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Addition of Branched-Chain Amino Acids Can Reverse Propyrisulfuron-Induced Acetolactate Synthase (ALS) and Grow....

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PHILIPP AGRIC SCIENTIST ISSN 0031-7454 Vol. 98 No. 4, 351–361 December 2015

Addition of Branched-Chain Amino Acids Can Reverse Propyrisulfuron-Induced Acetolactate Synthase (ALS) and Growth Inhibition in Three Rice Cultivars and Five Weed Species

Kevin C. Salamanez1,*, Aurora M. Baltazar2, Evelyn B. Rodriguez1, Marivic S. Lacsamana1, Abdelbagi M. Ismail3 and David E. Johnson3

1Institute of Chemistry, College of Arts and Sciences, University of the Philippines Los Baños, College, Laguna 4031, Philippines 2Crop Protection Cluster, College of Agriculture, University of the Philippines Los Baños, College, Laguna 4031, Philippines 3Crop and Environmental Sciences Division, International Rice Research Institute, Los Baños, Laguna, Philippines *Author for correspondence; e-mail: [email protected]; Tel.: +63 49 536 2220

Propyrisulfuron is a new pyrimidinylsulfonylurea with a fused heterocyclic moiety. Similar to older sulfonylureas, propyrisulfuron inhibits the activity of acetolactate synthase (ALS), the involved in branched-chain synthesis. Previous studies have shown that ALS activity, and consequently, the growth of susceptible plants was inhibited when treated with propyrisulfuron. This study was conducted to determine whether or not the addition of branched chain amino acids to the growth medium will alleviate the inhibition of ALS activity and the growth of propyrisulfuron-treated rice and weeds. Visual assessment of injury, shoot height, shoot biomass, in vivo ALS activity and ALS content showed that addition of 100 mg L-1 , and significantly reversed ALS inhibition and resulted in recovery from ALS and growth inhibition of the propyrisulfuron-treated rice and weeds. Recovery of the weeds, which were more susceptible than rice, was more pronounced than that of rice. Addition of all three amino acids resulted in greater reversal of ALS inhibition and better recovery compared with addition of only two amino acids.

Key Words: acetolactate synthase, Cyperus iria, Echinochloa colona, Echinochloa crus-galli, Leptochloa chinensis, Ludwigia hyssopifolia, propyrisulfuron, rice

Abbreviations: AHAS – acetohydroxyacid synthase, ALS – acetolactate synthase, CPCA – 1,1- cyclopropanedicarboxylic acid, DAT – days after treatment, I – isoleucine, L – leucine, SDS-PAGE – sodium dodecyl sulfate polyacrylamide gel electrophoresis, V – valine

INTRODUCTION move via the phloem to all plant parts (Gunsolus and Curran 1996; Naylor 2002). In 1975, Weeds reduce yields by approximately 10% in George Levitt discovered the sulfonylureas, a group of transplanted rice and by 20–30% in direct-seeded rice herbicides which inhibit acetolactate synthase (ALS) or (Rao et al. 2007). In most Asian countries, the most acetohydroxyacid synthase (AHAS). Sulfonylureas were common weed problems infesting rice are grasses such as introduced commercially in 1982 for the control of weeds Echinochloa crus-galli, Echinochloa colona, and infesting wheat and barley and are now used around the Leptochloa chinensis; broadleaf weeds including world in all primary agronomic crops (Poston 1999). Monochoria vaginalis, Ludwigia octovalvis and Generally, sulfonylureas are effective against annual Ludwigia hyssopifolia; and sedges including Cyperus broadleaves and sedges, however, the more recently difformis and Cyperus iria (Pancho and Obien 1995; developed sulfonylureas have activity against grasses and Caton et al. 2004). perennial weeds. These herbicides work by blocking the Herbicides provide an economic and effective way to enzyme acetolactate synthase (ALS) (syn. help manage weeds (Lingenfelter and Hartwig 2007). acetohydroxyacid synthase, AHAS), which produces Herbicide use has increasingly been adapted in Asia since branched-chain amino acids needed to synthesize many countries are facing labor shortages as more and necessary for plant growth. If ALS is inhibited, a more farm workers migrate from rural to urban areas continuous supply of essential proteins is hindered, thus, (Gianessi 2013). Selective herbicides inhibit specific the plant’s metabolic processes like cell division are shut plant physiological processes as target sites while crops down, resulting in mortality in susceptible plants (Mazur are relatively unharmed (Singh 2010). Herbicides that et al. 1987; Singh 2010). Zhou et al. (2007) found that prevent biosynthesis of amino acids are excellent in ALS inhibitors cause plant death due also to secondary controlling annual and perennial weeds because these effects such as buildup of 2-ketobutyrate or 2-

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aminobutyrate, depletion of intermediates in the pathways of critical processes and disruption of photosynthesis, transport and respiration processes. Some sulfonylurea derivatives with a fused heterocyclic moiety have herbicidal activity not only against broadleaves and sedges but also against grasses as well as sulfonylurea-resistant biotypes (Tanaka et al. 2006). Propyrisulfuron (Fig. 1) [1-(2-chloro-6- propylimidazo[1,2-b]pyridazin-3-ylsulfonyl)-3-(4,6- dimethoxypyrimidin-2-yl)urea] is a novel sulfonylurea herbicide with a fused heterocyclic moiety developed in 2008 and registered for use in rice in Japan in 2010 (Ikeda et al. 2011a). In their evaluation of herbicidal activities of new sulfonylureas with fused heterocyclic moieties using whole-plant and ALS enzyme assays, Ikeda et al. (2011b) showed that propyrisulfuron effectively controlled Echinochloa species (grasses), Schoenoplectus juncoides, Cyperus serotinus (sedge), and Monochoria vaginalis (broadleaf) at 70 and 140 g a.i. ha-1 with good selectivity to rice. Propyrisulfuron can also control weed biotypes that have developed resistance to the older sulfonylurea herbicides (Ikeda et al. 2011a; Kramer et al. 2012). Results of our previous study (Salamanez 2012) concurred with those of Tanaka et al. (2006) and Ikeda et al. (2011b), confirming the herbicidal activity of propyrisulfuron against the grasses Echinochloa crus- galli, Echinochloa colona and Leptochloa chinensis, the sedge Cyperus iria and the broadleaf weed Ludwigia Fig. 1. Structure of propyrisulfuron. hyssopifolia. We found that propyrisulfuron inhibited in vivo and in vitro ALS activity and reduced ALS content in concentrate (SC)] was obtained from Sumitomo these weeds, with a corresponding reduction in growth Chemicals, Inc. All studies were conducted at the (Salamanez 2012). laboratory and greenhouse facilities of the Weed Science In identifying that ALS inhibition is the site of action and Plant Physiology laboratories of the Crop and of sulfonylureas, previous studies have shown an indirect Environmental Sciences Division, International Rice method of determining the mechanism of ALS-inhibiting Research Institute (IRRI), Laguna, Philippines, from herbicides – that supplementation with the branched- December 2011 to December 2012. chain amino acids valine, isoleucine and leucine reversed growth inhibition in pea roots and seedlings treated with Seed Collection chlorsulfuron, an older sulfonylurea (Ray 1984; Tanaka Seeds of rice cultivars IR64, Azucena, and N22 were and Yoshikawa 1998). obtained from the TTChang Rice Genetic Resources This study was conducted to determine the effect of Center at IRRI. Seeds of the weed species Echinochloa addition of valine, isoleucine and leucine on in vivo ALS colona, Echinochloa crus-galli, Leptochloa chinensis, activity and growth of three rice varieties and five weed Ludwigia hyssopifolia and Cyperus iria were obtained species treated with propyrisulfuron. It aims to from the seed collection at the Weed Science Group at demonstrate if this newly developed sulfonylurea the Crop and Environmental Sciences Division at IRRI. (propyrisulfuron) will differ in older sulfonylureas, in terms of its effect when applied with branched-chain Preparation and Growing of Plants amino acids. Understanding the mechanism of action of Seeds of the three rice cultivars and the five weed species propyrisulfuron is essential in assessing herbicide mentioned above were incubated at 45 °C for 1 h. The compatibility and its use in herbicide rotation. seeds were sown at 1 cm deep in sterilized soil contained in plastic cups (100 mm x 100 mm x 70 mm). At 6 d after emergence, the plants were thinned to one plant per MATERIALS AND METHODS container. Plants were grown under natural light (6 am to 6 pm) and temperature (27 °C at night and 32 °C at Materials and Time and Place of the Study daytime) conditions in the greenhouse and watered daily. Unless otherwise stated, all analytical grade laboratory Pots were arranged in a completely randomized design reagents were purchased from Belman Compania with three replications per treatment. The plants were Incorporada (Belman Laboratories, Manila, Philippines). grown until they were used for greenhouse or laboratory Commercially formulated propyrisulfuron [10 soluble studies as shown in Figure 2.

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Treatment of Rice and Weeds with Propyrisulfuron ALS Enzyme Extraction and Amino Acids Leaf tissues from 6-d-old plants (two-leaf stage) treated At the two-leaf stage, the rice and weed seedlings were with 50 g a.i. ha-1 propyrisulfuron, with or without 10, 50 sprayed with 50 or 100 g a.i. ha-1 propyrisulfuron (Fig. 2) or 100 mg L-1 each of valine, isoleucine and leucine or using a research track sprayer (De Vries Manufacturing, 100 mg L-1 of valine and isoleucine, were used for Hollandale, MN) with 214 L ha-1 spray volume delivery enzyme extraction for the assay of in vivo ALS activity. and spray pressure at 140 kPa, fitted with flat fan nozzles This procedure was adapted from Yoon et al. (2003) and (Teejet 80015E, Spraying Systems Co., North Ave., Forlani et al. (1991). All laboratory operations were Schmale Rd., Wheaton, IL 60189). Five minutes after carried out at 0–4 °C with minimum exposure to sunlight. treatment with propyrisulfuron, 25 mL of 10, 50 or 100 The leaves were cut from the plant, thoroughly washed mg L-1 each of valine, isoleucine and leucine or 100 mg with tap water, cut into 2 mm pieces and placed in an ice L-1 of valine and isoleucine were added to each pot. bath (0–4 °C). The leaf pieces were suspended in 5 mL Untreated control plants (without amino acid and without g-1 of ice-cold standard buffer [20 mM potassium propyrisulfuron) were grown for comparison. Plant phosphate (pH 7.5) containing 20% by volume glycerol, height was measured and visual phytotoxicity ratings 1 mM magnesium chloride, 0.25 mM dithiothreitol (based on Rahman et al. 2011) were recorded at 2, 4 and (DTT), 0.1 mM (TPP) and 0.01 6 d after treatment (DAT). At 6 DAT, the seedlings were mM flavine adenine dinucleotide (FAD)]. To partially harvested and plant height and shoot fresh weight were purify the enzyme, the mixture was homogenized using a recorded. Teflon-in-glass potter homogenizer and 10 mg mL-1 of polyvinyl polypyrrolidone was added. The mixture was centrifuged at 20,000 g for 15 min and the supernatant containing the ALS was collected.

Fig. 2. Schematic diagram of greenhouse and laboratory studies. (ALS – acetolactate synthase, DAT – days after transplanting, V – valine, I – isoleucine, L – leucine).

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The extracts were analyzed for concentration gels were stored in zip-locked plastic wrappers to prevent using the method of Bradford (1976) and then in vivo them from drying up. ALS activities were determined. The enzyme ALS of To determine the molecular weight (kDa) of ALS each leaf sample from untreated plants was characterized subunits, a calibration curve was prepared by plotting the by sodium dodecyl sulfate polyacrylamide gel logarithm of the molecular weight of the standard electrophoresis (SDS-PAGE). proteins vs. the Rf value of each protein standard. The Rf value of each protein band was calculated by dividing the Assessment of ALS Concentration by the Bradford distance traveled by the sample by the distance traveled Assay by the tracking dye. The molecular weight of each ALS concentration in the extracts from the three rice protein subunit was determined from the calibration cultivars and five weed species was estimated by the curve. method of Bradford (1976) using bovine serum albumin (BSA) as standard. Crude protein extract (50 µL) from In vivo Acetolactate Synthase Assay each plant sample was pipetted into each microfuge tube This procedure was adapted from the methods developed then added with 850 µL distilled water and 100 µL of by Lovell et al. (1996), Volenberg et al. (2002), Bozic et Bradford reagent. The mixture was allowed to stand for 5 al. (2007), Uchino et al. (2007) and Maja and Branko min, then the absorbance at 595 nm was measured with a (2011). Plants sprayed with propyrisulfuron at 50 g a.i. UV-Vis Shimadzu 1800 spectrophotometer. ha-1 and then added with 10, 50 or 100 mg L-1 valine, leucine and isoleucine were used in this study. At 21 h Characterization of ALS Extracts using Voges- after herbicide treatment, 766 g ha-1 of 1,1- Proskauer Reaction cyclopropanedicarboxylic acid (CPCA) containing 0.25% The enzyme extracts from untreated plant samples were v/v non-ionic surfactant Tween 20 (sorbitan added with 0.05 mL of 6 N H2SO4. The reaction mixture monolaureate) was sprayed on leaves, which were then was heated at 30 °C for 15 min, and 0.5 mL of 0.5% (w/ harvested 3 h later, placed in an ice bath, then cut into v) creatine and 5% (w/v) 1-naphthol dissolved in 10% small pieces. Subsamples (about 0.1 g) were stored in a NaOH was added. Addition of H2SO4 allows −20 °C freezer for 24 h, then 3 mL distilled water was decarboxylation of acetolactate into acetoin. Upon added to each sample and thawed at 25 °C for 45 min, addition of α-naphthol and creatine in sodium hydroxide with vortexing every 15 min. solution, the red-colored α-naphthol-creatine-acetoin The decantate (3 mL) was then collected and treated complex is formed (λmax = 530 nm). To catalyze the with 50 µL 6N H2SO4 and mixed in a vortex mixer for 20 conversion of acetoin to diacetyl in the presence of s. Samples were incubated at 60 °C for 30 min to allow oxygen, α-naphthol is added. Diacetyl reacts with conversion of acetolactate to acetoin. A 1-mL aliquot of guanidine-containing compounds such as creatine in the creatine and α-naphthol solution at 0.09 and 0.9 w/v, presence of α-naphthol to form a pinkish red end product respectively, in 2 N NaOH was added to each sample and (Voges and Proskauer 1898). Thus, the formation of a red the solutions were vortexed for 10 s. Samples were then complex indicates the presence of ALS. incubated in a 60 °C water bath for 30 min to allow color development from colorless to red. The samples were Estimation of Molecular Weight of ALS Using SDS- subsequently cooled to ambient room temperature and PAGE centrifuged at 10,000 g for 5 min. Absorbance of the SDS-PAGE was carried out according to the method of supernatant was then measured at 530 nm using UV-Vis Yoon et al. (2003). Resolving gel (11%) and stacking gel Shimadzu 1800 spectrophotometer (Shimadzu Scientific were prepared in a 50-mL Erlenmeyer flask. Each ALS Instruments, Kyoto, Japan). Extracts from plants not extract (15 µL) was added to 15 µL of sample loading treated with herbicide or CPCA were used as background buffer [15 mM Tris (6.8), 10% glycerol, 0.05% checks for the spectrophotometric measurements. bromphenol blue, 0.4 M β-mercaptoethanol] in a 1.5 mL Absorption values were converted to µg acetoin using a Eppendorf tube. The tubes were placed in boiling water standard curve. bath for 5 min. The samples were allowed to cool to room temperature, then vortexed before loading into the Statistical Design and Data Analysis gel using 15 µL of each sample. Treatments were arranged in a completely randomized One lane was loaded with 4 µL of molecular weight design in the greenhouse and replicated three times. The standards (10–225 kDa). The setup was run until the experimental unit was one plant. Least significant tracking dye almost reached the bottom of the gel. The difference (LSD) and standard error (SE) values for shoot separating gel was carefully removed from the glass plate length and shoot biomass were computed using SAS and was placed in a plastic container. The gel was stained version 9.1.3 PROC MIXED (SAS Institute Inc., Cary, with Coomassie Brilliant Blue G250 (CBB) staining North Carolina). PROG REG of the same statistical solution. The container was set aside for 12 h. After software was used to determine correlation between ALS staining, the gel was immersed in a destaining solution activity and other growth parameters at the two-leaf [7% acetic acid (v/v) in 40% methanol] with gentle stage. For all statistical tests, statistical significance was shaking for 1 h. The de-staining solution was repeatedly set at P ≤ 0.05. changed until protein bands were clearly defined. The

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RESULTS AND DISCUSSION

Characterization of ALS from Rice and Weeds All extracts from rice and weeds formed a red-colored complex upon reaction with H2SO4 followed by addition of α-naphthol-creatine mixture in alkaline medium, which indicated the presence of ALS. Addition of H2SO4 resulted in decarboxylation of acetolactate to produce acetoin. To catalyze the conversion of acetoin to diacetyl in the presence of oxygen, α-naphthol was added. Diacetyl reacted with guanidine-containing compounds such as creatine in the presence of α-naphthol to form a pinkish red end-product (Voges and Proskauer 1898). The resulting red-colored α-naphthol-creatine-acetoin complex, which had absorbance maximum at 530 nm, indicates the presence of ALS in both rice and weeds. Further characterization of the extracted ALS was accomplished using SDS-PAGE. Two dominant bands Lane 1 (Echinochloa colona); 2 (Echinochloa crus-galli); 3 (Leptochloa were present at 35–74 kDa and in both rice and weed chinensis); 4 (Cyperus iria); 5 (Ludwigia hyssopifolia); 6 (IR-64, O. sativa seedling extracts (Fig. 3). The molecular weights of indica); 7 (Azucena; O. sativa japonica); 8 (N22, O. sativa aus). major subunits of ALS in the three rice cultivars and five Fig. 3. SDS PAGE electrophoretogram of crude aceto- weed species were obtained using a calibration curve. lactate synthase (ALS) extract. Grasses (E. colona, E. crus-galli and L. chinensis) had ALS subunit molecular weights at 42–74 kDa (Fig. 3). iria were reduced by 60–88% (Table 2). Shoot fresh These figures are similar to the values of 54.06 and 75.85 -1 weight was reduced by 62–96% at 50 g a.i. ha kDa obtained by Zein et al. (2010) for the ALS of E. -1 propyrisulfuron and by 75–99% at 100 g a.i. ha colona. propyrisulfuron for all the five weeds (Table 2). On the ALS subunits of C. iria had estimated molecular -1 other hand, rice cultivars treated with 50 g a.i. ha and weights of 35 and 54 kDa, while those of L. hyssopifolia -1 100 g a.i. ha propyrisulfuron had lower % reduction in were 44 and 54 kDa (Fig. 3). Rice cultivars had subunits heights (19–56%) and less % reduction in shoot fresh with molecular weights of 37–63 kDa (Fig. 3). Sikdar weight (22–74%). The results obtained for determining in and Kim (2010) constructed a phylogenetic analysis of vivo ALS activity were consistent with those of growth ALS in rice (Table 1) showing that rice has ALS subunit parameters, showing a significant reduction in weight with a molecular weight of 69.4 kDa and suggested that (P < 0.0001) in ALS activity by 42–46% for all weeds OsALS are divergent and grouped interestingly -1 and by 26–40% for rice cultivars at 50 g a.i. ha with ALS from Zoysia grass and Alopecurus propyrisulfuron (Table 2). myosuroides, the major weeds in cereal crops, among -1 Propyrisulfuron at 50 and 100 g a.i. ha caused several plant groups and far from bacterial and yeast significant reductions in plant height and shoot fresh ALS. weight (P < 0.0001) in both rice and weeds, with greater In general, most ALS contain both a catalytic subunit reductions in weeds than in rice (Tables 3 and 4), which (65 kDa) and a smaller regulatory subunit, which varies is also supported by the visual phytotoxicity ratings in size between 9 and 54 kDa, depending on the origin of (Table 3). Among the weeds, L. hyssopifolia was the the species (Table 1). In plant ALS, this regulatory -1 -1 most susceptible to 50 g a.i. ha and 100 g a.i. ha subunit stimulates activity and confers sensitivity to propyrisulfuron, showing 60–82% reduction in plant inhibition by the branched-chain amino acids (Lee and height and 95–97% reduction in shoot fresh weight Duggleby 2001). In bacteria, ALS isozymes have two (Table 2). These values corresponded to the highest % catalytic subunits of approximately 60 kDa as well as two reduction in ALS activity (46%) when L. hyssopifolia smaller, regulatory subunits (9–17 kDa) (Grimminger -1 was applied with 50 g a.i. ha propyrisulfuron (Table 2). and Umbarger 1979; Eoyang and Silverman 1984; In the three rice cultivars, N22 (aus) was to be the most Schloss et al. 1985; Joo and Kim 2001). In plants, the -1 -1 tolerant to 50 g a.i. ha and 100 g a.i. ha quaternary structure of ALS appears more diverse since propyrisulfuron, which was proven by the lowest the molecular mass of the non-denatured ALS varies reduction in shoot fresh weight (22–31%) and a significantly from 55 to 440 kDa in various species considerable reduction (31–41%) in plant height (Table (McCourt et al. 1987; Pang et al. 2003). 2). The tolerance of N22 as shown by plant height and

fresh weight conformed with the lowest reduction in ALS Effect of Propyrisulfuron on ALS Activity and activity (10.87%) when applied with 50 g a.i. ha-1 Growth of Rice and Weeds -1 -1 propyrisulfuron (Table 2). At 50 g a.i. ha and 100 g a.i. ha propyrisulfuron, the heights of E. colona, E. crus-galli and L. chinensis were reduced by 57–80% while those of L. hyssopifolia and C.

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Table 1. Molecular weight of acetolactate synthase (ALS) from different sources. Molecular Weight of Source Reference Subunits (kDA) 65 (catalytic subunit); Most ALS in plants Lee et al. (2011) 9-54 (regulatory subunit) Oryza sativa (rice) 69.4 (from phylogenetic analysis) Sikdar and Kim (2010) Hordeum vulgare (barley) 65 (ALS I and II) Shyur et al. (1988); Yoon et al. (2003) Echinocloa colona 54.06 and 75.85 Zein et al. (2010) 62 (2 large subunits); Serratia marcescens Joo and Kim (2001) 35 (2 small subunits) Saccharomyces cerevisiae (yeast) 75 ( two subunits) Poulsen and Stougaard (1989) Lactococcus lactis 55 Kisrieva et al. (1964) (isoenzyme I) 9.5 and 60 (hexamer) Eoyang and Silverman (1984) Grimminger and Umbarger (1979); Schloss et al. (1985); Joo Escherichia coli (isoenzyme III) 17.5 and 62 (oligomer) and Kim (2001) Pseudomonas aeruginosa 15 and 60 (hexadecamer) Schomburg and Salzmann (1990) Aerobacter aerogenes 58 (tetramer) Schomburg and Salzmann (1990) Salmonella typhimurium (isoenzyme II) 9.8 and 59 (tetramer) Schomburg and Salzmann (1990)

Table 2. Effect of propyrisulfuron on growth and acetolactate synthase (ALS) activity of rice cultivars and weeds. ALS Activity (μg acetoin Plant Height (mm plant-1) and Shoot Fresh Weight (g plant-1) and hr-1 g fresh wt foliage-1)

Reduction in Height (%) Reduction in Weight (%) and Reduction in ALS Plants Activity (%) Propyrisulfuron Propyrisulfuron Propyrisulfuron -1 -1 -1 (g a.i. ha ) (g a.i. ha ) (g a.i. ha ) Untreated 0 50 100 Untreated 0 50 100 Untreated 0 50 E. colona 250 (0) 97.5 (61) 75 (70) 4.00 (0) 0.16 (96) 0.04 (99) 15.21 8.88 (42) E. crusgalli 264 (0) 114 (57) 103 (61) 2.60 (0) 0.16 (94) 0.05 (98) 14.67 8.15 (44) L. chinensis 48 (0) 14 (71) 10 (80) 1.50 (0) 0.57 (62) 0.38 (75) 14 7.88 (44) C. iria 105 (0) 22 (79) 13 (88) 2.00 (0) 0.10 (95) 0.04 (98) 13.93 7.59 (46) L. hyssopifolia 20 (0) 8 (60) 4 (82) 1.70 (0) 0.09 (95) 0.05 (97) 13.69 7.38 (46) IR-64 281 (0) 228 (19) 208 (26) 3.10 (0) 1.80 (42) 1.52 (52) 14.64 10.16 (31) Azucena 298 (0) 161 (46) 131 (56) 2.40 (0) 0.89 (63) 0.62 (74) 14.42 8.63 (40) N22 278 (0) 192 (31) 164 (41) 2.90 (0) 2.26 (22) 2.00 (31) 14.67 10.87 (26) aNumbers in parentheses indicate percent reduction in height, weight or ALS activity based on non-treated control. Data are means from three replicates.

Table 3. Visual phytotoxicity rating of rice cultivars and weeds treated with propyrisulfuron and added exogenouly with valine, leucine and isoleucine. PLANTS TREATMENT E. col. E. crus. L. chin. C. iria L. hys. IR-64 Azuc. N22 10 ppm V, I, L 1 1 1 1 1 1 1 1 50 ppm V, I, L 1 1 1 1 1 1 1 1 100 ppm V, I, L 1 1 1 1 1 1 1 1 100 ppm V, I 1 1 1 1 1 1 1 1 Prop 50 g ai ha-1 4 4 2 5 5 1 1 1 Prop. 50 + 10 ppm V, I, L 4 4 1 4 4 1 1 1 Prop. 50 + 50 ppm V, I, L 3 3 1 3 3 1 1 1 Prop. 50 + 100 ppm V, I, L 1 1 1 1 1 1 1 1 Prop. 50 + 100 ppm V, I, 4 4 1 4 4 1 1 1 Prop. 100 g ai ha -1 5 5 2 5 5 1 1 1 Prop. 100 + 10 ppm V, I, L 4 4 2 5 5 1 1 1 Prop. 100 + 50 ppm V, I, L 4 4 1 5 5 1 1 1 Prop. 100 + 100 ppm V, I, L 3 3 1 4 4 1 1 1 Prop. 100 + 100 ppm V, I, 4 4 1 5 5 1 1 1 Prop. = propyrisulfuron; V, I, L = valine, isoleucine and leucine Rating scale (Rahman et al. 2011) : 1 = highly resistant; 2 = resistant; 3 = partly resistant; 4 = susceptible; 5 = highly susceptible Effect of Valine, Isoleucine and Leucine on Growth of 103% when treated with 50 g a.i. ha-1 propyrisulfuron Plants Treated with Propyrisulfuron with 100 ppm V, I, and L. The increase was from 43 to Addition of 50–100 ppm each of valine (V), leucine (L) 91% when treated with 100 g a.i. ha-1 propyrisulfuron and isoleucine (I) resulted in an increase in the shoot with 100 ppm V, I, and L (Table 4). Weeds exhibited 60– fresh weights of rice and weeds treated with 50–100 g a.i. 76% (50 g a.i. ha-1 propyrisulfuron with 100 ppm V, I, ha-1 propyrisulfuron compared with propyrisulfuron- and L) and 7–29% increase in shoot fresh weight (100 g treated rice and weeds but not with the amino acids a.i. ha-1 propyrisulfuron with 100 ppm V, I, and L) (Table (Table 4). Shoot fresh weights in rice increased by 87– 4).

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Table 4. Percent increase in shoot fresh weights of rice and weeds added with valine, leucine and isoleucine after treatment with propyrisulfuron. % Shoot Weight Increase Relative to Untreated* TREATMENT E. col. E. crus. L. chin. C. iria L. hys. IR-64 Azuc. N22 10 ppm V, I, L 101.8a 103.1a 100.9a 100.6a 102.3a 100.5a 101.1a 101.4a 50 ppm V, I, L 103.9a 106.0a 104.1a 56.8 103.6a 101.6a 102.0a 102.4a 100 ppm V, I, L 106.4a 107.9a 105.7a 58.7c 105.5a 104.4a 103.0a 102.9a 100 ppm V, I 106.4a 106.3a 120.9a 55.9c 112.7a 100.9a 103.0a 101.9a Prop 50 g ai ha-1 4.1e 6.1d 38.3d 5.3e 5.5de 58.2d 36.9d 78.2d Prop. 50 + 10 ppm V, I, L 4.9e 12.4d 26.9e 9.0d 11.0d 59.6d 40.1c 101.4a Prop. 50 + 50 ppm V, I, L 1.5f 20.1d 35.8de 19.5e 20.1d 66.6c 87.3b 102.4a Prop. 50 + 100 ppm V, I, L 60.4c 64.9c 76.1c 63.7b 61.3c 95.9ab 87.3b 102.9a Prop. 50 + 100 ppm V, I, 1.6f 19.8d 34.7de 19.2e 18.6d 70.4c 48.9c 101.9a Prop. 100 g ai ha-1 1.5f 2.2e 24.7f 2.1f 2.9e 48.7e 25.9e 69.5e Prop. 100 + 10 ppm V, I, L 8.9d 3.2e 20.6e 1.9f 3.0e 49.1e 27.8e 71.5e Prop. 100 + 50 ppm V, I, L 18.0e 3.9e 23.8e 3.1f 4.9e 59.9d 36.1d 82.6cd Prop. 100 + 100 ppm V, I, L 8.2d 7.7d 29.1e 9.5d 8.2d 70.7c 43.8c 90.9c Prop. 100 + 100 ppm V, I, 2.51f 3.6e 23.0e 2.6f 4.5e 61.3d 39.1cd 85.5d SEM 0.6174 0.7188 1.4379 0.5086 0.8995 1.3269 1.7091 0.4907 P-value <0.0001† <0.0001† <0.0001† <0.0001† <0.0001† <0.0001† <0.0001† <0.0001† Prop. = propyrisulfuron; V, I, L = valine, isoleucine and leucine * Data are means from three replicates. Means with same superscript in each column do not differ significantly at P ≤ 0.05. †Significant at P≤0.05.

Table 5. Percent increase in shoot height of rice and weeds added with valine, leucine and isoleucine after treatment with propyrisulfuron. % Shoot Height Increase Relative to Untreated* TREATMENT E. col. E. crus. L. chin. C. iria L. hys. IR-64 Azuc. N22 10 ppm V, I, L 101.9a 93.0a 96.7a 90.7a 101.5a 90.6abc 100.5a 90.2a 50 ppm V, I, L 106.1a 101.4a 104.0a 100.8a 102.9a 98.4ab 101.2a 99.2a 100 ppm V, I, L 107.7a 101.9a 117.6a 102.5a 117.4a 108.2ab 117.1a 105.4a 100 ppm V, I 102.6a 101.2a 114.7a 98.6a 100.7a 94.1ab 110.5a 97.7a Prop 50 g ai ha-1 39.4c 43.b 28.64c 20.6c 39.9c 80.8bc 54.1cd 69.2bc Prop. 50 + 10 ppm V, I, L 33.6c 48.4b 29.4c 18.9c 36.8c 85.6bc 60.6c 72.8bc Prop. 50 + 50 ppm V, I, L 63.0b 69.7b 63.4b 38.0b 69.6b 100.0ab 77.3b 99.6c Prop. 50 + 100 ppm V, I, L 56.0b 68.2b 69.8b 37.9b 65.2b 107.1ab 72.3b 90.0b Prop. 50 + 100 ppm V, I, 98.1a 98.5a 97.9a 96.1a 101.5a 120.5a 88.2b 123.1a Prop. 100 g ai ha -1 29.8c 38.9b 19.8c 12.4c 18.1d 74.4c 44.1d 58.9c Prop. 100 + 10 ppm V, I, L 30.7c 35.5b 19.6c 17.4c 26.1c 60.7c 51.9cd 73.1bc Prop. 100 + 50 ppm V, I, L 30.8c 37.1b 23.3c 20.4c 30.4c 75.7c 67.2d 83.8b Prop. 100 + 100 ppm V, I, L 32.5c 41.8b 28.5b 23.1c 43.5c 91.4abc 77.7bc 95.7ab Prop. 100 + 100 ppm V, I, 29.1c 40.38b 25.91c 21.3c 36.26c 62.83c 70.67b 70.63bc SEM 11.823 2.6858 11.1046 6.3267 5.8642 10.3403 9.0047 9.658 P-value 0.0005† <0.0001† 0.0002† <0.0001† <0.0001† 0.0794‡ 0.0007† 0.0059† Prop. = propyrisulfuron; V, I, L = valine, isoleucine and leucine * Data are means from three replicates. Means with same superscript in each column do not differ significantly at P ≤ 0.05. †Significant at P≤0.05. ‡Has the tendency to be significant at P≤0.05. Greater alleviative effect was observed when 100 fresh weight when supplemented with 100 ppm V, I, L at ppm V, I, and L was supplemented with 50 g a.i. ha-1 50 g a.i. ha-1 propyrisulfuron (76.1%) and at 100 g a.i. propyrisulfuron compared with 100 g a.i. ha-1 combined ha-1 propyrisulfuron (29.1%) (Table 4). In terms of shoot with the branched-chain amino acids. At 50–100 g a.i. height increase, E. colona had the highest among the ha-1 propyrisulfuron with the addition of 50–100 ppm weeds when treated with 100 ppm V, I, L and 50 g a.i. each of V, I, and L, shoot heights in rice increased by 72– ha-1 propyrisulfuron (98.1%) while E. crusgalli had the 107% while those in the weeds increased by 23–70% highest at 100 g a.i. ha-1 propyrisulfuron (41.8%) (Table (Table 5). 5). N22 had the highest alleviative effect in shoot fresh Compared with shoot weight, the alleviative effect weight among the rice cultivars treated with 100 ppm V, on shoot height was found both at 100 ppm V, I, L, and I, L and 50 g a.i. ha-1 propyrisulfuron (102.9%) and 100 g 100 ppm V, I in which plants were treated with 50 g a.i. a.i. ha-1 propyrisulfuron (90.9%) (Table 4). The highest ha-1 propyrisulfuron (Table 5). In the 100 g a.i. ha-1 increase in shoot height was also observed in N22 when propyrisulfuron treatment, a similar trend was observed treated with 100 ppm V, I, L at 50 g a.i. ha-1 both in shoot weight and height. A slight alleviation from propyrisulfuron (123.1%) and at 100 g a.i. ha-1 the effect of propyrisulfuron took place when 100 ppm V, propyrisulfuron (95.7%) (Table 5). I, L was added to the plants (Tables 4 and 5). Among the Recovery in terms of increased plant weight and weeds, L. chinensis showed the highest increase in shoot shoot height was less pronounced in the rice cultivars

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Reversal of Propyrisulfuron-Induced Growth Inhibition Kevin C. Salamanez et al. than in the weeds possibly because the rice seedlings exhibited less injury to propyrisulfuron compared with the weeds. Other studies have reported recovery of rice treated with cinosulfuron or bensulfuron when combined with 1 mM valine, leucine and isoleucine, compared with sulfonylurea-treated rice without amino acids (Sengnil et al. 1992; Park et al. 1993). Plant death occurred once the amino acid pools have decreased below a critical concentration (Scheel and Casida 1985; Naylor 2002).

Effect of Valine, Isoleucine and Leucine on ALS Activity of Plants Treated with Propyrisulfuron The in vivo ALS activity in the three rice cultivars and five weed species treated with 10, 50 or 100 ppm valine, leucine and isoleucine after treatment with 50 g a.i. ha-1 of propyrisulfuron also increased over those of the Fig. 4. Activity of acetolactate synthase (ALS) after herbicide-treated plants not treated with amino acids (Fig. application of varying amounts of valine, isoleucine and leucine in plants treated with 50 4). The highest increase in in vivo ALS activity was -1 observed in the herbicide-treated plants supplemented g a.i. ha of propyrisulfuron. R in the treatments refers to the rate of application of with 100 ppm valine, isoleucine, and leucine (Fig. 4). -1 The in vivo ALS activity of rice cultivars applied with 50 propyrisulfuron in g a.i. ha . Data are means -1 from three replicates. Vertical bars represent g a.i. ha of propyrisulfuron was 61–83 %. When rice standard error (SE) of the mean. cultivars were applied with 50 g a.i. ha-1 of propyrisulfuron and supplemented with 100 ppm valine, amino acids, therefore, the flow of carbon must be tightly isoleucine and leucine, % ALS activity was increased at regulated so that none of these amino acids becomes 84–97 % (Fig. 3). Among the rice cultivars, the highest limiting for plant growth (Singh et al. 1988). Different increase in in vivo ALS activity (97%) was observed in plants have varying amount of endogenous branched- N22 (Fig. 4). On the other hand, the selected weeds chain amino acids in their seeds and leaves (Table 6). Wu treated with 50 g a.i. ha-1 propyrisulfuron had 48–64% et al. (2000) reported that seeds of japonica rice ALS activity (Fig. 3). Supplementation of branched-chain contained 3,520 ppm of valine, 2,510 ppm of isoleucine amino acids resulted in an increase of 78–92% in ALS and 4,770 ppm of leucine at mature stage while indica activity (Fig. 4). rice seeds contained 3,690 ppm, 2,560 ppm and 4,730 Increase in in vivo ALS activity was higher in the ppm of valine, isoleucine and leucine, respectively. three rice cultivars (P < 0.0001) than in the five weed Addition of branched-chain amino acids to the growth species. The increase in ALS activity was lowest when medium of propyrisulfuron-treated seedlings is an only two of the amino acids (valine and isoleucine) were indirect determination of a possible target site of added at 100 ppm compared with the ALS activity in propyrisulfuron. By noting the effects of different plants treated with all three amino acids (Fig. 4). The concentrations of amino acids exogenously applied to combined effect of the three branched-chain amino acids each plant, a possible target site of the propyrisulfuron, (valine, isoleucine and leucine) in alleviating ALS which is ALS, was deduced from this study. When the inhibition was much greater than when only two amino three branched-chain amino acids were supplemented acids (valine and isoleucine) were added. In all plant with the herbicide, the inhibition in ALS activity is not populations, less (P< 0.0001) inhibition in in vivo ALS pronounced because plants can possibly utilize the activity was observed as the dosage of each herbicide supplementary additives. From the pathway (Fig. 5), was increased. plants could not biosynthesize the three branched-chain Sulfonylureas inhibit plant growth by inactivating the amino acids because of the inhibition of ALS by key enzyme in the biosynthesis of branched-chain amino propyrisulfuron. The addition of branched-chain amino acids (Mazur et al. 1987). ALS is involved in the first acids had a protective effect or alleviative effect. Hence, step in the synthesis of branched-chain amino acids such indirectly our study determined that a possible site of as leucine, isoleucine and valine (Fig. 4). For the action of propyrisulfuron is ALS. biosynthesis of valine and leucine, condensation of two Several studies have shown that symptoms of growth molecules of pyruvate occurs to form 2-acetolactate. For inhibition of plants treated with ALS inhibitors could be the formation of isoleucine, one molecule of pyruvate counteracted when leucine, isoleucine or valine was reacts with 2-ketobutyrate (Durner et al. 1991). ALS included in the growth media and that plants died once inhibitors starve affected plants of branched-chain amino the amino acid pools have decreased below a critical acids causing inhibition of DNA synthesis (Singh 2010), concentration. In rice and other plants treated with which eventually leads to the interference of cell sulfonylureas such as cinosulfuron, bensulfuron, and division, inhibition of plant growth and ultimately, plant chlorsulfuron, addition of valine, leucine and isoleucine death. The biosynthetic pathway of branched-chain resulted in marked recovery of shoot growth whereas the amino acids supplies the carbon for the three different growth of plants without amino acid supplementation

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Table 6. Endogenous amount of branched-chain amino acids in various rice cultivars and weeds. SEEDS LEAVES Harrold Watson Yeoh et al. Yeoh and Yeoh and Branched- Wu et al. (2000) and and Rajwar et (1986) Watson Watson Chain Amino (taxonomic mean) Nalejawa Creaser al. (1980) (taxonomic (1982) (1982) Acid (2012) (1975) mean) Japonica Indica American E. crus- O. sativa O. sativa E. crus- Cyperus E. colona Rice Rice Rice galli (rice) (rice) galli sp. Valine (ppm) 3,560 3,690 3,540 5,500 5,400 40,000-52,000 1,400 47,000 48,000 Isoleucine (ppm) 2,510 2,560 2,460 4,200 3,800 28,000-36,000 _ 34,000 37,000 Leucine (ppm) 4,770 4,730 4,620 1,000 8,100 88,000-93,000 1,800 91,000 99,000

SUMMARY AND CONCLUSION

Characterization of the ALS enzyme through SDS-PAGE in both rice and weeds showed two prominent protein bands with estimated molecular weights between 35 and 74 kDa. ALS subunits of weeds had molecular weights of 42–74 kDa for grasses (E. colona, E. crus-galli and L. chinensis) and 35–54 kDa for C. iria and L. hyssopifolia. Rice cultivars (IR64, Azucena and N22) contained ALS subunits with molecular weights between 37 and 63 kDa. The presence of ALS in the extract was also confirmed by the Voges-Proskauer reaction, which has a positive result of red complex formation. Inhibition of growth, shoot biomass, in vivo ALS activity and reduction in ALS concentration in propyrisulfuron-treated plants were alleviated with the addition of branched chain amino acids (valine, isoleucine and leucine). Amino acids at 100 ppm provided significant reversal (P<0.0001) of ALS activity inhibition in both rice and weeds treated with propyrisulfuron. The alleviation of ALS inhibition and growth were much more significant with supplementation Fig. 5. Biosynthesis of branched-chain amino acids in of all three amino acids to the propyrisulfuron-treated plants. rice and weeds compared with application of only two amino acids (valine and isoleucine). was inhibited (Ray 1984; Sengnil et al. 1992; Park et al. 1993; Shimizu et al. 1996). Tanaka and Yoshikawa (1998) observed the alleviating effect of branched-chain amino acids on the growth inhibition induced by ACKNOWLEDGMENTS imazosulfuron on rice seedlings, where high concentrations of valine, leucine and isoleucine (e.g. 100 This study is a portion of the M.S. thesis of Kevin C. ppm) reversed the inhibition of imazosulfuron to almost Salamanez, with research funding from the International the same degree as that of the herbicide-untreated plants. Rice Research Institute (IRRI). We thank Teodoro Migo, Similar to our results, these studies also showed that the Ofelia Namuco and Evangelina S. Ella for the technical protective effect was much greater when the three assistance, and Sumitomo Chemicals for the branched-chain amino acids were supplied together rather propyrisulfuron used in this study. than when only two of them (valine and isoleucine) were supplied to the herbicide-treated plants. Our data indicate that branched-chain amino acids at REFERENCES CITED 100 ppm provided a significant reversal of inhibition of ALS in plants treated with propyrisulfuron. Since BOZIC D, VRBNICANIN S, BARAC M, STEFANOVIC L. supplementation of branched-chain amino acids caused 2007. Determination of Johnson grass level of sensibility to nicosulfuron. Maydica 52: 271–275. reversal of propyrisulfuron-induced growth inhibition, it could indicate that ALS is a target site of propyrisulfuron, BRADFORD M. 1976. A rapid and sensitive method for the similar to results of other studies on sulfonylureas (Ikeda quantification of microgram quantities of protein utilizing et al. 2011a). the principle of protein-dye binding. Anal Biochem 72: 248–252.

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