Uptake, Translocation and Metabolism of the
Herbicide Molinate in Tobacco and Rice
Yar-Ning Hsieh,1 Li-Fei Liu2 & Yei-Shung Wang1*
1 Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan
2 Department of Agronomy, National Taiwan University, Taipei, Taiwan
(Received 19 August 1997 ; revised version received 5 January 1998 ; accepted 30 January 1998)
Abstract : Molinate, a selective herbicide, is used for the control of annual and perennial weeds in rice paddy Ðelds. This study was designed to assess the basis of the selective action of molinate between a susceptible broadleaf crop, tobacco, and a resistant graminaceous plant, rice. Experiments were conducted comparing plant growth under di†erent concentrations of molinate, determining the absorp-tion and translocaabsorp-tion of the herbicide in the plant and identifying the metabo-lites in suspension cells. Rice showed greater tolerance to molinate than tobacco. Leaves of tobacco showed retarded and distorted growth at 10 mg liter~1 of molinate 14 days after treatment, but rice leaves were una†ected at this concen-tration. Higher concentrations of molinate accumulating in the root of tobacco seedlings may inhibit root development and represent a signiÐcant factor in the herbicideÏs selective action. Seven and eight metabolites were found in tobacco and rice cells, respectively, with molinate sulfoxide and molinate sulfone present in both species. ( 1998 SCI
Pestic. Sci., 53, 149È154 (1998)
Key words : molinate ; uptake ; translocation ; metabolism ; tobacco ; rice
1 INTRODUCTION
Molinate (S-ethyl perhydroazepine-1-carbothioate) is extensively used as a selective herbicide to control ger-minating broad-leaves and grassy weeds in rice paddy Ðelds. Molinate is applied after water seeded or drilled rice is Ñooded and after the aquatic weed has emerged about 5È10 cm from the soil, and is at least two-thirds submerged by the water. Rates of application are from 2É2 to 3É4 kg ha~1. A number of studies have been published on the fate and selectivity of molinate. Molin-ate controls barnyardgrass (Echinochloa crus-galli (L.) Beauvois) in paddy rice Ðelds, and Chem et al.1 report-ed that barnyardgrass absorbreport-ed more molinate than did rice. Imai and Kuwatsuka2 found similar results,
deduc-* To whom correspondence should be addressed.
Contract/grant sponsor : Council of Agriculture of the Repub-lic of China.
Contract/grant number : 84-Science-2.4-Food-37(1)-01.
ing that molinate only moved acropetally in rice plants, but both acropetally and basipetally in barnyardgrass when applied to the basal part of the shoots, although the molinate showed whole-plant systemicity in both plants when applied to the roots. In a study on e†ect of thiocarbamate herbicides on fatty acid synthesis by potato, Bolton and Harwood3 found that thiocarba-mates reduce the amount of surface lipid of plants. Evi-dence suggesting that thiocarbamates inhibit one or more acyl-CoA elongases is largely indirect.4 Imai and Kuwatsuka5h8 studied the degradation of this herbicide in soil and its metabolism by soil microorganisms. Soderquist et al.9 reported that photodecomposition products of molinate were present in Ðeld water, and that volatilization of molinate from water was the primary mode of dissipation in the environment. Ross and Sava10 determined atmospheric residues at 48 cm above the water surface, Ðnding that the maximum mol-inate concentration was 48 lg m~3 on the day of appli-cation of 3É10 kg ha~1 to rice Ðelds. In a study in rice 149
1998 SCI. Pestic. Sci. 0031-613X/98/$17.50. Printed in Great Britain (
by Imai and Kuwatsuka,11 21% of the total molinate applied was detected in the soil and 1É6% in the rice plants at harvest, and of the molinate residues found in the rice plants, about 96% was in straw and 4% in the grains.
The present study was carried out to clarify the absorption and translocation of molinate in plants of tobacco, a susceptible broad-leaf, and rice. The metabo-lism of molinate in suspension cells tissue culture of both plants was also determined.
2 MATERIALS AND METHODS 2.1 Materials
[Ethyl-1-14C]molinate was provided by Asahi Chemical Industry Co., Ltd, Japan, and had a speciÐc activity of 107É3 MBq mmol~1 and a radiochemical purity of greater than 99%. Analytical grade molinate of greater than 99% purity was purchased from Riedel de Haen Co., Germany. Seeds of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) and rice (Oryza sativa L. cv. Tainan 5) were provided by the Department of Botany and Department of Agronomy, National Taiwan University, respectively.
2.2 Seedling preparation
Seeds of tobacco were soaked in ethanol] water (75] 25 by volume) for 1 min and sterilized with sodium hypochlorite solution (10 g liter~1) for 15 min. Heavy rice seeds were selected with sodium chloride solution (50 g liter~1 ; relative density 1É05)12 and then surface sterilized for 2 h with sodium hypochlorite (10 g liter~1), adding one drop of “TweenÏ-20. After washing three times with deionized water, the tobacco seeds were germinated and grown in glass bottles containing MS agar medium13 (50 ml) for several weeks at 25¡C under 3000 lux illumination (16 h day~1), and the rice seeds were germinated on a stainless steel net laid on the upper surface of liquid in glass bottles containing MS liquid medium for one week. Shoot apices of tobacco plants were excised and transplanted every three to Ðve weeks.
2.3 E†ect of molinate on growth of tobacco and rice plants
Tobacco plants (two weeks after transplantation, 5 cm in height) and rice plants (one week after germination, 7 cm in height) were transferred to MS liquid medium (complemented with sucrose and vitamins) containing 0, 1, 10 and 100 mg liter~1 of molinate, separately. After
incubating for 14 days, gross e†ects or whole plant growth were noted. The experiment was performed in duplicate.
2.4 Absorption and translocation of molinate by plants Rice seedlings and shoot apices from tobacco plants were removed and transferred to another glass bottle containing MS liquid medium (30 ml) and glass beads. The plants were Ðxed with glass beads. After roots had developed (seven days), [14C]molinate (0É11 MBq) was added to the medium. After incubating for two and seven days, whole plants were washed with distilled water containing acetone (1É0 ml liter~1) and “TweenÏ-20 (1É0 ml liter~1), Ðxed on a glass plate and then covered with X-ray Ðlm (20] 25 cm, Kodak X-Omat AR) at [ 20¡C for two months for autoradiography. The plant materials were then subjected to wet com-bustion and the radioactivity was quantiÐed. The test was performed in duplicate. Detailed methods are described elsewhere.14
For a thorough understanding of the relationship between molinate concentration and plant absorption and translocation, an experiment was also performed by adding 2É5, 5É0, 7É5 or 10É0 mg liter~1 of [14C]molinate to the medium and determining the radioactivity in the shoot and root after incubation for two days. Each test was performed in duplicate.
2.5 Suspension cultures of tobacco leaf cells and rice embryogenic cells
For callus preparation, whole tobacco leaves from beneath the shoot apices were sliced into pieces of 1È2 cm in length, and placed on MS agar medium con-taining 2-(1-naphthyl)acetic acid (NAA ; 1É0 mg liter~1) and 6-benzylaminopurine (BAP ; 0É1 mg liter~1) for callus induction. Induced callus was suspended and dis-persed in a MS liquid medium containing NAA,(0É5 mg liter~1) and BAP, (0É1 mg liter~1). The cultures were shaken at 100 rev min~1 at 25¡C. Homogeneous sus-pensions were obtained by Ðltering with a 1-mm Ðlter net. Subculture was performed every two weeks.
Rice embryogenic calli obtained from cultured imma-ture embryos of rice seed were transplanted and cul-tured in a 250-ml Ñask containing MS liquid medium (50 ml) supplemented with 2,4-D (10 lM) on a shaker (100 rev min~1). Homogeneous suspensions were obtained by Ðltering with a 1-mm Ðlter net. Subculture was performed every seven days.
2.6 Metabolism of molinate in tobacco and rice cell suspensions
Metobolism of molinate in tobacco and rice was exam-ined by culturing the cells in medium containing
[14C]molinate (10 mg liter~1). In Ðve 50 ml-Ñasks, [14C]molinate solution (0É037 M Bq) in ethanol was added to the suspension (10 ml ; approximate cell density 4] 105 ml~1) in each Ñask to give 10 mg liter~1 concentration of molinate. The cells were shaken at 25¡C under 3000 lux illumination and 50 rev min~1. After incubating for 3, 6, 12, 24 and 48 h, one Ñask was taken and the suspension was centrifuged (1000g, Sigma 2K-15 12139 rotor) to sediment the cells. The cells were collected, washed with MS liquid medium (5 ml) and again centrifuges. The percentage of radioactivity remaining in the culture medium was determined. Col-lected cells were added to 70% acetone (10 ml), homog-enized and extracted three times. The extracts were combined, diluted with water and then extracted with hexane three times. The procedure for determining mol-inate and its metabolites is shown in Fig. 1, a method used previously by Imai and Kuwatsuka.2 The radioac-tivity of the residues was determined by the wet com-bustion method.14 Metabolites were analyzed by co-chromatography with authentic standards. Hexane and ether extracts were concentrated and co-chromatographed. Polar conjugates were hydrolysed with hydrochloric acid (1 M) at 70¡C for 2 h.
The metabolites molinate sulfoxide [1-ethyl-sulÐnylcarbonyl perhydroazepine] and molinate sulfone
Fig. 1. Extraction of molinate and its metabolites from tobacco and rice cells.
[1-ethylsulfonylcarbonyl perhydroazepine] were pre-pared by the method of Soderquist et al.9 and identiÐed by GC-MS and FT-IR (Bio-red model FTS-7). GC-MS analysis was performed by using Shimadzu GC-14A interfaced to a QP1100EX mass detector equipped with a Supelco PTE 5 fused silica capillary column (30 m] 0É25 mm ID, Ðlm thickness 0É25 lm). Helium was used as the carrier gas with a Ñow rate of 3 ml min~1 with split ratio 1 : 50. The injection port temperature was 200¡C ; interface was 250¡C ; oven tem-perature was held at 100¡C for 5 min, then programmed to 250¡C at 10¡C min~1, and maintained at the Ðnal temperature of 250¡C for 20 min. Following identiÐca-tion by GC-MS, the retenidentiÐca-tion times were found to be 18É9 min (molinate sulfoxide) and 16É9 min (molinate sulfone), both compounds displaying base peak m/e 126 (C
6H12NCO).
Thin layer chromatography was performed on silica gel 60F (Merck 5715) using hexane] acetone (1 ] 1
254
by volume) as developing solvent and metabolites were identiÐed at 254 nm with a UV lamp. TheR values of
f
molinate, molinate sulfoxide and molinate sulfone were 0É90, 0É55 and 0É83, respectively.
3 RESULTS AND DISCUSSION
E†ects of di†erent concentrations of molinate on the growth of tobacco and rice plants were compared after incubation for 14 days. Growth of tobacco shoots was not a†ected by 1 mg liter~1 of molinate, but leaf distor-tion and growth inhibidistor-tion were found at 10 mg liter~1. At a concentration of 100 mg liter~1, the older leaves showed necrosis and a brownish colour. Growth of tobacco roots was slightly inhibited by 1 mg liter~1 of molinate, and when the concentration exceed 10 mg liter~1, root development was greatly inhibited. The growth of rice plants was inhibited and the leaves become a brownish colour at a concentration of 100 mg liter~1. Rice plants showed more tolerance to molinate than tobacco. The lengths of shoots and roots of both species after treatment with di†erent concentrations of molinate are shown in Table 1.
Autoradiograms (not shown) and tissue combustion of [14C]molinate-treated plants indicated that, in tobacco, more [14C] derived from molinate remained in the root than translocated to the shoot after two days incubation, but most of the radioactivity was trans-located to the shoot at seven days, when expressed on a dpm g~1 basis. By contrast, in rice radioactivity trans-located more rapidly to the shoot at two days after incubation. Table 2 shows the plant distribution of [14C] derived from molinate. No obvious di†erence was found for total [14C] absorption between the two species ; about 0É59% (0É17% in root and 0É42% in shoot) and 0É55% (0É2% in root and 0É35% in shoot) of the total activity being absorbed by tobacco and rice,
TABLE 1
Length of Tobacco and Rice Shoots and Roots after Treatment with Di†erent Con-centrations of Molinate
Organ length (cm) (^SD)
Concentration T obacco Rice
of molinate
(mg liter~1) Shoot Root Shoot Root
Control 4É03 (^0É40) 6É38 (^0É26) 17É00 (^0É90) 5É27 (^0É25) 1 5É03 (^0É20) 5É44 (^0É37) 19É01 (^1É22) 6É02 (^0É95) 10 3É39 (^0É23) 2É20 (^1É35) 18É44 (^0É14) 5É54 (^0É27) 100 1É68 (^0É06) 0 6É80 (^0É80) 4É06 (^0É40)
respectively, after incubation for seven days. Chem et al.1 and Imai and Kuwatsuka2 had noted that E. crus-galli absorbed more molinate and rice absorbed rela-tively less molinate and therefore this may be the reason for molinateÏs selectivity. The higher level of radioac-tivity in the roots of tobacco compared with those of rice may indicate that a critical level of molinate or its metabolites in tobacco roots may be a factor in the selective activity of the herbicide. Figure 2 clariÐes this point and shows the relationship between [14C]molinate concentration and accumulation by tobacco and rice plants when incubated for two days. The results showed that the higher the concentration of molinate present in culture solution, the more molinate was absorbed by both species. However, most of the molinate absorbed was translocated to the shoot in rice plants and only a small amount remained in root, regardless of the concentration. On the other hand, most of molinate absorbed by tobacco accumulated in the root, only a small amount being translocated to the shoot.
After exposing cell suspensions to [14C]molinate for two days, the herbicide and its metabolites were deter-mined. Table 3 lists the percentages of radioactivity dis-tribution in di†erent extraction fractions after di†erent incubation periods. In rice cells, more than 60% of the radioactivity derived from [14C] molinate was found in
the aqueous residue fraction after 48 h, implying con-version to polar metabolites. Rice cells were able to metabolize molinate to polar metabolites more rapidly than tobacco cells. The relatively non-polar metabolites (hexane fraction) accounted for 15È30% in rice cells and were lower than that in tobacco cells. After acid
hydro-Fig. 2. Relationship between [14C]molinate concentration and accumulation by tobacco and rice plants after two days. TABLE 2
Absorption and Translocation of Radioactivity Derived from [14C]molinate in Tobacco and Rice Plants
T obacco Rice
Incubation
(days) Root Shoot Root Shoot
Radioactivity 2 2664 (^51) 2758 (^37) 3605 (^33) 3912 (^32) in plant (dpm) 7 11 040 (^229) 28 026 (^186) 12 950 (^59) 23 364 (^147) Plant weight 2 0É01 (^0É000) 0É07 (^0É007) 0É07 (^0É007) 0É03 (^0É000) (dry, g) 7 0É02 (^0É000) 0É18 (^0É007) 0É05 (^0É000) 0É06 (^0É007) dpm g~1 2 266 400 (^5100) 39 400 (^4000) 51 500 (^5200) 130 400 (^1100) 7 552 000 (^11 400) 155 700 (^6100) 259 000 (^1200) 339 400 (^46 700) 14C 2 0É04 (^0É001) 0É04 (^0É001) 0É05 (^0É001) 0É06 (^0É000) absorbed (%) 7 0É17 (^0É003) 0É42 (^0É003) 0É20 (^0É001) 0É35 (^0É002)
TABLE 3
Distribution of Radioactivity in Di†erent Extract Fractions when Tobacco and Rice Suspension Cells Were Incubated in [14C]Molinate for Di†erent Periods
Radioactivity recovered (%) Supernatant following extraction in
70% acetone After hydrolysis
Ether extract Ether extract
Incubation Matrix Hexane Aqueous T otal
time residues extract Neutral Basic Acidic Acidic Basic residue radioactivity
(h) (8)a (1) (2) (3) (4) (5) (6) (7) (dpm) Tobacco 3 0É3 45É1 6É4 5É1 2É5 1É2 0É8 38É5 174 200 6 0É2 27É9 9É0 5É9 3É3 1É7 1É0 51É0 157 400 12 0É3 23É4 9É6 6É0 3É4 1É7 1É6 53É9 140 300 24 0É1 40É0 4É8 3É0 2É0 2É0 0É8 47É2 220 700 48 0É2 29É2 7É0 5É6 2É6 1É4 1É3 52É6 181 100 Rice 3 0É2 26É8 20É7 7É4 2É7 1É0 1É5 39É6 155 500 6 0É2 30É4 18É0 4É5 2É3 0É9 0É8 42É9 179 600 12 0É2 25É4 14É1 4É2 2É4 1É1 1É3 51É2 173 600 24 0É4 20É4 10É5 4É2 3É2 1É1 1É7 58É4 148 200 48 0É2 15É0 10É4 7É9 3É6 1É2 1É6 60É1 184 700
a The number corresponds to the fractions in Fig. 1.
lysis, 14C-compounds in the ether extracts showed no signiÐcant di†erent between tobacco and rice cell, the sum of residues in the acidic and basic ether extracts totalling 1É7 to 3É3% of total radioactivity found in the cell. These results imply that only small amounts of [14C]molinate were conjugated as glycosides or other complexes.
When the 70% acetone extract was separated by thin layer chromatography, in addition to the parent molin-ate, eight (in tobacco) and 10 (in rice) spots were
observed on chromatographic plates. Most of these metabolites were found in cells of both species, such as molinate sulfone,(R 0É83), molinate sulfoxide, 0É55)
f (Rf
and several unknown compounds with R values 0É86, f
0É79, 0É72, 0É68 and at the origin. However, tobacco cells also formed unknown compounds withR values
f 0É76 and 0É03, and in rice cells, additional metabolites were found at R 0É95, 0É37 and 0É18. There are
there-f
fore signiÐcant di†erences in the metabolites produced by rice and tobacco cells. The aqueous layers (after acid
TABLE 4
Change in Amounts of [14C]Molinate and Its Metabolites in Tobacco and Rice Cells and in Culture Medium with Incu-bation Time
Amount found (%)
T obaccoa Ricea
In culture
Incubation Metabolitesc Metabolitesc mediumb
time
(h) Molinate MSO MSO
2 Others Molinate MSO MSO2 Others T obacco Rice
3 23É0 1É6 3É0 72É4 18É6 2É3 3É8 75É3 81É8 83É9
6 11É8 1É5 3É0 83É7 14É0 1É8 3É5 80É7 85É7 83É0
12 10É8 2É0 3É0 84É2 9É3 2É0 3É4 85É3 86É5 83É2
24 9É7 1É3 1É7 87É3 10É3 2É6 2É8 84É3 81É0 85É9
48 6É8 1É4 2É8 89É0 6É5 1É3 2É0 90É2 84É2 85É9
a % of total radioactivity in cells. b % of total radioactivity applied.
c MSO : molinate sulfoxide ;MSO molinate sulfone. 2:
analysis, fraction 7 in Fig. 1) contained only two spots, an unknown compound atR 0É72 and activity retained
f at the origin.
The amounts of molinate and the major metabolites, the sulfoxide and the sulfone of molinate, in cells of both species were determined (Table 4). The percentages of the radioactivity for the other metabolites in the cell and in the cell-culture Ñuid were determined separately and are shown in Table 4. Several papers15h17 have reported that thiocarbamate herbicides must be metab-olized in the plant to thiocarbamate sulfoxides to exhibit herbicidal activity, but this is disputed by Jablonkai and Hatzios.18 Imai and Kuwatsuka2 have proposed a metabolic pathway for molinate degrada-tion in plants. They reported that molinate initially formed molinate sulfoxide, and this was then conju-gated with glutathione or oxidized to molinate sulfone. In this study the di†erences in metabolism of molinate between rice and tobacco are minimal and do not appear to be a signiÐcant factor in selectivity. However, studies on metabolism in whole plants are required to clarify the role of metabolism further.
ACKNOWLEDGEMENTS
The authors wish to express their thanks to Asahi Chemical Industry Co., Ltd, Japan, for providing 14C-labelled molinate. This research was supported by the Council of Agriculture of the Republic of China. Grant No. : 84-Science-2.4-Food-37(1)-01.
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