suggested that trifluralin and colchicine affect cellular metabolism by different mechanisms (4, 9, 10). The effect of trifluralin on plant metabolism is dependent upon plant species, concentration, plant organ, and time (2, 24). Effects on nucleic acid metabolism may be a key to trifluralin’s mode of action (1). However, trifluralin effects on RNA and DNA synthesis remain contradictory (19. 27, 30). Evidence suggests that trifluralin may bind to the DNA template, inhibiting synthesis (25). Protein synthesis and amino acid composition may be altered by trifluralin (17, 19, 30). Effects of trifluralin on organelle phosphorylation and oxygen consumption has been examined. The herbicide inhibited I Abbreviations used: trifluralin, 2.6-dinitro-,Y.N-dipropyl-p-toluidine; chlorophenoxy) acetic acid; PEP. vate: DNP. 2.Cdinitrophenol.
n.u,u-trifluoro2.4.D.(2,4-diphosphoenol pyru-
201 004%3575/81/030201-08%02.00/O CopyrIght All rights
C 1981 by Academic Press. Inc. of reproduction in any form reserved.
202
SLOAN
the level of adenosine phosphates energy charge in carrot callus tissue. MATERIALS
Culture
conditions of carrots (Daucus
AND
AND
and
METHODS
and growth. Taproots carota L.) were ob-
tained locally. No distinctions were made between varieties in the subsequent procedures. Carrot callus was isolated by a modified procedure of Komamine et al. (13-15). Carrot roots were surface sterilized in absolute ethanol for 2 min, rinsed, and resterilized in 20% clorox for 30 min. Transverse sections were excised, cut into 2-mm sections, and cultured on modified Murashige and Skoog medium (22) containing 0.9% agar, 0.9% agar supplemented with 2,4-D (1 mg/ml), or 0.9% agar supplemented with 2,4-D (1 mg/ml) and kinetin (0.1 mg/ ml). The pH was adjusted to 6 with 1 N NaOH before autoclaving (20 min, l/O5 kg/ cmz). Subcultures were made at monthly intervals and cultures were maintained in the dark at 23 to 25°C. These cultures were used in the subsequent studies. Stock solutions of technical trifluralin were prepared in pesticide-grade acetone and Millipore filtered. Serial dilutions were prepared so that to each 125ml flask containing 50 ml of medium was added 0.25 ml regardless of the concentration. Each control flask contained 0.25 ml acetone. Trifluralin was added to autoclaved, but cooled, medium prior to solidification. Fresh herbicide solutions were made for each medium preparation and designated Day 0. DNP was prepared and handled in a similar manner. Tissue was harvested every 3 days for a period of 18 days; all experiments had three replicates and were repeated at least once (data represent an average of six determinations). Flasks were incubated in the dark at 23 + 2°C. Dry weight was determined as described by Gamborg and Wetter (6). Respiration. Respiration was determined by standard manometric techniques utilizing a Gibson differential respirometer. Respiratory rate was determined at 30°C for
CAMPER
1.5 hr with readings taken every 10 min. Conditions were those of Komamine et al. (13-15). Extraction
and assay of total adenylates.
Extraction of ATP was conducted by a modified procedure as described by Karl and Holm-Hanson (11). A 0.03- to 0.05-g sample was removed, immersed immediately in 5 ml of boiling Tris buffer (0.02 M, pH 7.75) for 1 min, and immediately homogenized in a tissue grinder. The sample was boiled for 1 min and brought to a final volume of 10 ml with Tris buffer. The suspension was centrifuged for 10 min at 13,000g (Beckman Model J 2-21). The supernatant was decanted and stored frozen (-4°C) for analysis. Enzymatic determination of total adenylates was performed as described by Karl and Holm-Hanson (11) and Kimmich, Randles, and Brand (12) as modified by the authors. Fire fly extract was reconstituted as prescribed by Sigma Chemical Company. For ATP determinations, 0.4 ml of the sample (or ATP standards) was added to a test tube containing 0.1 ml of a 100 mM TrisHCl (pH 7.4), 35 m&I KCl, and 6 n&I MgC12. Enzymatic conversion of ADP+ATP was determined by adding 0.4 ml of the sample to 0.1 ml of a 100 mM Tris-HCl (pH 7.4), 35 mM KCl, and 6 mM MgCl, containing 0.5 mM PEP, and 20 pg pyruvate kinase. Enzymatic determination of AMP-+ATP was conducted in the same manner as ADP with the addition of 25 pg of myokinase to the reaction buffer. All samples were incubated at 30°C for 30 min and placed in a boiling water bath for 2 min to deactivate the enzymes. The samples were diluted to 2 ml with Tris buffer (0.02 M, pH 7.75) to read 5,000 to 150,000 cpm on a Beckman LS1OOC (3H-channel, 0.1 min, 2% error). The amount of ATP was determined from a standard curve conducted daily. Levels of ADP and AMP were calculated from the difference of these measured values. The energy charge was calculated by the following equation as described by Atkinson (3): (ATP + ‘/ ADP)/(ATP + ADP + AMP).
TRIFLURALIN
AND
RESULTS
Data reported in this study (e.g., fresh and dry weight, adenylates levels, etc.) were not measured until Day 3 of the experiments. Preliminary studies showed that up to 3 days from initial culturing were required for stabilized growth due to tissue source, adjustment to media, and possibly other factors. Growth of untreated tissue increased with time (Fig. 1, control). All concentrations of trifluralin tested significantly inhibited fresh weight gain by the callus tissue (Fig. 1). Growth at highest levels tested (lo-’ and lo-” M) was not different: similar trends were evident for IO-“,
CALLUS
203
TISSUE
10-7, and lop8 M. Significant differences in growth rates can be seen between the higher trifluralin levels (lo-“ and IO-” M) and the lower ones (lOpci, lo-‘. and IO-” M). Similar results were observed with dry weight gain (Fig. 2). Respiratory activity of the control remained essentially constant during the test period; however, there was a trend toward decreased 0, consumption with incubation time (Fig. 3). Trifluralin at concentrations lo-” and lo-” M significantly reduced oxygen uptake as compared to the control throughout the test period. Initially the respiratory rate of lo-” M-treated tissue was
Trifluralln
0
3
6
9
12
15
18 0
3
6
9
12
15
18
204
SLOAN
AND
CAMPER
LEGEND: 1o-4
M-
10-5
M ----_
10-6
M ---_-
lo-'
M --M ---_
10-e o-
CULTURE FIG.
treuted
2. Effect
of trij7urulin
on dry weight
GROWTH TIME
(DAYS)
(g k SE) uccumulation
of carrot
callus
tissue.
Control
(O),
(0).
less than the control until Day 6, thereafter it was not different from the control. Oxygen consumption by tissue treated with 10m7 and 10eE M trifluralin was lower than the control except at Days 12 and 18 (lOpR A4 only). The effect of DNP on oxygen consump-
01
31
6I
tion was determined for comparison with trifluralin. Oxygen consumption by the control tissue increased from Days 3 to 9 and decreased thereafter (Fig. 4). DNP at 5 x IO-” A4 significantly decreased oxygen consumption through Day 9 after which oxygen uptake could not be detected. The
I9
1 12
I 15
ia
CULTURE GROWTH TIME (DAYS) FIG.
Control
3. Effect of frifluralin (0). trrated (0).
on oqvgen
uptukr
(~1 OJIO
minlg
fresh
wjt k SE) by carrot
cullus
tissue.
TRIFLURALIN
AND
CALLUS
205
TISSUE
SO-
IOG 0
tissue.
Cnnlrol
of dinitrophenol ( 0). rretrted (0).
3
6 Culture
on oxygen
9 Growth
rrptuke
concentration of 10-j M significantly stimulated oxygen uptake on Days 9, 12, and 15; whereas 10m7 M significantly stimulated oxygen consumption on Days 12 and 15 only. The level of ATP in the control increased from Days 3 to 9 and decreased thereafter (Fig. 5). Concentrations of ADP and AMP remained essentially at a constant level throughout the test period in the untreated tissue. ATP levels were higher in tissue treated with lop4 and 10e5 M trifluralin than in the control except at Day 9. The level of ATP in tissue treated with 10m6, lo-‘, and lo-* M trifluralin did not show a definite trend. Similarly, the levels of ADP and AMP were not significantly affected by treatment with trifluralin except that the level of AMP was higher than the control from 12-18 days. The energy charge of the treated tissue paralleled that of the control from Days 3 to 15; however, it was lower than the control on Day 15 (Table 1). At Day 9 an exception to this statement can be seen for tissue treated with 10e4 M herbicide. DISCUSSION
The growth data show that carrot callus is susceptible to trifluralin. At Day 6 trifluralin concentrations required to reduce
Time
12 (days)
(p.1 O.JlO
minlg
15
,fresh wt I
18
SE)
fresh and dry weight gain by 30% were <10-x and 5.5 x lO-‘j M, respectively. Throughout the test period fresh weight was more sensitive to the herbicide (e.g., I,, for fresh weight at 18 days was 5.5 x lo-” M and for dry weight, 9.5 x lO-‘j M). This response might suggest that the cellular expansion phase of growth is inhibited to a greater extent than is the synthesis of cellular material (dry weight). Effects of trifluralin on mitochondrial activities reported in the literature are extremely variable. Feeny (5) observed no effect on oxygen consumption in excised oat coleoptiles. In isolated mitochondria, oxygen consumption in soybeans, a resistant plant, was inhibited more than a susceptible plant, sorghum (23). In Zpomoeu, a resistant species, oxygen uptake was stimulated (18). Since resistant plants are more susceptible to trifluralin action, the role that mitochondrial responses play in selectivity is unknown. In isolated mitochondria from corn seedlings germinated in the presence of trifluralin, oxidative phosphorylation was uncoupled (21). This stimulatory effect was confounded by the stronger inhibitory effect of the electron transport chain (20). The state of inhibition was not relieved by dinitrophenol as it was by oligomycin (21). Wang et al. (29) observed that trifluralin’s
206
SLOAN
I
II
I
03
coNTm
T
10dM
6
9
12
15
IO
AND
It
16•M
I
IO-‘M
3
9
6
TIME FIG.
fresh (.--.).
CAMPER
5. Effecf
of triflurulin on concentrations WI c SE) of currot callus tissue. ATP (-),
effect on mitochondria was similar to oligomycin, an electron transfer inhibitor. Trifluralin at a concentration of 10V6 M stimulated oxygen consumption by carrot callus on Day 12, whereas the other concentrations remained inhibitory in our study. Dinitrophenol at low concentrations (lop5 and lo-’ M) had some stimulatory effects on oxygen consumption; DNP at lop5 was inhibitory resulting in complete cessation of 0, consumption by Day 12. Triflura-
12
15
I9
3
6
9
12
15
1B
(days)
of ATP, ADP
ADP, AMP, and toiul (---), AMP (---),
udcnylutes (nglmlig total adenylates
lin does not give an inhibition profile similar to DNP. Thus, in this system, the mechanism of inhibition of 0, consumption by trifluralin is unclear. Interpretation of results obtained with ATP, ADP, AMP, and total adenylates is difficult. In the control tissue both total adenylates and ATP decreased with time; however, ADP and AMP levels remained constant. ADP and AMP concentrations also maintained at a steady state at all levels
TRIFLURALIN
Treatment time (days)
Energy 0
AND
charge
(i
CALLUS
SE)
at trifluralin
0.69 0.78
f 0.03 I 0.04
0.68 0.81
2 0.04 k 0.03
0.77 0.83
0.82
-c 0.05
0.68
c 0.03
0.76
+ 0.03 1-.0.03 t 0.03
12 15
0.74 0.73
2 0.02 I 0.03
0.76 0.61
+ 0.02 + 0.03
0.73 20.64
k 0.06 -t 0.08
in duckweed and in low levels compared to ATP levels (16). In the presence of trifluralin no clear trend was observed in its effects on ATP, ADP, and AMP (except with ATP levels at concentrations of 1O-4 and 10-j M herbicide). AMP levels after 9 days were higher than the control which may suggest an enhanced degradative activity and more AMP released into the cellular pool. Total adenylates were generally higher than the control after 12 days. Part of this increase may have been derived from the increase in AMP levels. Similar variation in nucleic acid metabolism in tissue treated with trifluralin has been observed (30). An examination of energy charge data showed that only in the later stages of the growth period followed was there an effect of trifluralin. These values may be indicative of a general decline in cellular activities which may have been enhanced by trifluralin. Carrot callus tissue was used as a model system in this study to examine various effects of trifluralin. Considerable variation in responses at the molecular level was encountered which may be related to resistance of carrots observed in the field. Oxygen consumption was impaired in treated tissue: however, ATP levels were not drastically affected. Trifluralin has been classed as a multisite inhibition affecting both electron transfer and phosphorylation of ATP. Our data tend to support this concept.
0.68 0.85 0.78 0.7.5 0.59
..~~
IO i
2 0.02 ? 0.02 + 0.01 t 0.05 2 0.08
IO ”
0.74
k 0.04
0.07
f 0.06
0.84
r 0.05
0.81
0.76 0.74 0.59
r 0.06 rt 0.03 t 0.05
0.70 0.69 0.61
-t 0.03 2 0.04 i 0.03 ? 0.04
REFERENCES 1. F. M. Ashton and A. S. Crafts, “The Mode of Action of Herbicide5.” Academic Press, New York, 1973. 2.
3.
F. M. Ashton. W. B. Duke, herbicides.
and 4.
0.
T. de Villiers. R. K. Gleen. and Localization of sites of action of Pt,.\tic,. Bioc~11em. Ph~\io/. 7. 1112
(1977). D. E. Atkinson. Its
“Cellular
5.
F. W. oat
Kelleber.
6.
7.
Culture Methods,” tory. Saskatoon. R. E. Gruenhagan
8.
hypocotyls. D. He55 and
Northetrst and
herbicides
on
IO.
12,
in
171 ( 197.5).
C‘otrttd
Wetter,
Cot$
“Plant
of oat
20.
Ti5\ue
Prairie Regional LaboraSaskatchewan, 1975. and D. E. Moreland. Effects of ATP
levels
in
excised
of dividing plant of cotton (Go.ts.vpiu~~
L. ‘Acala 4-42’), J. Cdl. Sci. D. Hess and D. Bayer. Binding trifluralin to Ch/~rrn~lk,mo,iu.\ .I. (‘rli. Sci. 24, 351 (1977). D. Hess. The influence of the on flagella E.rp. Ceil
against
on the growth of exci5ed
Wed
1.. R.
Margrulis.
soybean
Wred St,;. 19. 3 19 (I971 ). D. Bayer. The effect of trifluralin
the ultrastructure root meristems 9.
C~lohim
Effect of trifluralin and respiration
roots, Prw. 595 t 1966). 0. L. Gamborg
L.
New
is active morphogenesi\
oral
c~o/rru/u.v,
Press.
and
trifluralin
Feeny. seedlings
Metabolism
Academic
The herbicide microtubular-based
Serltor
Energy
Regulation.”
York. 1977. S. Banerjee. J. K.
on
cells
of the
/icr.sitiuni
IS, 429 (1974). of the
herbicide
flagella herbicide
tubulin. trifluralin
regeneration in Ch/o,n~c/,,nfo,fir.\. Rrs. 119. 99 (1979).
1 I.
D. M. Karl and 0. Holm-Hanson. ATP. ADP and AMP determination5 in water samples and algal cultures. it7 “Phycological Methods.” pp. 197 -206. Cambridge Univ. Press. London/New
12.
G. A.
York. ACKNOWLEDGMENTS
(M)
10~~6
3 6 9
Trifluralin was generously ucts Co. Contribution No. tural Experiment Station.
concentration
, o-.Y
IO-’
207
TISSUE
Assay AMP
provided by Elanco Prod1850 of the S. C. Agricul13.
1978. Kimmich.
J.
Randles.
and
J. S.
Brand.
of picomole amounts of ATP, ADP and using the luciferase enzyme system. Atfcri. Bioc.hrm. 69. I87 (297.5). A. Komamine. Y. Morohashi. and M. Shimokori-
208
SLOAN AND CAMPER
yama, Changes in respiratory metabolism in tissue cultures of carrot root. Planf Cell. Physiol. 12, 73 (1969). 14. A. Komamine, T. Shimizu, H. Ashihara, and M. Shimokoriyama. The mechanism of changes in respiratory activity during callus formation in carrot root slices culture, Plutnt Cell. Physiol. 13, 821 (1972). 15. A. Komamine and T. Shimizu. Changes in some enzyme activities and respiration in the early stages of callus formation in a carrot root tissue culture, Physiol. Plufll. 33, 47 (1975). 16. T. Kondo and H. Nakashima. Content of adenosine phosphate compounds in a long day plant duckweed, f.ir?rniu gibha L. 63, under different light and nutritional conditions, Phvsiol.
Piant.
33, 47 (1979).
17. B. E. Lapade and B. L. Mercado, Morphological and physical responses of crops and weeds to trifluralin in VI. Free amino acid content of the rice seedlings. Philipp. Agr. 55, 239 (1971). 18. B. L. Mercado and J. N. Sierra, Respiration in resistant and susceptible seedlings treated with trifluralin. Philipp. Agr. 54, 115 (1970). 19. D. E. Moreland, S. S. Malhotia. R. D. Gruenhagen, and E. H. Shokrah. Effects of herbicides on RNA and protein synthesis, Weed Sci. 17, 556 (1969). 20. D. E. Moreland. F. S. Farmer, and G. G. Hussey. Inhibition of photosynthesis and respiration by substituted 2,6-dinitroaniline herbicides. I. Effects on chloroplasts and mitochondrial activities. Pesfic. Biochem. Physiol. 2,342 (1972). 21. D. E. Moreland. F. S. Farmer, and G. G. Hussey. Inhibition of photosynthesis and respiration by
22. 23.
24. 25.
substituted 2,6-dinitroaniline herbicides. II. Effects on responses in excised plant tissues and treated seedlings, Pesfic. Biochrm. Physiol. 2. 354 (1972). T. Murashige and F. Skoog. A revised media for rapid growth and bioassays with tobacco callus tissue, Physiol. Plum. 15, 473 (1962). M. S. Negi, H. H. Funderburk, Jr., D. P. Schultz. and D. E. Davies, The effect of trifluralin and nitralin on mitochondria activities, Weed Sci. 16, 83 (1968). S. J. Parka and Q. F. Sober, The physiology and mode of action of the dinitroaniline herbicide, Weed Sri. 25, 79 (1977). D. Penner and R. W. Early, Action of trifluralin on chromatin activity in corn and soybeans, Weed Sri.
20. 364 (1972).
26. S. J. Robinson. C. F. Yocum, H. Ikuma, and F. Hayoski, Inhibition of chloroplasts electron transport reactions by trifluralin and diallate, P/UII~ Physiol.
60, 840 (1977).
27. D. D. Schultz. H. H. Funderburk, Jr.. and N. S. Negi, The effect of trifluralin on growth, morphology. and nucleic acid synthesis, Plunt Physiol.
43, 265 (1968).
28. R. E. Talbert, Effect of trifluralin on soybean root development, Proc. Saurhern Weed Conf 18, 652 (1965). 29. B. Wang, S. Grooms, and R. E. Frans, Response of soybean mitochondria to substituted dinitroaniline herbicides, Weed Sci. 22, 64 (1974). 30. L. W. Young and N. D. Camper, Trifluralin effects on tobacco callus tissue: Mitosis and selected metabolic effects, Pesfic. Biochrm. Ph.vsioi.
12, 117 ( 1979).