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The propanil hydrolyzing enzyme aryl acylamidase in the wild rices of genus Oryza
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
The Propanil Hydrolyzing
38, 26-33 (1990)
Enzyme Aryl Acylamidase of Genus Oryza
CHENJIANJUN Division of Environmental
in the Wild Rices
AND SHOOICHI MATSUNAKA
Science, The Graduate School of Science and Technology, Kobe University, Rokkodai, Nada-ku, Kobe 657, Japan Received February 20, 1990; accepted May 24, 1990
The propanil hydrolyzing enzyme aryl acylamidase I (aryl acylamine amidohydrolase, EC 3.5.1.13) in the wild rices of genus Oryza was investigated from both the quantitative and qualitative aspects. An accurate distribution level of this enzyme in genus Oryza was established. Ten species with genomes AA, BB, CC, and BBCC showed comparable enzyme activity with that of 0. sativa. As an exception, 0. collina with the genome CC showed a low enzyme activity in strong contrast to 0. oficinalis and 0. eichingeri with the same genome. Three species with the genome CCDD showed enzyme activity which was related to their foliar morphological characters. The wide leaf strains showed low, while the narrow leaf strains had high activity. Two species with genome EE and FF, respectively, had no enzyme activity. Six species with unidentified genome types showed no or only low enzyme activity. The herbicidal effect of propanil on the wild rices depends essentially on the enzyme activity. It was demonstrated that at least seven species in genus Oryza can be selectively controlled by propanil. Temperature was found to affect enzyme activity in the Oryza plants. Growth of some wild rices at 2O-25°C during summer increased activity up to 20 times of the control at 3O”C, while protein and chlorophyll contents decreased. Qualitative studies revealed significant differences among the species in the enzyme optimum temperature, relative activity against propanil analogues, and the Michaelis constant, indicating that the enzyme has been modified in some species of genus Oryza. The data also show that the enzyme will serve as a useful marker for the understanding of phylogenetic traits as well as propanil susceptibility of the Oryza plants. Q 1990 Academic Press, Inc.
chloroacetanilide (2,5-DCAA) and 2,3dichloroacetanilide, but not propanil. This enzyme is widely distributed in higher plants, including barnyard grass (Echinochola crusgalli L.), and does not seem to be an enzyme specific to plant species. AA111 hydrolyzes propanil as well as AA1 does; however, it exhibits significant differences in its molecular weight and substrate specificity in addition to different solubility compared to AAI. It was found in parsley (Petroselium sativum), honewort (Cryptotaenia japonica), and tulip (Tulipa gesneriana). So far, AA1 has been demonstrated as a key enzyme in the detoxification of the herbicide propanil in rice plants (14). It catalyzes propanil hydrolytically into 3,4dichloroaniline (DCA) and propionic acid, protecting rice plants from propanil phytotoxicity. Since most weeds have no AAI, or have such an enzyme but with significantly lower activity compared to that in culti-
INTRODUCTION
Akatsuka (1) has studied higher plant enzyme aryl acylamidases and classified them into three groups according to their properties. The first is the particle-bound type such as propanil (3’,4’-dichloropropionanilide) hydrolyzing enzyme in rice (Oryza sativa L.) which possesses broad substrate specificity characterized as aryl acylamidase I (AAI).’ The second and third are the soluble types. Depending upon their ability to hydrolyze propanil, they were characterized as aryl acylamidase II (AAII) or aryl acylamidase III (AAIII). AA11 can hydrolyze some anilide compounds, e.g., 2,5di’ Abbreviations
used: AAI, aryl acylamidase I; 2,5DCA, 3,4-dichloroaniline; 2,3-DCPA, 2’,3’-dichloropropionanilide; TCA, trichloroacetic acid; p-DACA, p-dimethylaminocinnamaldehyde; MLC, minimal lethal concentration.
DCAA, 2,5-dichloroacetanilide;
26 0048-3575190 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
ARYL
ACYLAMIDASE
vated rice plants (1,2,5), the herbicide propanil, when used in the rice fields, exhibits a remarkable selectivity between cultivated rice and weeds. Even barnyard grass, which can not easily be distinguished visually from rice in the early stages of growth, is readily killed by propanil (6). The fact that AA1 with high activity appears only in rice plants and that its physiological role is unknown lead us to focus our attention on wild rices which often appear in cultivated rice fields and are closely related to 0. sativa in view of their systematics. It is still unclear whether they possess the enzyme AA1 and whether they can-and in this case to what extent-be selectively controlled by propanil. Studies on this enzyme in wild rices of genus Oryza are not only very important for wild rice control by propanil but also of great interest regarding the systematics and phylogenetics of the Oryza plants. Therefore the present investigation was undertaken to characterize AA1 in the wild rices of genus Oryza from both the quantitative and qualitative aspects in order to determine its distribution and level in this genus. The experimental results are expected to play an important role in wild rice control by propanil and provide some evidence for making systematic and phylogenetic inferences in genus Oryza. MATERIALS
AND
METHODS
Plant Materials
One hundred and two strains of wild rice covering 22 species in genus Oryza were supplied by H. Morishima of the National Institute of Genetics in Mishima, Japan, and planted individually in plastic pots (l/ 5000~) under greenhouse conditions. Balanced fertilizers were supplied periodically to keep them growing in the best nutritive state. Old leaves were cut off monthly. Chemicals
Propanil and DCA with a purity of 99% were obtained commercially from Wako
IN
27
Oryza
Pure Chemical Industry Co., Ltd. Propanil emulsion with 35% a.i. was purchased from Nissan Chemical Industry Co., Ltd. Propanil analogues 2’,3’-dichloropropionanilide (2,3-DCPA) and 2,5-DCAA with a purity of 96% were synthesized by the authors in their laboratory. Preparation
of Enzyme
Solutions
For the quantitative studies, 0.5 g of newly expanded leaves from each plant was ground with a pestle in a chilled mortar containing 0.2 g of quartz sand and 7.0 ml of 13 mM phosphate buffer (pH 7.0). The macerate was first squeezed through four layers of gauze and then centrifuged at IOOOg for 10 min to remove the remaining debris. The supernatant served as a crude enzyme solution for the enzyme assay (Tables 1 and 2 and Fig. 1). For the qualitative studies, fresh leaves of each species were sampled, washed, and homogenized in a high-speed blender (10 min at 15,000 rpm) with 13 n&f phosphate buffer (pH 7.0) containing 1% (w/w) sodium L-ascorbate and 20% (w/w) polyvinylpyrolidone. The homogenate was first squeezed through four layers of gauze and then centrifuged at 1OOOgfor 10 min. The supernatant was further centrifuged at 20,OOOg for I hr. After the assay of the enzyme activity in both supernatant and precipitate, the supernatant was discarded and the precipitate was treated with 75% prechilled acetone (-20°C) to remove the chlorophyll. The chlorophyll-free enzyme precipitate was collected by centrifugation at 10,OOOg for 20 min at 0°C. This pellet was washed twice with 0.05 M phosphate buffer (pH 7.0) and resuspended in the same buffer containing 0.1% (w/w) Triton X-100 for enzyme solubilization. The suspension was stirred on a magnetic device for 18 hr and centrifuged at 20,OOOg for 1 hr. The supematant, in which the enzyme specific activity was 20 to 40 times of that in the homogenates, was employed in the enzyme qualitative studies. Unless stated otherwise, all the operations were carried out at between 0 and 4°C.
28
CHEN
AND
Enzyme Assay
The standard enzyme assay consisted of 1 ml of the enzyme solution and 1 ml of 0.4 mkf propanil containing 1% ethanol. This reaction mixture was incubated at 37°C for 30 min. The reaction was terminated by the addition of 2 ml of 10% trichloroacetic acid (TCA). Controls were prepared by adding TCA to the enzyme solution prior to the addition of propanil. The mixture was centrifuged, and the supernatant was used for the DCA determination by combining it with a 0.1% p-dimethylaminocinnamaldehyde (p-DACA):ethanol solution (1:2, v/v). After the mixture stood for 15 min, the optical density was measured at 540 nm with a Hitachi spectrophotometer (U-3200) and converted into a DCA concentration. The enzyme activity was defined as production of micromoles DCA per 30 min per milligram protein. Since p-DACA is unsuitable for detecting 2,3-DCA and 2,5-DCA, the method of Goto and Sato (7) was then employed in the studies of relative enzyme activity against propanil analogues. The experiments were conducted with three replicates. Determination Chlorophyll
of Protein and Concentration
The protein concentration was determined as described by Bradford (8) using crystalline bovine serum albumin as standard. The chlorophyll concentration was determined as described by Arnon (9). Determination of Optimum and Temperature
pH
Five types of phosphate buffers (pH 5.5, 6, 7, 8, and 8.5) were employed to determine the enzyme optimum pH. The complete reaction mixture contained 250 p,mol buffer, 0.4 pmol propanil, 150 p.g Triton X100-soluble enzyme, and distilled water to a total volume of 2.0 ml. The reaction was incubated at 37°C for 1 hr and terminated by adding TCA. To determine the enzyme optimum temperature, the reaction mixture,
MATSUNAKA
which consisted of 250 p,mol buffer, pH 7.0, 0.4 prnol propanil, 150 pg Triton X100-soluble enzyme, and distilled water to a total volume of 2.0 ml was used. The reaction was incubated at 20, 30,40,50,60, and 70°C for 1 hr and terminated by adding TCA. Control preparation and DCA determination were conducted as described in the method for the enzyme assay. Evaluation of Propunit Wild Rices
Effect on
Since most of the wild rices in genus type, having a high regenerating ability of excised stem segments, but a low ability of seed-setting and seed propagation (10, ll), the plant synchronization was conducted through multitransplanting. Juvenile plants were divided equally into several parts and transplanted separately in plastic pots. About 1 month after transplanting, the most synchronized plants were chosen and sprayed with propanil emulsions of different concentrations. After 10 days, the shoots of each plant were cut and their dry weights recorded. The propanil concentration which caused 50% dry-weight reduction of control was defined as the minimal lethal concentration (MLC) of propanil in that plant. Oryza are of the perennial
RESULTS
Quantitative
Studies of the Enzyme
The distribution of aryl acylamidase in genus Oryza is shown in Table 1. The enzyme activity in each species is indicated in percentage compared to that of 0. sativa. As shown in the table, out of 22 tested species, 18 had enzyme activity, and only 4 had no enzyme activity. Most of the species in section Oryzae showed strong or intermediate AA1 activity, while no species in section Angustifoliae showed such activity, and species in the other sections showed considerably low activity. The AA1 activity in wild rices also differs significantly with regard to the genomes. 0.
ARYL ACYLAMIDASE TABLE Distribution
of the Propanil
Section species
Hydrolyzing
Enzyme
29
IN Oryza 1
Aryl
Acylamidase
Genome
Enzyme activity
AA AA AA AA AA EE cc cc cc BB,BBCC BBCC BBCC CCDD CCDD CCDD CCDD CCDD
100 (94-107) 85 (69-98) 128 (105-143) 120 (106-138) 117 (114-132) No activity 90 (84-100) 105 (68-150) 31 (21-40) 75 (68-95) 73 (65-80) 118 (106130) 40 (37-45) 102 (g&120) 43 (37-46) 98 (75-l 12) 45 (28-58)
in Rice Plants
of
(%)
Genus
Oryza
No. of tested strains
Ovzae sativa rufipogon longistaminata glaberrima breviligulata australiensis eichingeri officinalis collina punctata maiabarensis minuta alta (WL) alta (NL) latifolia (WL) latifolia (NL) grandiglumis Granulatae meyeriana Ridleyanae ridkyi longiglumis Angustifoliae brachyantha perrieri tisseranti Coarctutae coarctata ---
(WL)
?? 1777 . ????
FF ?? ??
????
+++ +++ +++ +++ +++ +++ +++ + ++ ++ +++ + +++ + +++ +
21 (12-30)
+
27 (13-32) 22 (13-28)
+ +
No activity No activity No activity
-
8 (5-13)
Trace
15 14 8 2 2 2 4 10 1 9 3 3 1 2 6 5 4
Nore. The enzyme assay was carried out three times with a interval of more than 4 weeks; the mean value was recorded. Data in parentheses indicate the minimal and maximal values. WL, broad leaf; NL, narrow leaf. + + + . high (more than 80); + f , intermediate (S&80); + , low (less than 50); - , no activity.
sativa, 0. rufipogon, 0. longistaminata, 9. glaberrima, and 0. breviligulata with ge-
nome AA showed strong AAI activity.
0.
punctata, 0. malabarensis, and 0. minuta with genomes BB or BBCC; 0. offkinalis and 0. eichingeri with genome CC also
showed comparable AAI activity to that of 0. sativa. As an exception, 0. collina with genome CC showed low AA1 activity in strong contrast to the other two species with the same genome. 0. alta, 0. latifolia, and 0. grandiglumis showed AAI activity which was related to their foliar morphological characters. The strains with wide leaves (>2 cm) always showed low activity,
while the ones with narrow leaves (<2 cm) showed an activity as high as in 0. sativa (leaf width, <2 cm). On the other hand, 0. australiensis and 0. brachyantha with genomes EE and FF, respectively, had no AA1 activity. The other seven species with unidentified genomes had no or only very low AA1 activity. In addition, it was found that temperature affects the activity of the propanil hydrolyzing enzyme in the Oryza plants. This is most striking in one strain of 0. ridleyi which exhibits a seasonal change of the enzyme activity (12). As depicted in Fig. 1,
CHENANDMATSUNAKA
30
ent rates onto wild rices, it was demonstrated that the propanil effect on wild rices in genus Oryza depends essentially on the AA1 activity. Figure 2 shows typical patterns of dry-weight reduction of the wild rices 10 days after propanil application. As depicted in the figure, the tendency of dryweight reduction caused by propanil reflects well the significant difference of AA1 activity among the five species. Table 2 reveals the enzyme activity and the MLC of propanil on 15 species of wild rices in genus Oryza. From the table, it becomes very clear that the stronger the AA1 activity, the higher the MLC required. There is a high correlation between AA1 activity and MLC (Y = 0.95). Four species (0. australiensis, 0. brachyantha, 0. perrieri, and 0. tisseranti), which had no AA1 activity, were readily killed by propanil as ordinary weeds with a practical application rate (0.35%). Since 0. sativa is resistant to propanil up to 1.75%, it is evident that at least seven species in genus Oryza, which had no or only considerably low activity, can be selectively controlled by propanil. Qualitative Studies of the Enzyme 0 I--J”1
10 20 30 40 50 60 70 Days after incubation .____ I_____ Aug
._____ I____ Sq,.-l
FIG. 1. Effect of environmental temperatures during growth period on the activity (a), specific activity (b) of the propanil hydrolyzing enzyme aryl acylamidase, and chlorophyll content (c) in 0. ridleyi. An enzyme unit is defined here as the amount of enzyme required to produce I pmol DCA per minute under the assay condition used. Specific activity is dejkted as the number of enzyme units per milligram protein. 20°C (A); 25°C (0); 30°C (a) (control).
the incubation of this strain at lower temperatures (20-2X) for 60 days during summer increased the activity up to 20 times of the control at 30°C while the protein and chlorophyll contents decreased to some extent. Phytotoxicity
of Propanil on Wild Rices
By spraying propanil
emulsion
at differ-
The properties of the propanil hydrolyzing enzyme aryl acylmidase in the genus Oryza are summarized in Table 3. Centrifugation at 20,OOOg for 1 hr of the homoge-
0 0.25 0.5 0.75 1.0 1.25 1.5 Propanil
concentration
1.75
(%)
FIG. 2. Effect of propanil on the dry weight of wild rices in genus Oryza. Transplanted (I month) plants were sprayed with proanil and harvested IO days after application. 0. sativa (0); 0. punctata (0); 0. collina (A); 0. ridleyi (A); 0. australiensis (W).
ARYL
ACYLAMIDASE
TABLE 2 The Propanil Hydrolyzing Enzyme Aryl Acylamidase Activity and the Minimal Lethal Concentration (MLC) of Propanil in the Wild Rices of Genus Oryza
Species 0. 0. 0. 0. 0. 0.
sutiva officinalis punctata collina grandiglumis latifolia Narrow leaf
AA cc BBCC cc CCDD CCDD
Enzyme activity m
MLC r%l
100 110 78 31 43
1.75 1.75 1.5 1 .o 1.25
31
IN Oryza
wild rice species of genus Oryza. When compared to propanil as lOO%, the relative hydrolysis of 2,5-DCAA and 2,3-DCPA was significantly different among the species, demonstrating a qualitative diversity of this enzyme in the wild rices. DISCUSSION
The present studies investigated the propanil hydrolyzing enzyme aryl acylamidase in all the species of genus Oryza (13) except 1.75 97 1.0 0. schlechteri and 0. augustifolia. The Wide leaf 38 0. alto CCDD quantitative studies established accurate Wide leaf 40 1.0 1711 0. longiglumis 25 0.75 enzyme distribution in this genus. Concem3313 0.5 0. ridieyi 29 ing the species which showed low enzyme ?1?9 0.2 0. coarctaara 11 0. nusrraliensis EE No activity 0.05 activity, it was suspected that more enzyme 0. brochyanthn FF No activity 0.3 inhibitors were present in their crude en0. tisseranti ?? No activity 0.3 ?? 0.25 zyme solutions or that higher rates of DCA 0. per&vi No activity conjugation by glucose affected measurable Note. Y = 0.284 + 0.015x. r = 0.95 (n = 15). The enzyme enzyme activity. Thus a comparative examactwity was assayed immediately before the prbpanil appiication. MLC was determined as described under Materials and ination of the AA1 inhibitors and conjuMethods gated DCA in the crude enzyme solution was conducted during the assay. However, there was no significant difference of these nate resulted in more than 80% of the total enzyme activity in the precipitate. No ex- two factors among the species (data not ceptions were found in the precipitation shown), indicating that their low enzyme pattern among the tested species. It is evi- activity is probably controlled inherently. dent that this enzyme in the wild rices ap- This assumption was confirmed by the propears to be a particle or membrane-bound panil effects in vivo on the wild rices. As enzyme, as characterized by Frear and Still shown in Fig. 2 and Table 2, the dry-weight (4) and Akatsuka (1) for the enzyme iso- reduction of wild rices following propanil lated from cultivated rice plants. In addi- application depends essentially on their tion, it was also found that the enzyme pH AA1 activity. The species which had low optimum at 7.0 was true for all the tested AA1 activity were seriously affected by species. However, regarding the enzyme propanil. There is a high correlation beoptimum temperature, a significant differtween the AA1 activity and MLC (Y = ence was found among the species. Most 0.95). The minor variation might have resulted from differences in the surface strucspecies showed the enzyme optimum temperature at 5O”C, while in two species the ture of the wild rices which possibly affects value was 40°C. This difference was even the propanil uptake. However, it is of interreflected in the same species of 0. ridleyi, est to note the difference of MLC between exhibiting a intraspecific variation. Kinetic 0. australiensis and the other three species studies determined the Michaelis constant which had no AA1 activity. Although no (K,) of the enzyme. Except for 0. coarcenzyme activity was detected in any of the tata, there is only a minor difference among four species, there might be a great differthe species in the K,,, values which are ence concerning the cause. Considering the similar to that of AA1 in cultivated rice origin of 0. austruliensis (Australia), the disjunction of the continents in the early plants (1). Figure 3 illustrates the relative enzyme Cretaceous period, which has played a maactivity against propanii analogues in 13 jor role in the differentiation of rice species
32
CHEN AND MATSUNAKA TABLE 3 Properties of the Propanil Hydrolyzing Enzyme Aryl Acylamidase in the Wild Rices of Genus Otyza Section Species
Oryzae sativa rufpogon longistaminata officinalis collina punctata alta latifolia grandiglumis Ridleyanae longiglumis ridleyi ridleyi Coarctatae coarctata
Genome
Particlebound
PH optimum
Optimum temp. (“C)
AA AA AA cc cc BBCC CCDD CCDD CCDD
Yes Yes Yes Yes Yes Yes Yes Yes Yes
7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0
50 50 50 50 50 50 50 50 50
3.5 4.0 4.1 5.0 5.7 4.9 5.1 5.0 5.6
7777 . ... ???? ????
Yes Yes Yes
7.0 7.0 7.0
50 50 40
7.1 6.3 8.0
????
Yes
7.0
40
25.0
(14), probably resulted in the absence of AA1 in this species. On the other hand, in the other three species, it is more likely that the aryl acylamidase activity was just led to a undetectable level by some factors such as genetic mutation during the long evolutionary process. Clearly, further investiga-
10 Relative
20
enzyme activity
30
40
50
against 2,5-DCAA
60 (%)
FIG. 3. Twenty-nine wild rice strains of 13 species in genus Oryza scattered by the relative enzyme activity against propanil analogues. The relative activity was defined as enzyme-hydrolyzed propanil analogues compared to propanil as 100%. Data represent the mean value of three replicates.
-
Michaelis constant (lo-4 M)
tions are needed to confirm this hypothesis. In addition to the quantitative difference of the propanil-hydrolyzing enzyme aryl acylamidase among the wild rice species, we also demonstrated qualitative diversity of this enzyme in genus Oryza. The variation in the enzyme optimum temperature, the relative enzyme activity, and the Km values indicate that AA1 probably has been modified in some species of the genus Oryza. Our previous studies (12, 15) indicated the possible role of AA1 for determining the interspecific relationship in Oryza. This is further confirmed by the present investigation. As depicted in Fig. 3, the allocation of 13 species according to their relative enzyme activity against 2,5-DCAA and 2,3-DCPA reflects well their genetic variation (16, 17) during the evolutionary process. By taking advantage of its quantitative and qualitative diversities, AA1 will serve as a good marker for making systematic and phylogenetic inferences in genus Oryza. Our detailed investigation of this enzyme in wild rices will provide more information at the molecular level in a future publication. In contrast to aryl acylamidase of other
ARYL
ACYLAMIDASE
plants (18, 19), the rice plant enzyme appears to be associated with particulate cell constituents, representing a particle-bound enzyme. Hirase and Matsunaka (20) studied its physiological role by comparing cultivated rice plants with a propanilsusceptible mutant rice, suggesting that AA1 plays an important role in the nitrogen metabolism of rice plants. Wild rices provide us suitable material to study the physiological aspects of this enzyme. ACKNOWLEDGMENT
The authors thank Professor H. Morishima of the National Institute of Genetics in Mishima, Japan, for generously providing us with the wild rices. REFERENCES
1. T. Akatsuka, Purification of aryl acylamidase I, II, III from higher plants and selectivity of propanil, Weed Res. (Japan) 24, 55 (1979). [In Japanese] 2. C. C. Still and 0. Kuzirian, Enzyme detoxification of 3’,4’-dichloropropionanilide in rice and barnyardgrass, a factor in herbicide selectivity, Nature (London) 216, 799 (1967). 3. R. Y. Yih, D. H. McRae, and H. F. Wilson, Mechanism of selective action of 3’,4’-dichloropropionanilide, Phnt Physiol. 43, 1291 (1968). 4. D. S. Frear and G. G. Still, The metabolism of 3’,4’-dichloropropionanilide in rice plants. Partial purification and properties of an aryl acylamidase from rice, Phytochemistry 72, 913 (1968). 5. R. E. Hoagland and G. Graf, Enzymatic hydrolysis of herbicides in plants, Weed Sci. 20, 303 (1972). 6. R. J. Smith, 3’,4’-Dichloropropionanilide for control of barnyardgrass in rice, Weeds 9, 318 (1961). 7. M. Goto and R. Sato, Microanalysis of propanil and MCPCA. Pestic. Tech. 10, 16 (1964). [In Japanese]
IN
Oryza
33
8. M. M. Bradford, Rapid and sensitive method for the quantification of proteins using the principle of protein-dye binding. Anal. Biochem. 72, 248 (1976). 9. D. I. Amon, Copper enzyme in isolated chloroplasts. Plant Physiol. 24, 1 (1949). 10. H. Morishima, Y. Sano, and H. I. Oka, Differentiation of perennial and annual types due to habitat condition in the wild rice Oryza perennis. Plant Syst. Evol. 144, 119 (1984). Il. H. Morishima, Wild progenitors of cultivated rice and their population dynamics, in “Rice Genetics,” pp. 3-14. IRRI, Manila, 1986. 12. J. J. Chen and S. Matsunaka, Distribution of propanil hydrolyzing enzyme aryl acylamidase 1 in genus Oryza, Proc. Conf. Asian-Pacific Weed Sci. Sot., 12th 2, 479 (1989). 13. H. Morishima, Species relationships and the search for ancestors, in “Biology of Rice” (S. Tsunoda and N. Takahashi, Eds.), pp. 3-30, Japan Sci. Sot. Press, Tokyo/Elsevier. Amsterdam, 1984. 14. T. T. Chang, Crop history and genetic conservation: Rice-A case study, Iowa State .I. Res. 59, 425-455, 4w95 (1985). 15. S. Matsunaka and Y. Aoyama, Distribution of propanil hydrolyzing enzyme (rice aryl acylamidase I) in Oryza genus, Proc. Conf. AsianPacific Weed Sci. Sot., 8th 352 (1981). 16. S. Sampath, The Genus Oryza: Its taxonomy and species interrelationships, Oryza 1, 1 (1962). 17. T. Katayama and W. Onizuka, Intersectional F, plants from Oryza sativa x 0. ridleyi and 0. sativa X 0. meyeriana, Jpn. J. Genet. 54(l). 43 (1979). 18. R. E. Hoagland and G. Graf, An aryl acylamidase from tulip which hydrolyzes 3’,4’-dichloropropionanilide, Phytochemistry 11, 521 (1972). 19. R. E. Hoagland, Reversal of EPTC inhibition of aryl acylamidase activity by oximes, Pestir,. Biochem. Physiol. 34, 69 (1989). 20. K. Hirase and S. Matsunaka, Nitrogen Metabolism in a propanil susceptible rice mutant, Proc. Conf. Asian-Pacific Weed Sci. Sot., 12th 2,405 (1989).
BIOCHEMISTRY
AND
PHYSIOLOGY
The Propanil Hydrolyzing
38, 26-33 (1990)
Enzyme Aryl Acylamidase of Genus Oryza
CHENJIANJUN Division of Environmental
in the Wild Rices
AND SHOOICHI MATSUNAKA
Science, The Graduate School of Science and Technology, Kobe University, Rokkodai, Nada-ku, Kobe 657, Japan Received February 20, 1990; accepted May 24, 1990
The propanil hydrolyzing enzyme aryl acylamidase I (aryl acylamine amidohydrolase, EC 3.5.1.13) in the wild rices of genus Oryza was investigated from both the quantitative and qualitative aspects. An accurate distribution level of this enzyme in genus Oryza was established. Ten species with genomes AA, BB, CC, and BBCC showed comparable enzyme activity with that of 0. sativa. As an exception, 0. collina with the genome CC showed a low enzyme activity in strong contrast to 0. oficinalis and 0. eichingeri with the same genome. Three species with the genome CCDD showed enzyme activity which was related to their foliar morphological characters. The wide leaf strains showed low, while the narrow leaf strains had high activity. Two species with genome EE and FF, respectively, had no enzyme activity. Six species with unidentified genome types showed no or only low enzyme activity. The herbicidal effect of propanil on the wild rices depends essentially on the enzyme activity. It was demonstrated that at least seven species in genus Oryza can be selectively controlled by propanil. Temperature was found to affect enzyme activity in the Oryza plants. Growth of some wild rices at 2O-25°C during summer increased activity up to 20 times of the control at 3O”C, while protein and chlorophyll contents decreased. Qualitative studies revealed significant differences among the species in the enzyme optimum temperature, relative activity against propanil analogues, and the Michaelis constant, indicating that the enzyme has been modified in some species of genus Oryza. The data also show that the enzyme will serve as a useful marker for the understanding of phylogenetic traits as well as propanil susceptibility of the Oryza plants. Q 1990 Academic Press, Inc.
chloroacetanilide (2,5-DCAA) and 2,3dichloroacetanilide, but not propanil. This enzyme is widely distributed in higher plants, including barnyard grass (Echinochola crusgalli L.), and does not seem to be an enzyme specific to plant species. AA111 hydrolyzes propanil as well as AA1 does; however, it exhibits significant differences in its molecular weight and substrate specificity in addition to different solubility compared to AAI. It was found in parsley (Petroselium sativum), honewort (Cryptotaenia japonica), and tulip (Tulipa gesneriana). So far, AA1 has been demonstrated as a key enzyme in the detoxification of the herbicide propanil in rice plants (14). It catalyzes propanil hydrolytically into 3,4dichloroaniline (DCA) and propionic acid, protecting rice plants from propanil phytotoxicity. Since most weeds have no AAI, or have such an enzyme but with significantly lower activity compared to that in culti-
INTRODUCTION
Akatsuka (1) has studied higher plant enzyme aryl acylamidases and classified them into three groups according to their properties. The first is the particle-bound type such as propanil (3’,4’-dichloropropionanilide) hydrolyzing enzyme in rice (Oryza sativa L.) which possesses broad substrate specificity characterized as aryl acylamidase I (AAI).’ The second and third are the soluble types. Depending upon their ability to hydrolyze propanil, they were characterized as aryl acylamidase II (AAII) or aryl acylamidase III (AAIII). AA11 can hydrolyze some anilide compounds, e.g., 2,5di’ Abbreviations
used: AAI, aryl acylamidase I; 2,5DCA, 3,4-dichloroaniline; 2,3-DCPA, 2’,3’-dichloropropionanilide; TCA, trichloroacetic acid; p-DACA, p-dimethylaminocinnamaldehyde; MLC, minimal lethal concentration.
DCAA, 2,5-dichloroacetanilide;
26 0048-3575190 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
ARYL
ACYLAMIDASE
vated rice plants (1,2,5), the herbicide propanil, when used in the rice fields, exhibits a remarkable selectivity between cultivated rice and weeds. Even barnyard grass, which can not easily be distinguished visually from rice in the early stages of growth, is readily killed by propanil (6). The fact that AA1 with high activity appears only in rice plants and that its physiological role is unknown lead us to focus our attention on wild rices which often appear in cultivated rice fields and are closely related to 0. sativa in view of their systematics. It is still unclear whether they possess the enzyme AA1 and whether they can-and in this case to what extent-be selectively controlled by propanil. Studies on this enzyme in wild rices of genus Oryza are not only very important for wild rice control by propanil but also of great interest regarding the systematics and phylogenetics of the Oryza plants. Therefore the present investigation was undertaken to characterize AA1 in the wild rices of genus Oryza from both the quantitative and qualitative aspects in order to determine its distribution and level in this genus. The experimental results are expected to play an important role in wild rice control by propanil and provide some evidence for making systematic and phylogenetic inferences in genus Oryza. MATERIALS
AND
METHODS
Plant Materials
One hundred and two strains of wild rice covering 22 species in genus Oryza were supplied by H. Morishima of the National Institute of Genetics in Mishima, Japan, and planted individually in plastic pots (l/ 5000~) under greenhouse conditions. Balanced fertilizers were supplied periodically to keep them growing in the best nutritive state. Old leaves were cut off monthly. Chemicals
Propanil and DCA with a purity of 99% were obtained commercially from Wako
IN
27
Oryza
Pure Chemical Industry Co., Ltd. Propanil emulsion with 35% a.i. was purchased from Nissan Chemical Industry Co., Ltd. Propanil analogues 2’,3’-dichloropropionanilide (2,3-DCPA) and 2,5-DCAA with a purity of 96% were synthesized by the authors in their laboratory. Preparation
of Enzyme
Solutions
For the quantitative studies, 0.5 g of newly expanded leaves from each plant was ground with a pestle in a chilled mortar containing 0.2 g of quartz sand and 7.0 ml of 13 mM phosphate buffer (pH 7.0). The macerate was first squeezed through four layers of gauze and then centrifuged at IOOOg for 10 min to remove the remaining debris. The supernatant served as a crude enzyme solution for the enzyme assay (Tables 1 and 2 and Fig. 1). For the qualitative studies, fresh leaves of each species were sampled, washed, and homogenized in a high-speed blender (10 min at 15,000 rpm) with 13 n&f phosphate buffer (pH 7.0) containing 1% (w/w) sodium L-ascorbate and 20% (w/w) polyvinylpyrolidone. The homogenate was first squeezed through four layers of gauze and then centrifuged at 1OOOgfor 10 min. The supernatant was further centrifuged at 20,OOOg for I hr. After the assay of the enzyme activity in both supernatant and precipitate, the supernatant was discarded and the precipitate was treated with 75% prechilled acetone (-20°C) to remove the chlorophyll. The chlorophyll-free enzyme precipitate was collected by centrifugation at 10,OOOg for 20 min at 0°C. This pellet was washed twice with 0.05 M phosphate buffer (pH 7.0) and resuspended in the same buffer containing 0.1% (w/w) Triton X-100 for enzyme solubilization. The suspension was stirred on a magnetic device for 18 hr and centrifuged at 20,OOOg for 1 hr. The supematant, in which the enzyme specific activity was 20 to 40 times of that in the homogenates, was employed in the enzyme qualitative studies. Unless stated otherwise, all the operations were carried out at between 0 and 4°C.
28
CHEN
AND
Enzyme Assay
The standard enzyme assay consisted of 1 ml of the enzyme solution and 1 ml of 0.4 mkf propanil containing 1% ethanol. This reaction mixture was incubated at 37°C for 30 min. The reaction was terminated by the addition of 2 ml of 10% trichloroacetic acid (TCA). Controls were prepared by adding TCA to the enzyme solution prior to the addition of propanil. The mixture was centrifuged, and the supernatant was used for the DCA determination by combining it with a 0.1% p-dimethylaminocinnamaldehyde (p-DACA):ethanol solution (1:2, v/v). After the mixture stood for 15 min, the optical density was measured at 540 nm with a Hitachi spectrophotometer (U-3200) and converted into a DCA concentration. The enzyme activity was defined as production of micromoles DCA per 30 min per milligram protein. Since p-DACA is unsuitable for detecting 2,3-DCA and 2,5-DCA, the method of Goto and Sato (7) was then employed in the studies of relative enzyme activity against propanil analogues. The experiments were conducted with three replicates. Determination Chlorophyll
of Protein and Concentration
The protein concentration was determined as described by Bradford (8) using crystalline bovine serum albumin as standard. The chlorophyll concentration was determined as described by Arnon (9). Determination of Optimum and Temperature
pH
Five types of phosphate buffers (pH 5.5, 6, 7, 8, and 8.5) were employed to determine the enzyme optimum pH. The complete reaction mixture contained 250 p,mol buffer, 0.4 pmol propanil, 150 p.g Triton X100-soluble enzyme, and distilled water to a total volume of 2.0 ml. The reaction was incubated at 37°C for 1 hr and terminated by adding TCA. To determine the enzyme optimum temperature, the reaction mixture,
MATSUNAKA
which consisted of 250 p,mol buffer, pH 7.0, 0.4 prnol propanil, 150 pg Triton X100-soluble enzyme, and distilled water to a total volume of 2.0 ml was used. The reaction was incubated at 20, 30,40,50,60, and 70°C for 1 hr and terminated by adding TCA. Control preparation and DCA determination were conducted as described in the method for the enzyme assay. Evaluation of Propunit Wild Rices
Effect on
Since most of the wild rices in genus type, having a high regenerating ability of excised stem segments, but a low ability of seed-setting and seed propagation (10, ll), the plant synchronization was conducted through multitransplanting. Juvenile plants were divided equally into several parts and transplanted separately in plastic pots. About 1 month after transplanting, the most synchronized plants were chosen and sprayed with propanil emulsions of different concentrations. After 10 days, the shoots of each plant were cut and their dry weights recorded. The propanil concentration which caused 50% dry-weight reduction of control was defined as the minimal lethal concentration (MLC) of propanil in that plant. Oryza are of the perennial
RESULTS
Quantitative
Studies of the Enzyme
The distribution of aryl acylamidase in genus Oryza is shown in Table 1. The enzyme activity in each species is indicated in percentage compared to that of 0. sativa. As shown in the table, out of 22 tested species, 18 had enzyme activity, and only 4 had no enzyme activity. Most of the species in section Oryzae showed strong or intermediate AA1 activity, while no species in section Angustifoliae showed such activity, and species in the other sections showed considerably low activity. The AA1 activity in wild rices also differs significantly with regard to the genomes. 0.
ARYL ACYLAMIDASE TABLE Distribution
of the Propanil
Section species
Hydrolyzing
Enzyme
29
IN Oryza 1
Aryl
Acylamidase
Genome
Enzyme activity
AA AA AA AA AA EE cc cc cc BB,BBCC BBCC BBCC CCDD CCDD CCDD CCDD CCDD
100 (94-107) 85 (69-98) 128 (105-143) 120 (106-138) 117 (114-132) No activity 90 (84-100) 105 (68-150) 31 (21-40) 75 (68-95) 73 (65-80) 118 (106130) 40 (37-45) 102 (g&120) 43 (37-46) 98 (75-l 12) 45 (28-58)
in Rice Plants
of
(%)
Genus
Oryza
No. of tested strains
Ovzae sativa rufipogon longistaminata glaberrima breviligulata australiensis eichingeri officinalis collina punctata maiabarensis minuta alta (WL) alta (NL) latifolia (WL) latifolia (NL) grandiglumis Granulatae meyeriana Ridleyanae ridkyi longiglumis Angustifoliae brachyantha perrieri tisseranti Coarctutae coarctata ---
(WL)
?? 1777 . ????
FF ?? ??
????
+++ +++ +++ +++ +++ +++ +++ + ++ ++ +++ + +++ + +++ +
21 (12-30)
+
27 (13-32) 22 (13-28)
+ +
No activity No activity No activity
-
8 (5-13)
Trace
15 14 8 2 2 2 4 10 1 9 3 3 1 2 6 5 4
Nore. The enzyme assay was carried out three times with a interval of more than 4 weeks; the mean value was recorded. Data in parentheses indicate the minimal and maximal values. WL, broad leaf; NL, narrow leaf. + + + . high (more than 80); + f , intermediate (S&80); + , low (less than 50); - , no activity.
sativa, 0. rufipogon, 0. longistaminata, 9. glaberrima, and 0. breviligulata with ge-
nome AA showed strong AAI activity.
0.
punctata, 0. malabarensis, and 0. minuta with genomes BB or BBCC; 0. offkinalis and 0. eichingeri with genome CC also
showed comparable AAI activity to that of 0. sativa. As an exception, 0. collina with genome CC showed low AA1 activity in strong contrast to the other two species with the same genome. 0. alta, 0. latifolia, and 0. grandiglumis showed AAI activity which was related to their foliar morphological characters. The strains with wide leaves (>2 cm) always showed low activity,
while the ones with narrow leaves (<2 cm) showed an activity as high as in 0. sativa (leaf width, <2 cm). On the other hand, 0. australiensis and 0. brachyantha with genomes EE and FF, respectively, had no AA1 activity. The other seven species with unidentified genomes had no or only very low AA1 activity. In addition, it was found that temperature affects the activity of the propanil hydrolyzing enzyme in the Oryza plants. This is most striking in one strain of 0. ridleyi which exhibits a seasonal change of the enzyme activity (12). As depicted in Fig. 1,
CHENANDMATSUNAKA
30
ent rates onto wild rices, it was demonstrated that the propanil effect on wild rices in genus Oryza depends essentially on the AA1 activity. Figure 2 shows typical patterns of dry-weight reduction of the wild rices 10 days after propanil application. As depicted in the figure, the tendency of dryweight reduction caused by propanil reflects well the significant difference of AA1 activity among the five species. Table 2 reveals the enzyme activity and the MLC of propanil on 15 species of wild rices in genus Oryza. From the table, it becomes very clear that the stronger the AA1 activity, the higher the MLC required. There is a high correlation between AA1 activity and MLC (Y = 0.95). Four species (0. australiensis, 0. brachyantha, 0. perrieri, and 0. tisseranti), which had no AA1 activity, were readily killed by propanil as ordinary weeds with a practical application rate (0.35%). Since 0. sativa is resistant to propanil up to 1.75%, it is evident that at least seven species in genus Oryza, which had no or only considerably low activity, can be selectively controlled by propanil. Qualitative Studies of the Enzyme 0 I--J”1
10 20 30 40 50 60 70 Days after incubation .____ I_____ Aug
._____ I____ Sq,.-l
FIG. 1. Effect of environmental temperatures during growth period on the activity (a), specific activity (b) of the propanil hydrolyzing enzyme aryl acylamidase, and chlorophyll content (c) in 0. ridleyi. An enzyme unit is defined here as the amount of enzyme required to produce I pmol DCA per minute under the assay condition used. Specific activity is dejkted as the number of enzyme units per milligram protein. 20°C (A); 25°C (0); 30°C (a) (control).
the incubation of this strain at lower temperatures (20-2X) for 60 days during summer increased the activity up to 20 times of the control at 30°C while the protein and chlorophyll contents decreased to some extent. Phytotoxicity
of Propanil on Wild Rices
By spraying propanil
emulsion
at differ-
The properties of the propanil hydrolyzing enzyme aryl acylmidase in the genus Oryza are summarized in Table 3. Centrifugation at 20,OOOg for 1 hr of the homoge-
0 0.25 0.5 0.75 1.0 1.25 1.5 Propanil
concentration
1.75
(%)
FIG. 2. Effect of propanil on the dry weight of wild rices in genus Oryza. Transplanted (I month) plants were sprayed with proanil and harvested IO days after application. 0. sativa (0); 0. punctata (0); 0. collina (A); 0. ridleyi (A); 0. australiensis (W).
ARYL
ACYLAMIDASE
TABLE 2 The Propanil Hydrolyzing Enzyme Aryl Acylamidase Activity and the Minimal Lethal Concentration (MLC) of Propanil in the Wild Rices of Genus Oryza
Species 0. 0. 0. 0. 0. 0.
sutiva officinalis punctata collina grandiglumis latifolia Narrow leaf
AA cc BBCC cc CCDD CCDD
Enzyme activity m
MLC r%l
100 110 78 31 43
1.75 1.75 1.5 1 .o 1.25
31
IN Oryza
wild rice species of genus Oryza. When compared to propanil as lOO%, the relative hydrolysis of 2,5-DCAA and 2,3-DCPA was significantly different among the species, demonstrating a qualitative diversity of this enzyme in the wild rices. DISCUSSION
The present studies investigated the propanil hydrolyzing enzyme aryl acylamidase in all the species of genus Oryza (13) except 1.75 97 1.0 0. schlechteri and 0. augustifolia. The Wide leaf 38 0. alto CCDD quantitative studies established accurate Wide leaf 40 1.0 1711 0. longiglumis 25 0.75 enzyme distribution in this genus. Concem3313 0.5 0. ridieyi 29 ing the species which showed low enzyme ?1?9 0.2 0. coarctaara 11 0. nusrraliensis EE No activity 0.05 activity, it was suspected that more enzyme 0. brochyanthn FF No activity 0.3 inhibitors were present in their crude en0. tisseranti ?? No activity 0.3 ?? 0.25 zyme solutions or that higher rates of DCA 0. per&vi No activity conjugation by glucose affected measurable Note. Y = 0.284 + 0.015x. r = 0.95 (n = 15). The enzyme enzyme activity. Thus a comparative examactwity was assayed immediately before the prbpanil appiication. MLC was determined as described under Materials and ination of the AA1 inhibitors and conjuMethods gated DCA in the crude enzyme solution was conducted during the assay. However, there was no significant difference of these nate resulted in more than 80% of the total enzyme activity in the precipitate. No ex- two factors among the species (data not ceptions were found in the precipitation shown), indicating that their low enzyme pattern among the tested species. It is evi- activity is probably controlled inherently. dent that this enzyme in the wild rices ap- This assumption was confirmed by the propears to be a particle or membrane-bound panil effects in vivo on the wild rices. As enzyme, as characterized by Frear and Still shown in Fig. 2 and Table 2, the dry-weight (4) and Akatsuka (1) for the enzyme iso- reduction of wild rices following propanil lated from cultivated rice plants. In addi- application depends essentially on their tion, it was also found that the enzyme pH AA1 activity. The species which had low optimum at 7.0 was true for all the tested AA1 activity were seriously affected by species. However, regarding the enzyme propanil. There is a high correlation beoptimum temperature, a significant differtween the AA1 activity and MLC (Y = ence was found among the species. Most 0.95). The minor variation might have resulted from differences in the surface strucspecies showed the enzyme optimum temperature at 5O”C, while in two species the ture of the wild rices which possibly affects value was 40°C. This difference was even the propanil uptake. However, it is of interreflected in the same species of 0. ridleyi, est to note the difference of MLC between exhibiting a intraspecific variation. Kinetic 0. australiensis and the other three species studies determined the Michaelis constant which had no AA1 activity. Although no (K,) of the enzyme. Except for 0. coarcenzyme activity was detected in any of the tata, there is only a minor difference among four species, there might be a great differthe species in the K,,, values which are ence concerning the cause. Considering the similar to that of AA1 in cultivated rice origin of 0. austruliensis (Australia), the disjunction of the continents in the early plants (1). Figure 3 illustrates the relative enzyme Cretaceous period, which has played a maactivity against propanii analogues in 13 jor role in the differentiation of rice species
32
CHEN AND MATSUNAKA TABLE 3 Properties of the Propanil Hydrolyzing Enzyme Aryl Acylamidase in the Wild Rices of Genus Otyza Section Species
Oryzae sativa rufpogon longistaminata officinalis collina punctata alta latifolia grandiglumis Ridleyanae longiglumis ridleyi ridleyi Coarctatae coarctata
Genome
Particlebound
PH optimum
Optimum temp. (“C)
AA AA AA cc cc BBCC CCDD CCDD CCDD
Yes Yes Yes Yes Yes Yes Yes Yes Yes
7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0 7.0
50 50 50 50 50 50 50 50 50
3.5 4.0 4.1 5.0 5.7 4.9 5.1 5.0 5.6
7777 . ... ???? ????
Yes Yes Yes
7.0 7.0 7.0
50 50 40
7.1 6.3 8.0
????
Yes
7.0
40
25.0
(14), probably resulted in the absence of AA1 in this species. On the other hand, in the other three species, it is more likely that the aryl acylamidase activity was just led to a undetectable level by some factors such as genetic mutation during the long evolutionary process. Clearly, further investiga-
10 Relative
20
enzyme activity
30
40
50
against 2,5-DCAA
60 (%)
FIG. 3. Twenty-nine wild rice strains of 13 species in genus Oryza scattered by the relative enzyme activity against propanil analogues. The relative activity was defined as enzyme-hydrolyzed propanil analogues compared to propanil as 100%. Data represent the mean value of three replicates.
-
Michaelis constant (lo-4 M)
tions are needed to confirm this hypothesis. In addition to the quantitative difference of the propanil-hydrolyzing enzyme aryl acylamidase among the wild rice species, we also demonstrated qualitative diversity of this enzyme in genus Oryza. The variation in the enzyme optimum temperature, the relative enzyme activity, and the Km values indicate that AA1 probably has been modified in some species of the genus Oryza. Our previous studies (12, 15) indicated the possible role of AA1 for determining the interspecific relationship in Oryza. This is further confirmed by the present investigation. As depicted in Fig. 3, the allocation of 13 species according to their relative enzyme activity against 2,5-DCAA and 2,3-DCPA reflects well their genetic variation (16, 17) during the evolutionary process. By taking advantage of its quantitative and qualitative diversities, AA1 will serve as a good marker for making systematic and phylogenetic inferences in genus Oryza. Our detailed investigation of this enzyme in wild rices will provide more information at the molecular level in a future publication. In contrast to aryl acylamidase of other
ARYL
ACYLAMIDASE
plants (18, 19), the rice plant enzyme appears to be associated with particulate cell constituents, representing a particle-bound enzyme. Hirase and Matsunaka (20) studied its physiological role by comparing cultivated rice plants with a propanilsusceptible mutant rice, suggesting that AA1 plays an important role in the nitrogen metabolism of rice plants. Wild rices provide us suitable material to study the physiological aspects of this enzyme. ACKNOWLEDGMENT
The authors thank Professor H. Morishima of the National Institute of Genetics in Mishima, Japan, for generously providing us with the wild rices. REFERENCES
1. T. Akatsuka, Purification of aryl acylamidase I, II, III from higher plants and selectivity of propanil, Weed Res. (Japan) 24, 55 (1979). [In Japanese] 2. C. C. Still and 0. Kuzirian, Enzyme detoxification of 3’,4’-dichloropropionanilide in rice and barnyardgrass, a factor in herbicide selectivity, Nature (London) 216, 799 (1967). 3. R. Y. Yih, D. H. McRae, and H. F. Wilson, Mechanism of selective action of 3’,4’-dichloropropionanilide, Phnt Physiol. 43, 1291 (1968). 4. D. S. Frear and G. G. Still, The metabolism of 3’,4’-dichloropropionanilide in rice plants. Partial purification and properties of an aryl acylamidase from rice, Phytochemistry 72, 913 (1968). 5. R. E. Hoagland and G. Graf, Enzymatic hydrolysis of herbicides in plants, Weed Sci. 20, 303 (1972). 6. R. J. Smith, 3’,4’-Dichloropropionanilide for control of barnyardgrass in rice, Weeds 9, 318 (1961). 7. M. Goto and R. Sato, Microanalysis of propanil and MCPCA. Pestic. Tech. 10, 16 (1964). [In Japanese]
IN
Oryza
33
8. M. M. Bradford, Rapid and sensitive method for the quantification of proteins using the principle of protein-dye binding. Anal. Biochem. 72, 248 (1976). 9. D. I. Amon, Copper enzyme in isolated chloroplasts. Plant Physiol. 24, 1 (1949). 10. H. Morishima, Y. Sano, and H. I. Oka, Differentiation of perennial and annual types due to habitat condition in the wild rice Oryza perennis. Plant Syst. Evol. 144, 119 (1984). Il. H. Morishima, Wild progenitors of cultivated rice and their population dynamics, in “Rice Genetics,” pp. 3-14. IRRI, Manila, 1986. 12. J. J. Chen and S. Matsunaka, Distribution of propanil hydrolyzing enzyme aryl acylamidase 1 in genus Oryza, Proc. Conf. Asian-Pacific Weed Sci. Sot., 12th 2, 479 (1989). 13. H. Morishima, Species relationships and the search for ancestors, in “Biology of Rice” (S. Tsunoda and N. Takahashi, Eds.), pp. 3-30, Japan Sci. Sot. Press, Tokyo/Elsevier. Amsterdam, 1984. 14. T. T. Chang, Crop history and genetic conservation: Rice-A case study, Iowa State .I. Res. 59, 425-455, 4w95 (1985). 15. S. Matsunaka and Y. Aoyama, Distribution of propanil hydrolyzing enzyme (rice aryl acylamidase I) in Oryza genus, Proc. Conf. AsianPacific Weed Sci. Sot., 8th 352 (1981). 16. S. Sampath, The Genus Oryza: Its taxonomy and species interrelationships, Oryza 1, 1 (1962). 17. T. Katayama and W. Onizuka, Intersectional F, plants from Oryza sativa x 0. ridleyi and 0. sativa X 0. meyeriana, Jpn. J. Genet. 54(l). 43 (1979). 18. R. E. Hoagland and G. Graf, An aryl acylamidase from tulip which hydrolyzes 3’,4’-dichloropropionanilide, Phytochemistry 11, 521 (1972). 19. R. E. Hoagland, Reversal of EPTC inhibition of aryl acylamidase activity by oximes, Pestir,. Biochem. Physiol. 34, 69 (1989). 20. K. Hirase and S. Matsunaka, Nitrogen Metabolism in a propanil susceptible rice mutant, Proc. Conf. Asian-Pacific Weed Sci. Sot., 12th 2,405 (1989).