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.     

Genetic control of a cytochrome P450 metabolism-based herbicide resistance mechanism in Lolium rigidum

The dynamics of herbicide resistance evolution in plants are influenced by many factors, especially the biochemical and genetic basis of resistance. Herbicide resistance can be endowed by enhanced rates of herbicide metabolism because of the activity of cytochrome P450 enzymes, although in weedy plants the genetic control of cytochrome P450-endowed herbicide resistance is poorly understood. In this study we have examined the genetic control of P450 metabolism-based herbicide resistance in a well-characterized Lolium rigidum biotype. The phenotypic resistance segregation in herbicide resistant and susceptible parents, F1, F2 and backcross (BC) families was analyzed as plant survival following treatment with the chemically unrelated herbicides diclofop-methyl or chlorsulfuron. Dominance and nuclear gene inheritance was observed in F1 families when treated at the recommended field doses of both herbicides. The segregation values of P450 herbicide resistance phenotypic traits observed in F2 and BC families was consistent with resistance endowed by two additive genes in most cases. In obligate out-crossing species such as L. rigidum, herbicide selection can easily result in accumulation of resistance genes within individuals.     

Is the cost of herbicide resistance expressed in the breakdown of the relationships between characters? A case study using synthetic-auxin-resistant Arabidopsis thaliana mutants

A mutation endowing herbicide resistance is often found to induce a parallel morphological or fitness penalty. To test whether such 'cost' of resistance to herbicides is expressed through lower resource acquisition, changes in resource allocation, or both, is of ecological significance. Here, we analysed 12 morphological traits in 900 plants covering three herbicide resistance mutations at genes AUX1 , AXR1 and AXR2 in the model species Arabidopsis thaliana . Comparing these 2,4-D herbicide-resistant homozygous (RR) and heterozygous (RS) plants to homozygous susceptible (SS) plants, this analysis estimates the dominance level of the resistance allele on morphology. We also demonstrated that the herbicide resistance cost was primarily expressed as a change in resource acquisition (62.1-94% of the analysed traits). Although AUX1 , AXR1 and AXR2 genes act in the same metabolic pathway of auxin response, each resistance factor was found to have its own unique signature in the way the cost was expressed. Furthermore, no link was observed between the absolute fitness penalty and the respective modifications of resource acquisition and/or resource allocation in the resistant plants. These results and their implications for herbicide resistance spread and establishment are discussed.     

拟南芥AtUNE12基因的耐盐功能初探

bHLH转录因子家族成员在植物生长发育、生理代谢及非生物胁迫响应过程中起重要作用。本研究选取拟南芥抗逆相关bHLH转录因子家族中AtUNE12基因为研究对象,对其进行耐盐功能初探。首先构建AtUNE12基因的植物过表达载体（pROKⅡ-AtUNE12）,通过农杆菌介导的浸花法转化拟南芥,利用qRT-PCR技术检测获得T₃代AtUNE12过表达转基因植株。在盐胁迫下,分析过表达AtUNE12与野生型拟南芥长势、根长及鲜重;比较过表达AtUNE12与野生型植株的电解质渗透率、失水率、MDA含量、POD与SOD活性及H₂O₂含量,鉴定AtUNE12基因是否具有耐盐能力。结果表明：过表达AtUNE12基因降低了拟南芥植株的失水率、电解质渗透率及MDA含量,保护细胞膜结构的完整性;增强了POD与SOD活性,降低了拟南芥植株内的H₂O₂含量,进而增强拟南芥植株的ROS清除能力,从而提高拟南芥的耐盐能力。     

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.     

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.     

Specialized binary vector for plant transformation: expression of the Arabidopsis thaliana AHAS gene in Nicotiana tabacum.

We constructed a cosmid vector, pOCA18, designed for transferring plant genomic libraries from Agrobacterium tumefaciens to plants. Clones from a genomic library of Arabidopsis thaliana DNA in pOCA 18 were propagated stably in both Escherichia coli and A. tumefaciens. Clones from the pOCA18 A. thaliana library were used to construct transgenic Nicotiana tabacum plants; the DNA inserts were transferred intact in 10 out of 16 transgenic N. tabacum plants examined but were partially deleted in six others. Transgenic N. tabacum plants constructed with a mutant A. thaliana acetohydroxy acid synthase gene (from the pOCA18 library) that encodes an enzyme resistant to the herbicide chlorsulfuron were resistant to chlorsulfuron. A statistical analysis indicated that if the A. thaliana library contains 10(7) members and if 10(7) A. tumefaciens transconjugants containing the library were used to transform plant cells, then 2 x 10(4) transformed plant cells must be generated to have a 95% probability of constructing a transgenic plant carrying a specific DNA sequence from the A. thaliana library.     

Alteration of β-tubulin in Nicotiana plumbaginifolia confers resistance to amiprophos-methyl

 A Nicotiana plumbaginifolia plant (apm5^r) resistant to amiprophos-methyl (APM), a phosphoro-amide herbicide, was isolated from protoplasts prepared from leaves of haploid plants. Genetic analysis revealed that the resistance is coded for by a dominant nuclear mutation and is associated with the increased stability of cortical microtubules. Two-dimensional polyacrylamide-gel electrophoresis, combined with immunoblotting using anti-tubulin monoclonal antibodies, showed that part of the β-tubulin in the resistant plant possessed lower isoelectric points than the β-tubulin of susceptible wild-type plants. These results provide evidence that the resistance to APM is associated with a mutation in a β-tubulin gene. The APM-resistant line showed cross-resistance to trifluralin, a dinitroaniline herbicide, suggesting a common mechanism of resistance between these two classes of herbicides. Received: 26 January 1997 / Accepted: 17 February 1998     

SulE, a sulfonylurea herbicide de-esterification esterase from Hansschlegelia zhihuaiae S113

De-esterification is an important degradation or detoxification mechanism of sulfonylurea herbicide in microbes and plants. However, the biochemical and molecular mechanisms of sulfonylurea herbicide de-esterification are still unknown. In this study, a novel esterase gene, sulE, responsible for sulfonylurea herbicide de-esterification, was cloned from Hansschlegelia zhihuaiae S113. The gene contained an open reading frame of 1,194 bp, and a putative signal peptide at the N terminal was identified with a predicted cleavage site between Ala37 and Glu38, resulting in a 361-residue mature protein. SulE minus the signal peptide was synthesized in Escherichia coli BL21 and purified to homogeneity. SulE catalyzed the de-esterification of a variety of sulfonylurea herbicides that gave rise to the corresponding herbicidally inactive parent acid and exhibited the highest catalytic efficiency toward thifensulfuron-methyl. SulE was a dimer without the requirement of a cofactor. The activity of the enzyme was completely inhibited by Ag(+), Cd(2+), Zn(2+), methamidophos, and sodium dodecyl sulfate. A sulE-disrupted mutant strain, ΔsulE, was constructed by insertion mutation. ΔsulE lost the de-esterification ability and was more sensitive to the herbicides than the wild type of strain S113, suggesting that sulE played a vital role in the sulfonylurea herbicide resistance of the strain. The transfer of sulE into Saccharomyces cerevisiae BY4741 conferred on it the ability to de-esterify sulfonylurea herbicides and increased its resistance to the herbicides. This study has provided an excellent candidate for the mechanistic study of sulfonylurea herbicide metabolism and detoxification through de-esterification, construction of sulfonylurea herbicide-resistant transgenic crops, and bioremediation of sulfonylurea herbicide-contaminated environments.     

细胞色素P450与除草剂抗性转基因植物

介绍了除草剂代谢有关的细胞色素P450基因及其应用,已从动植物体中分离具有除草剂代谢活性的细胞色素P450基因,通过转基因方法,成功培育出抗除草剂的转基因植物。     

化学除草剂对农田生态系统野生植物多样性的影响

农田生物多样性是全球生物多样性的重要组成部分, 除草剂的大量施用对其产生了严重影响。本文综述了化学除草剂对农田生态系统中野生植物多样性的影响, 并分析归纳了其影响机制。除草剂的施用会使敏感植物减少, 抗药性植物增多, 从而改变农田生态系统中的野生植物物种组成, 并使其趋同化, 降低遗传多样性和物种多样性, 以致植物功能群单一化, 群落稳定性下降。除草剂的主要影响机制是杀死植物或改变其生长代谢、抗性、繁殖等, 改变生境, 并与人为因素、环境因素等产生协同影响。不同种类的除草剂影响程度不同, 且不同物种间、不同群落间的响应也存在差异。我国化学除草剂使用量持续增长, 应加强除草剂对野生植物多样性的影响及其机制研究, 重视除草剂使用历史记录和野生植物的长期监测, 以及除草剂使用规范和相关政策法律研究, 更好地保护我国农田生态系统中的生物多样性。     

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.     

The ABC transporter SpTUR2 confers resistance to the antifungal diterpene sclareol

PDR5-like proteins represent one group of the ABC superfamily of transporters. Members of this group are present in plants and, due to the function of PDR5-related proteins in fungi in the excretion of xenobiotics (including antifungal agents), it has been proposed that they might play a similar role in plants in the response to and detoxification of herbicides and fungicides. However, until now no functional data has been presented showing an altered plant response to any herbicide or fungicide as a result of manipulating the expression of a PDR5-like gene in plants. In this paper, we show that the plant SpTUR2 PDR5-like ABC transporter is localised to the plasma membrane and that expression of this protein in Arabidopsis leads to the acquisition of resistance to the diterpenoid antifungal agent sclareol. These data both define a possible endogenous substrate for this transporter and highlight the potential of manipulating plant chemical resistance via modulating the expression of specific PDR5-like transporters.     

The plasma membrane-bound phospholipase Ddelta enhances freezing tolerance in Arabidopsis thaliana

Freezing injury is a major environmental limitation on the productivity and geographical distribution of plants. Here we show that freezing tolerance can be manipulated in Arabidopsis thaliana by genetic alteration of the gene encoding phospholipase Ddelta (PLDdelta), which is involved in membrane lipid hydrolysis and cell signaling. Genetic knockout of the plasma membrane-associated PLDdelta rendered A. thaliana plants more sensitive to freezing, whereas overexpression of PLDdelta increased freezing tolerance. Lipid profiling revealed that PLDdelta contributed approximately 20% of the phosphatidic acid produced in wild-type plants during freezing, and overexpression of PLDdelta increased the production of phosphatidic acid species. The PLDdelta alterations did not affect the expression of the cold-regulated genes COR47 or COR78 or alter cold-induced increases in proline or soluble sugars, suggesting that the PLD pathway is a unique determinant of the response to freezing and may present opportunities for improving plant freezing tolerance.     

Paraquat resistance of transgenic tobacco plants over-expressing the Ochrobactrum anthropi pqrA gene

Transgenic tobacco plants over-expressing the Ochrobactrum anthropi pqrA gene, which encodes a membrane transporter mediating resistance to paraquat, were generated. Transgenic plants displayed higher resistance against paraquat than wild-type plants, as estimated by plant viability, ion leakage and chlorophyll loss, but no resistance against other active oxygen generators, such as H2O2 and menadione. Moreover, lower levels of paraquat accumulated in transgenic plants, compared to wild-type plants, indicating that the PqrA protein detoxifies paraquat either via increased efflux or decreased uptake of the herbicide, but not by removing active oxygen species. The results collectively demonstrate that the bacterial paraquat resistance gene, pqrA, can be functionally expressed in plant cells, and utilized for the development of paraquat-resistant crop plants.     

Weed response to herbicides: regional-scale distribution of herbicide resistance alleles in the grass weed Alopecurus myosuroides

Effective herbicide resistance management requires an assessment of the range of spatial dispersion of resistance genes among weed populations and identification of the vectors of this dispersion. In the grass weed Alopecurus myosuroides (black-grass), seven alleles of the acetyl-CoA carboxylase (ACCase) gene are known to confer herbicide resistance. Here, we assessed their respective frequencies and spatial distribution on two nested geographical scales (the whole of France and the French administrative district of C?te d'Or) by genotyping 13 151 plants originating from 243 fields. Genetic variation in ACCase was structured in local populations at both geographical scales. No spatial structure in the distribution of resistant ACCase alleles and no isolation by distance were detected at either geographical scale investigated. These data, together with ACCase sequencing and data from the literature, suggest that evolution of A. myosuroides resistance to herbicides occurred at the level of the field or group of adjacent fields by multiple, independent appearances of mutant ACCase alleles that seem to have rather restricted spatial propagation. Seed transportation by farm machinery seems the most likely vector for resistance gene dispersal in A. myosuroides.     

Mutant genes of plant tubulins as selective marker genes for genetic engineering

This review describes different approaches to employment of new marker genes in selection of transformed plant cells, which are based on the use of mutant tubulin genes from natural plant biotypes and, in prospect, induced plant mutants. The results of studies of plant (biotypes, mutants) resistance to herbicides with antimicrotubular mode of action at molecular and cellular levels were summarized. The reports on the transfer and expression of mutant tubulin genes conferring resistance to amiprophosmethyl (phosphorothioamidate herbicide) and trifluralin (dinitroaniline herbicide) from corresponding Nicotiana plumbaginifolia mutants in related and remote plant species by somatic hybridization methods were analyzed. The results of experiments on transformation of monocotyledonous and dicotyledonous plants by mutant α-tubulin gene conferring resistance to dinitroanilines are described to test the possibility of its use as a marker gene and simultaneously obtaining dinitroaniline-resistant plants.     

A role for glutathione transferases functioning as glutathione peroxidases in resistance to multiple herbicides in black-grass

Black-grass (Alopecurus myosuroides) is a major weed of wheat in Europe, with several populations having acquired resistance to multiple herbicides of differing modes of action. As compared with herbicide-susceptible black-grass, populations showing herbicide cross-resistance contained greatly elevated levels of a specific type I glutathione transferase (GST), termed AmGST2, but similar levels of a type III GST termed AmGST1. Following cloning and expression of the respective cDNAs, AmGST2 differed from AmGST1 in showing limited activity in detoxifying herbicides but high activities as a glutathione peroxidase (GPOX) capable of reducing organic hydroperoxides. In contrast to AmGST2, other GPOXs were not enhanced in the herbicide-resistant populations. Treatment with a range of herbicides used to control grass weeds in wheat resulted in increased levels of hydroperoxides in herbicide-susceptible populations but not in herbicide-resistant plants, consistent with AmGST2 functioning to prevent oxidative injury caused as a primary or secondary effect of herbicide action. Increased AmGST2 expression in black-grass was associated with partial tolerance to the peroxidizing herbicide paraquat. The selective enhancement of AmGST2 expression resulted from a constitutively high expression of the respective gene, which was activated in herbicide-susceptible black-grass in response to herbicide safeners, dehydration and chemical treatments imposing oxidative stress. Our results provide strong evidence that GSTs can contribute to resistance to multiple herbicides by playing a role in oxidative stress tolerance in addition to detoxifying herbicides by catalysing their conjugation with glutathione.