Genetic mapping of non-target-site resistance to a sulfonylurea herbicide (Envoke®) in Upland cotton (Gossypium hirsutum L.)

Acetolactate synthase (ALS) is responsible for a rate-limiting step in the synthesis of essential branched-chain amino acids. Resistance to ALS-inhibiting herbicides, such as trifloxysulfuron sodium (Envoke^®), can be due to mutations in the target gene itself. Alternatively, plants may exhibit herbicide tolerance through reduced uptake and translocation or increased metabolism of the herbicide. The diverse family of cytochrome P450 proteins has been suggested to be a source of novel herbicide metabolism in both weed and crop plants. In this study we generated a mapping population between resistant and susceptible cotton (Gossypium hirsutum L.) cultivars. We found that both cultivars possess identical and sensitive ALS sequences; however, the segregation of resistance in the F2 progeny was consistent with a single dominant gene. Here we report the closely linked genetic markers and approximate physical location on chromosome 20 of the source of Envoke herbicide susceptibility in the cotton cultivar Paymaster HS26. There are no P450 proteins in the corresponding region of the G. raimondii Ulbr. genome, suggesting that an uncharacterized molecular mechanism is responsible for Envoke herbicide tolerance in G. hirsutum. Identification of this genetic mechanism will provide new opportunities for exploiting sulfonylurea herbicides for management of both weeds and crop plants.     

Role of tryptophanyl residues in tobacco acetolactate synthase.

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine. ALS is the target of three classes of herbicides, the sulfonylureas, the imidazolinones, and the triazolopyrimidines. Five mutants (W266F, W439F, W490F, W503F, and W573F) of the ALS gene from Nicotiana tabacum were constructed and expressed in Escherichia coli, and the enzymes were purified. The W490F mutation abolished the binding affinity for cofactor FAD and inactivated the enzyme. The replacement of Trp573 by Phe yielded a mutant ALS resistant to the three classes of herbicides. The other three mutations, W266F, W439F, and W503F, did not significantly affect the enzymatic properties and the sensitivity to the herbicides. These results indicate that the Trp490 residue is essential for the binding of FAD and that Trp573 is located at the herbicide binding site. The data also suggest that the three classes of herbicides bind ALS competitively.     

Nucleotide substitutions in the acetolactate synthase genes of sulfonylurea-resistant biotypes of Monochoria vaginalis (Pontederiaceae)

Some point mutations in acetolactate synthase (ALS) confer resistance to ALS-inhibiting herbicides in weeds. To clarify the evolution of the herbicide resistance of Monochoria vaginalis, a weed in rice fields in Japan, the nucleotide sequences of four genes encoding ALS were surveyed in five sulfonylurea-resistant (SU-R) and five sulfonylurea-susceptible (SU-S) biotypes. In the ALS1 gene, two SU-R biotypes showed nucleotide substitutions changing Pro197 to Ser and Leu, respectively. In a different gene, ALS3, three other SU-R biotypes showed either of the two nonsynonymous nucleotide substitutions seen in ALS1. Only two biotypes geographically located distantly from each other shared the same mutation conferring SU resistance in the same gene. These patterns of nucleotide substitutions indicate that the SU-R phenotype was acquired independently by different biotypes. Nucleotide diversity values of the genes showing SU-R mutations were higher than those of ALS2 lacking any SU-R mutation and of a putative pseudogene, ALS4. This result suggests that the maintenance of nucleotide variability within target genes provides an opportunity for the evolution of SU-R phenotypes by herbicide-driven selection for mutations conferring resistance.     

Simplified adenine base editors improve adenine base editing efficiency in rice

Adenine base editors (ABEs) have been exploited to introduce targeted adenine (A) to guanine (G) base conversions in various plant genomes, including rice, wheat and Arabidopsis. However, the ABEs reported thus far are all quite inefficient at many target sites in rice, which hampers their applications in plant genome engineering and crop breeding. Here, we show that unlike in the mammalian system, a simplified base editor ABE‐P1S (Adenine Base Editor‐Plant version 1 Simplified) containing the ecTadA*7.10‐nSpCas9 (D10A) fusion has much higher editing efficiency in rice compared to the widely used ABE‐P1 consisting of the ecTadA‐ecTadA*7.10‐nSpCas9 (D10A) fusion. We found that the protein expression level of ABE‐P1S is higher than that of ABE‐P1 in rice calli and protoplasts, which may explain the higher editing efficiency of ABE‐P1S in different rice varieties. Moreover, we demonstrate that the ecTadA*7.10‐nCas9 fusion can be used to improve the editing efficiency of other ABEs containing SaCas9 or the engineered SaKKH‐Cas9 variant. These more efficient ABEs will help advance trait improvements in rice and other crops.     

A novel mutated acetolactate synthase gene conferring specific resistance to pyrimidinyl carboxy herbicides in rice

Acetolactate synthase (ALS) is the first common enzyme in the biosynthetic pathway of branched-chain amino acids. Mutations of specific amino acids in ALS have been known to confer resistance to ALS-inhibiting herbicides such as sulfonylureas and pyrimidinyl carboxy (PC) herbicides. However, mutations conferring exclusive resistance to PC have not yet been reported to date. We selected PC resistant rice calli, which were derived from anther culture, using one of the PCs, bispyribac-sodium (BS), as a selection agent. Two lines of BS-resistant plants carrying a novel mutation, the 95th Glycine to Alanine (G95A), in ALS were obtained. In vitro ALS activity assay indicated that the recombinant protein of G95A-mutated ALS (ALS-G95A) conferred highly specific resistance to PC herbicides. In order to determine if the ALS-G95A gene could be used as a selection marker for rice transformation, the ALS-G95A gene was connected to ubiquitin promoter and introduced into rice. PC resistant plants containing integrated ALS-G95A gene were obtained after selection with BS as a selection agent. In conclusion, novel G95A mutated ALS gene confers highly specific resistant to PC-herbicides and can be used as a selection marker.     

Molecular breeding of a novel herbicide-tolerant rice by gene targeting

We have previously reported the production of a rice cell line tolerant to the acetolactate synthase (ALS)-inhibiting herbicide bispyribac (BS), and demonstrated that the BS-tolerant phenotype was due to a double mutation in the rice ALS gene. We further indicated that while changing either of the two amino acids (W548 L or S627I) individually resulted in a BS-tolerant phenotype, conversion of both amino acids simultaneously conferred increased tolerance to BS. As the BS-tolerant cell line had lost the ability to regenerate during two years of tissue culture selection, we attempted to introduce these two point mutations into the rice ALS gene via gene targeting (GT). Using our highly efficient Agrobacterium-mediated transformation system in rice, we were able to regenerate 66 independent GT rice plants from 1500 calli. Furthermore, two-thirds of these plants harbored the two point mutations exclusively, without any insertion of foreign DNA such as border sequences of T-DNA. The GT plants obtained in the present study are therefore equivalent to non-GM herbicide-tolerant rice plants generated by conventional breeding approaches that depend on spontaneous mutations. Surprisingly, GT rice homozygous for the modified ALS locus showed hyper-tolerance to BS when compared to BS-tolerant plants produced by a conventional transgenic system; ALS enzymatic activity in plants homozygous for the mutated ALS gene was inhibited only by extremely high concentrations of BS. These results indicate that our GT method has successfully created novel herbicide-tolerant rice plants that are superior to those produced by conventional mutation breeding protocols or transgenic technology.     

Distinct non-target site mechanisms endow resistance to glyphosate,ACCase and ALS-inhibiting herbicides in multiple herbicide-resistant <Emphasis Type="Italic">Lolium rigidum</Emphasis>

This study investigates mechanisms of multiple resistance to glyphosate, acetyl-coenzyme A carboxylase (ACCase) and acetolactate synthase (ALS)-inhibiting herbicides in two Lolium rigidum populations from Australia. When treated with glyphosate, susceptible (S) plants accumulated 4- to 6-fold more shikimic acid than resistant (R) plants. The resistant plants did not have the known glyphosate resistance endowing mutation of 5-enolpyruvylshikimate-3 phosphate synthase (EPSPS) at Pro-106, nor was there over-expression of EPSPS in either of the R populations. However, [¹⁴C]-glyphosate translocation experiments showed that the R plants in both populations have altered glyphosate translocation patterns compared to the S plants. The R plants showed much less glyphosate translocation to untreated young leaves, but more to the treated leaf tip, than did the S plants. Sequencing of the carboxyl transferase domain of the plastidic ACCase gene revealed no resistance endowing amino acid substitutions in the two R populations, and the ALS in vitro inhibition assay demonstrated herbicide-sensitive ALS in the ALS R population (WALR70). By using the cytochrome P450 inhibitor malathion and amitrole with ALS and ACCase herbicides, respectively, we showed that malathion reverses chlorsulfuron resistance and amitrole reverses diclofop resistance in the R population examined. Therefore, we conclude that multiple glyphosate, ACCase and ALS herbicide resistance in the two R populations is due to the presence of distinct non-target site based resistance mechanisms for each herbicide. Glyphosate resistance is due to reduced rates of glyphosate translocation, and resistance to ACCase and ALS herbicides is likely due to enhanced herbicide metabolism involving different cytochrome P450 enzymes.     

Efficient transformation of wheat by using a mutated rice acetolactate synthase gene as a selectable marker

Acetolactate synthase (ALS) is a target enzyme for many herbicides, including sulfonylurea and imidazolinone. We investigated the usefulness of a mutated ALS gene of rice, which had double point mutations and encoded an herbicide-resistant form of the enzyme, as a selectable marker for wheat transformation. After the genomic DNA fragment from rice containing the mutated ALS gene was introduced into immature embryos by means of particle bombardment, transgenic plants were efficiently selected with the herbicide bispyribac sodium (BS). Southern blot analysis confirmed that transgenic plants had one to more than ten copies of the transgene in their chromosomes. Adjustment of the BS concentration combined with repeated selection effectively prevented nontransgenic plants from escaping herbicide selection. Measurement of ALS activity indicated that transgenic plants produced an herbicide-resistant form of ALS and therefore had acquired the resistance to BS. This report is the first to describe a selection system for wheat transformation that uses a selectable marker gene of plant origin.     

Focus on Weed Control: A Novel Rice Cytochrome P450 Gene,CYP72A31, Confers Tolerance to Acetolactate Synthase-Inhibiting Herbicides in Rice and Arabidopsis

Target-site and non-target-site herbicide tolerance are caused by the prevention of herbicide binding to the target enzyme and the reduction to a nonlethal dose of herbicide reaching the target enzyme, respectively. There is little information on the molecular mechanisms involved in non-target-site herbicide tolerance, although it poses the greater threat in the evolution of herbicide-resistant weeds and could potentially be useful for the production of herbicide-tolerant crops because it is often involved in tolerance to multiherbicides. Bispyribac sodium (BS) is an herbicide that inhibits the activity of acetolactate synthase. Rice (Oryza sativa) of the indica variety show BS tolerance, while japonica rice varieties are BS sensitive. Map-based cloning and complementation tests revealed that a novel cytochrome P450 monooxygenase, CYP72A31, is involved in BS tolerance. Interestingly, BS tolerance was correlated with CYP72A31 messenger RNA levels in transgenic plants of rice and Arabidopsis (Arabidopsis thaliana). Moreover, Arabidopsis overexpressing CYP72A31 showed tolerance to bensulfuron-methyl (BSM), which belongs to a different class of acetolactate synthase-inhibiting herbicides, suggesting that CYP72A31 can metabolize BS and BSM to a compound with reduced phytotoxicity. On the other hand, we showed that the cytochrome P450 monooxygenase CYP81A6, which has been reported to confer BSM tolerance, is barely involved, if at all, in BS tolerance, suggesting that the CYP72A31 enzyme has different herbicide specificities compared with CYP81A6. Thus, the CYP72A31 gene is a potentially useful genetic resource in the fields of weed control, herbicide development, and molecular breeding in a broad range of crop species.The mechanism of herbicide tolerance can be classified roughly into two groups: target-site and non-target-site herbicide tolerance (Powles and Yu, 2010). Target-site herbicide tolerance is caused by the prevention of herbicide binding to the target enzyme, caused by point mutations occurring in the latter. It is relatively easy to elucidate the molecular mechanisms of target-site herbicide tolerance, because it is regulated mostly by a single gene encoding a target enzyme harboring point mutations. On the other hand, non-target-site herbicide tolerance is caused by reduction to a nonlethal dose of herbicide reaching the target enzyme, caused by mechanisms such as activation of herbicide detoxification, decrease of herbicide penetration, and herbicide compartmentation in plant cells (Yuan et al., 2007). Among these mechanisms, the oxidization of herbicides by endogenous cytochrome P450 monooxygenase is thought to be a major pathway in plants (Werck-Reichhart et al., 2000; Siminszky, 2006; Powles and Yu, 2010). From the point of view of weed control, non-target-site herbicide tolerance is a greater threat to crop production and in the evolution of herbicide-resistant weeds, because it is often involved in resistance to multiherbicides that inhibit different target proteins, including never-used and potential plant growth regulators (Yuan et al., 2007; Powles and Yu, 2010). Conversely, it is expected that multiherbicide-tolerant crops could be produced easily by the application of non-target-site herbicide tolerance. Moreover, information gained from study of the molecular mechanisms of non-target-site herbicide tolerance can be applied to the research and development of novel herbicides and plant growth regulators.Acetolactate synthase (ALS; also known as acetohydroxy acid synthase) plays a key role in the biosynthesis of branched-chain amino acids such as Val, Leu, and Ile in many organisms. ALS is the primary target site for at least four classes of herbicides: sulfonylurea, imidazolinone, pyrimidinyl carboxylates, and triazolopyrimidine herbicides (Shimizu et al., 2002, 2005). These herbicides can inhibit ALS activity, resulting in plant death caused by a deficiency of branched-chain amino acids. ALS-inhibiting herbicides control many weed species in addition to exhibiting high selectivity in major crops and low toxicity to mammals, which lack the branched-chain amino acid biosynthetic pathway. However, various mutations in ALS that confer ALS-inhibiting herbicide tolerance have been found in many weeds (Shimizu et al., 2005; Powles and Yu, 2010). Similar mutations in ALS have also been reported in crops (Shimizu et al., 2005). To date, crops that show tolerance to ALS-inhibiting herbicides have been produced by various approaches, such as conventional mutation breeding, conventional transformation, and pinpoint mutagenesis via gene targeting based on information obtained from analyses of ALS mutants (Shimizu et al., 2005; Endo and Toki, 2013). On the other hand, weeds that show tolerance to ALS-inhibiting herbicides by cytochrome P450-mediated detoxification have also been reported (Powles and Yu, 2010). However, compared with target-site herbicide tolerance, little is known of the molecular mechanism of herbicide metabolism mediated by cytochrome P450. In rice (Oryza sativa), an herbicide-sensitive mutant has been produced by γ-ray irradiation (Zhang et al., 2002). This mutant showed 60-fold higher sensitivity to bensulfuron-methyl (BSM), a sulfonylurea herbicide, compared with wild-type rice (Pan et al., 2006). Genetic mapping and complementation tests revealed that a cytochrome P450, CYP81A6, is involved in BSM tolerance (Pan et al., 2006). As far as we know, this is the only example of the isolation and characterization of a cytochrome P450 gene involved in nontarget herbicide tolerance in rice.Bispyribac sodium (BS), a pyrimidinyl carboxylate herbicide, is effective in controlling many annual and perennial weeds, with excellent selectivity on direct-seeded rice (Shimizu et al., 2002). Recently, it was reported that japonica rice varieties show higher sensitivity to BS compared with indica rice varieties at the early stages of plant growth (Ohno et al., 2008; Taniguchi et al., 2010). A mutated ALS gene confers BS tolerance in plants including rice (Shimizu et al., 2005; Endo and Toki, 2013). However, the deduced amino acid sequences were shown to be highly conserved among japonica and indica rice varieties, and ALS levels of sensitivity to BS were similar in japonica and indica rice varieties (Taniguchi et al., 2010). These results suggest the possibility that indica rice varieties might show higher tolerance to BS due to the acquisition of nontarget herbicide tolerance.In this study, we isolated and characterized a novel cytochrome P450 gene, CYP72A31, involved in BS tolerance in rice. We also demonstrated that overexpression of CYP72A31 confers tolerance to ALS-inhibiting herbicides, including BS and BSM, in Arabidopsis (Arabidopsis thaliana).     

Cytochrome P450 CYP81A10v7 in Lolium rigidum confers metabolic resistance to herbicides across at least five modes of action

Rapid and widespread evolution of multiple herbicide resistance in global weed species endowed by increased capacity to metabolize (degrade) herbicides (metabolic resistance) is a great threat to herbicide sustainability and global food production. Metabolic resistance in the economically damaging crop weed species Lolium rigidum is well known but a molecular understanding has been lacking. We purified a metabolic resistant (R) subset from a field evolved R L. rigidum population. The R, the herbicide susceptible (S) and derived F₂ populations were used for candidate herbicide resistance gene discovery by RNA sequencing. A P450 gene CYP81A10v7 was identified with higher expression in R vs. S plants. Transgenic rice overexpressing this Lolium CYP81A10v7 gene became highly resistant to acetyl-coenzyme A carboxylase- and acetolactate synthase-inhibiting herbicides (diclofop-methyl, tralkoxydim, chlorsulfuron) and moderately resistant to hydroxyphenylpyruvate dioxygenase-inhibiting herbicide (mesotrione), photosystem II-inhibiting herbicides (atrazine and chlorotoluron) and the tubulin-inhibiting herbicide trifluralin. This wide cross-resistance profile to many dissimilar herbicides in CYP81A10v7 transgenic rice generally reflects what is evident in the R L. rigidum. This report clearly showed that a single P450 gene in a cross-pollinated weed species L. rigidum confers resistance to herbicides of at least five modes of action across seven herbicide chemistries.     

Allelic mutations in acetyl-coenzyme A carboxylase confer herbicide tolerance in maize

Summary The genetic relationship between acetyl-coenzyme A carboxylase (ACCase; EC 6.4.1.2.) activity and herbicide tolerance was determined for five maize (Zea mays L.) mutants regenerated from tissue cultures selected for tolerance to the ACCase-inhibiting herbicides, sethoxydim and haloxyfop. Herbicide tolerance in each mutant was inherited as a partially dominant, nuclear mutation. Allelism tests indicated that the five mutations were allelic. Three distinguishable herbicide tolerance phenotypes were differentiated among the five mutants. Seedling tolerance to herbicide treatments cosegregated with reduced inhibition of seedling leaf ACCase activity by sethoxydim and haloxyfop demonstrating that alterations of ACCase conferred herbicide tolerance. Therefore, we propose that at least three, and possible five, new alleles of the maize ACCase structural gene (Acc1) were identified based on their differential response to sethoxydim and haloxyfop. The group represented by Acc1-S1, Acc1-S2 and Acc1-S3 alleles, which had similar phenotypes, exhibited tolerance to high rates of sethoxydim and haloxyfop. The Acc1-H1 allele lacked sethoxydim tolerance but was tolerant to haloxyfop, whereas the Acc1-H2 allele had intermediate tolerance to sethoxydim but was tolerant to haloxyfop. Differences in tolerance to the two herbicides among mutants homozygous for different Acc1 alleles suggested that sites on ACCase that interact with the different herbicides do not completely overlap. These mutations in maize ACCase should prove useful in characterization of the regulatory role of ACCase in fatty acid biosynthesis and in development of herbicide-tolerant maize germplasm.Cooperative investigation of the Minnesota Agriculture Experiment Station and the U.S. Department of Agriculture, Agricultural Research Service. Supported in part by a grant from BASF Corporation and a University of Minnesota Doctoral Dissertation Fellowship to LCM. Minnesota Agricultural Experiment Station Publication No. 19,056Mention of a trademark, vendor, or proprietary product does not constitute a guarantee or warranty of the product by University of Minnesota or the USDA, and does not imply its approval to the exclusion of other products or vendors that may also be suitable     

Amino acid residues conferring herbicide tolerance in tobacco acetolactate synthase

Acetolactate synthase (ALS) is the common enzyme in the biosynthetic pathways leading to valine, leucine, and isoleucine in plants and microorganisms. ALS is the target site of several classes of structurally unrelated herbicides including sulfonylureas, imidazolinones, and triazolopyrimidines. To identify the residues conferring herbicide tolerance in tobacco ALS, site-directed mutagenesis for three residues, Ala121, Pro187 and Ser652, was performed. Mutant A121T showed strong resistance to Londax (a sulfonylurea) and Cadre (an imidazolinone), while mutant S652T was resistant only to Cadre. The S652N mutation abolished the binding affinity of FAD, and inactivated the enzyme. Double mutation of Ala121 and Ser652 with Thr yielded a mutant highly tolerant to Londax, Cadre, and TP (a triazolopyrimidine sulfonamide), but has enzymatic properties similar to those of wild-type. Substitution of Pro187 with Ser resulted in the enzyme highly susceptible to oxidation and fragmentation. These results suggest that two residues Ala121 and Ser652 are potent residues conferring herbicide resistance in tobacco ALS, and that double mutation of Ala121 and Ser652 by Thr can confer stronger tolerance to Londax, Cadre, and TP.     

Herbicide resistance of transgenic rice plants expressing human CYP1A1

Cytochrome P450 monooxygenases (P450s) metabolize herbicides to produce mainly non-phytotoxic metabolites. Although rice plants endogenously express multiple P450 enzymes, transgenic plants expressing other P450 isoforms might show improved herbicide resistance or reduce herbicide residues. Mammalian P450s metabolizing xenobiotics are reported to show a broad and overlapping substrate specificity towards lipophilic foreign chemicals, including herbicides. These P450s are ideal for enhancing xenobiotic metabolism in plants. A human P450, CYP1A1, metabolizes various herbicides with different structures and modes of herbicide action. We introduced human CYP1A1 into rice plants, and the transgenic rice plants showed broad cross-resistance towards various herbicides and metabolized them. The introduced CYP1A1 enhanced the metabolism of chlorotoluron and norflurazon. The herbicides were metabolized more rapidly in the transgenic rice plants than in non-transgenic controls. Transgenic rice plants expressing P450 might be useful for reducing concentrations of various chemicals in the environment.     

Herbicide resistant rice (Oryza sativa L.): Global implications for weedy rice and weed management*

Rice cultivars resistant to broad‐spectrum herbicides have been developed and their commercial release is imminent, especially for imidazolinone and glufosinate resistant varieties in the USA and Latin America. Glyphosate‐resistant rice should follow within a few years. Rice growers throughout the world could benefit from the introduction of herbicide‐resistant rice cultivars that would allow in‐crop, selective control of weedy Oryza species. Other perceived benefits are the possibility to control ‘hard‐to‐kill’ weed species and weed populations that have already evolved resistance to herbicides currently used in rice production, especially those of the Echinochloa species complex. Weed management could also be improved by more efficient post‐emergence control. Introduction of herbicide resistant rice could also bring areas heavily infested with weedy rice that have been abandoned back to rice production, allow longer term crop rotations, reduce consumption of fossil fuels, promote the replacement of traditional chemicals by more environmentally benign products, and provide more rice grain without adding new land to production. There are also concerns, however, about the impact of releasing herbicide‐resistant rice on weed problems. Of most concern is the possibility of rapid transfer of the resistance trait to compatible weedy Oryza species. Development of such herbicide resistant weedy rice populations would substantially limit the chemical weed management options for farmers. Herbicide‐resistant rice volunteers also could become problematic, and added selection pressure to weed populations could aggravate already serious weed resistance problems. Because of the risk of weedy Oryza species becoming resistant to broad‐spectrum herbicides, mitigating measures to prevent gene flow, eventually attainable by both conventional breeding and molecular genetics, have been proposed. With commercialisation of the first herbicide resistant varieties planned for 2001, these mitigating measures will not be available for use with this first generation of herbicide resistant rice products. Release of herbicide resistant rice should depend on a thorough risk assessment especially in areas infested with con‐specific weedy rice or intercrossing weedy Oryza species. Regulators will have to balance risks and benefits based on local needs and conditions before allowing commercialisation of herbicide‐resistant rice varieties. If accepted, these varieties should be considered as components of integrated weed management, and a rational herbicide use and weedy rice control should be promoted to prevent losing this novel tool.     

Cross-Resistance of a Chlorsulfuron-Resistant Biotype of Stellaria media to a Triazolopyrimidine Herbicide

A biotype of Stellaria media (L.) Vill. has been identified that is highly resistant to the herbicide chlorsulfuron. Resistance is due to an altered acetolactate synthase (ALS) that is much less sensitive to chlorsulfuron than the ALS from the susceptible (S) biotype. The S biotype was extremely sensitive to D489 (N-[2,6-dichlorophenyl]-5,7-dimethyl-1,2,4-triazolo[1,5a] pyrimidine-2-sulfonamide), a member of a new class of triazolopyrimidine herbicides, while the chlorsulfuron-resistant biotype exhibited complete cross-resistance at both the whole plant and enzyme levels. ALS activity of the S biotype was reduced by approximately 90% in the presence of 0.1 micromolar D489, while that of the R biotype was reduced by less than 10%. This result suggests that the two herbicides share a common binding site on ALS. Only very slight cross-resistance at the ALS level was found to imazamethabenz, an imidazolinone herbicide.     

水稻品种多样性遗传分析与稻瘟病控制

以2个籼型杂交稻——汕优63(A)和汕优22(B)、2个地方糯稻品种——黄壳糯(C)和紫糯(D)和3个粳稻品种——合系41(E)、楚粳12(F)和8126(G)为材料进行抗病基因同源序列(Resistance Gene Analogue,RGA)遗传分析。结果表明,杂交稻品种间以及粳稻品种间的抗性遗传较为相似,其相似系数分别为0．86和0．84。糯稻品种间以及糯稻、杂交稻和粳稻间的抗性遗传差异较大,相似系数为0．45。聚类分析表明,RGA结果与品种的系谱来源相吻合,与品种的田间抗性基本一致。根据品种的抗性遗传差异、农艺性状和经济性状的不同,在云南籼稻区的建水和石屏县以及温暖粳稻区的泸西县分别选用5种(A／C、A／D、B／C、B／D和A／B)和2种(E／C和E／F／G)不同的品种组合进行品种多样性混合间栽控制稻瘟病田间试验,结果表明,抗性遗传差异大(相似性：0．45～0．77)的5个品种混合间栽组合对稻瘟病有极为显著的控制效果,尤其是在混合间栽中高度感病的优质地方稻品种稻瘟病的发病率、病情指数均有极显著的下降,防治效果达54．47％～92．18％;遗传差异较小(相似性：0．84～0．90)的2个混栽组合混栽对稻瘟病的控制效果不明显,稻瘟病的防治效果在15．12％～25．54％。此外,品种抗性遗传和株高差异大的品种组合具有显著的增产效果,与品种净栽相比,平均增产539．0～900．0kg／ha,增幅5．57％-10．38％;品种抗性遗传和株高相似的品种组合没有增产效果。     

Analysis of Substrate Specificity of Pig CYP2B22 and CYP2C49 towards Herbicides by Transgenic Rice Plants

We introduced two novel types of pig (Sus scrofa) cytochrome P450, CYP2B22 and CYP2C49, into rice plants (Oryza sativa L. cv. ‘Nipponbare’) to produce herbicide-tolerant plants and to confirm the metabolic activities of the cytochrome P450 species. In germination tests, both types of transgenic plants showed tolerance to various herbicides with different modes of action. CYP2B22 rice plants showed tolerance towards 12 herbicides including chlortoluron (100 μM), amiprofos-methyl (2.5 μM), pendimethalin (10 μM), metolachlor (2.5 μM), and esprocarb (20 μM). CYP2C49 rice plants showed tolerance towards 13 herbicides, including chlortoluron (100 μM), norflurazon (0.5 μM), amiprofos-methyl (2.5 μM), alachlor (0.8 μM), and isoxaben (1 μM). The herbicide tolerance was considered to reflect the substrate specificity of the introduced P450 species. We used ¹⁴C-labeled metolachlor and norflurazon to confirm the P450 activity in the transgenic rice plants. The herbicides were metabolized more quickly in the transgenic rice plants than in the nontransgenic rice plants. Therefore, CYP2B22 and CYP2C49 rice plants became more tolerant to various herbicides than nontransgenic control plants because of accelerated metabolism of the herbicides by the introduced P450 species. Assuming that public and commercial acceptance is forthcoming, these transgenic rice plants may become useful tools for the breeding of herbicide-tolerant crops.     

Selectable tolerance to herbicides by mutated acetolactate synthase genes integrated into the chloroplast genome of tobacco

Strategies employed for the production of genetically modified (GM) crops are premised on (1) the avoidance of gene transfer in the field; (2) the use of genes derived from edible organisms such as plants; (3) preventing the appearance of herbicide-resistant weeds; and (4) maintaining transgenes without obstructing plant cell propagation. To this end, we developed a novel vector system for chloroplast transformation with acetolactate synthase (ALS). ALS catalyzes the first step in the biosynthesis of the branched amino acids, and its enzymatic activity is inhibited by certain classes of herbicides. We generated a series of Arabidopsis (Arabidopsis thaliana) mutated ALS (mALS) genes and introduced constructs with mALS and the aminoglycoside 3'-adenyltransferase gene (aadA) into the tobacco (Nicotiana tabacum) chloroplast genome by particle bombardment. Transplastomic plants were selected using their resistance to spectinomycin. The effects of herbicides on transplastomic mALS activity were examined by a colorimetric assay using the leaves of transplastomic plants. We found that transplastomic G121A, A122V, and P197S plants were specifically tolerant to pyrimidinylcarboxylate, imidazolinon, and sulfonylurea/pyrimidinylcarboxylate herbicides, respectively. Transplastomic plants possessing mALSs were able to grow in the presence of various herbicides, thus affirming the relationship between mALSs and the associated resistance to herbicides. Our results show that mALS genes integrated into the chloroplast genome are useful sustainable markers that function to exclude plants other than those that are GM while maintaining transplastomic crops. This investigation suggests that the resistance management of weeds in the field amid growing GM crops is possible using (1) a series of mALSs that confer specific resistance to herbicides and (2) a strategy that employs herbicide rotation.     

Repression of Acetolactate Synthase Activity through Antisense Inhibition (Molecular and Biochemical Analysis of Transgenic Potato (Solanum tuberosum L. cv Desiree) Plants)

Acetolactate synthase (ALS), the first enzyme in the biosynthetic pathway of leucine, valine, and isoleucine, is the biochemical target of different herbicides. To investigate the effects of repression of ALS activity through antisense gene expression we cloned an ALS gene from potato (Solanum tuberosum L. cv Desiree), constructed a chimeric antisense gene under control of the cauliflower mosaic virus 35S promoter, and created transgenic potato plants through Agrobacterium tumefaciens-mediated gene transfer. Two regenerants revealed severe growth retardation and strong phenotypical effects resembling those caused by ALS-inhibiting herbicides. Antisense gene expression decreased the steady-state level of ALS mRNA in these plants and induced a corresponding decrease in ALS activity of up to 85%. This reduction was sufficient to generate plants almost inviable without amino acid supplementation. In both ALS antisense and herbicide-treated plants, we could exclude accumulation of 2-oxobutyrate and/or 2-aminobutyrate as the reason for the observed deleterious effects, but we detected elevated levels of free amino acids and imbalances in their relative proportions. Thus, antisense inhibition of ALS generated an in vivo model of herbicide action. Furthermore, expression of antisense RNA to the enzyme of interest provides a general method for validation of potential herbicide targets.