氨基酸生物合成抑制剂类除草剂作用机理及耐除草剂转基因植物研究进展

氨基酸是植物体内必不可少的物质,在植物的生长代谢中发挥着重要作用。与动物不同,植物的氨基酸供给全部靠自身来合成,一旦植物的氨基酸合成受阻,植物便难以继续生存。因此,植物氨基酸合成中的关键酶一直是新型除草剂研发中重要的靶标酶。在目前已经商品化的除草剂中,通过抑制植物氨基酸生物合成中的关键酶活性而发生作用的除草剂占很大比重;与此同时,随着植物转基因技术的不断发展完善,大批耐氨基酸生物合成抑制剂类除草剂转基因植物相继问世,成为了耐除草剂类转基因植物的主体。本文综述了常用的耐氨基酸生物合成抑制剂类除草剂、作用机理及耐除草剂转基因植物的研究进展。     

Impairment of carbon metabolism induced by the herbicide glyphosate

The herbicide glyphosate reduces plant growth and causes plant death by inhibiting the biosynthesis of aromatic amino acids. The objective of this work was to determine whether glyphosate-treated plants show a carbon metabolism pattern comparable to that of plants treated with herbicides that inhibit branched-chain amino acid biosynthesis. Glyphosate-treated plants showed impaired carbon metabolism with an accumulation of carbohydrates in the leaves and roots. The growth inhibition detected after glyphosate treatment suggested impaired metabolism that impedes the utilization of available carbohydrates or energy at the expected rate. These effects were common to both types of amino acid biosynthesis inhibitors. Under aerobic conditions, ethanolic fermentative metabolism was enhanced in the roots of glyphosate-treated plants. This fermentative response was not related to changes in the respiratory rate or to a limitation of the energy charge. This response, which was similar for both types of herbicides, might be considered a general response to stress conditions.     

Phytotoxic and Metabolic Effects of Exogenous Quinate on Pisum sativum L.

Quinate (1,3,4,5-tetrahydroxycyclohexanecarboxylate) is a compound synthesized in plants through a side branch of the shikimate biosynthesis pathway. Plants treated with herbicides that inhibit amino acid biosynthesis (branched-chain and aromatic) accumulate quinate in their leaves. The objective of this study was to evaluate whether quinate mimics the effects of herbicides in plants. In pea plants, exogenous application of quinate through the nutrient solution was compared with leaf spraying at a concentration of 4 and 400 mM, respectively, and evaluated in parallel to the effects of herbicides. The analysis facilitated an assessment of the phytotoxicity and potential use of quinate as a natural herbicide. The application of quinate through the nutrient solution, but not the spray, was lethal, although both treatments affected plant growth. Quinate was absorbed and translocated to other plant organs remote from the application site, and an increase in the levels of aromatic amino acids and caffeic acid (that is, compounds located after quinate in the shikimate biosynthesis pathway) was detected, which indicates that quinate was metabolized and incorporated into the shikimate pathway. Exogenous application of quinate affected the carbohydrate content in the leaves and roots in a way similar to the toxic effects of herbicides. The phytotoxic effects of quinate reported in this study suggest that this compound deregulates the shikimate pathway and mimics some physiological effects described in the mode of action of herbicides inhibiting amino acid biosynthesis.     

谷子叶绿体乙酰辅酶A羧化酶cDNA的克隆和禾本科除草剂靶位点的序列分析

乙酰辅酶A羧化酶是一个生物素羧化酶,它所催化的反应是脂肪酸生物合成中的第一个植物叶绿体中的乙酰辅酶A羧化酶是两类禾本科除草剂的靶蛋白.从抗除草剂拿捕净和感拿捕净的谷子(SetariaitalicaBeauv.)中克隆了两个乙酰辅酶A羧化酶的全长cDNA,分别命名为foxACC-R和foxACC-S,它们推导的蛋白质均编码2 321个氨基酸,然而在第1 780个氨基酸处,foxACC-R编码亮氨酸,而foxACC-S编码异亮氨酸.采用生物信息学方法,我们推断这个cDNA编码的是叶绿体中的乙酰辅酶A羧化酶,并预测了它的功能域和保守区.通过这两个cDNA编码的氨基酸序列与其他乙酰辅酶A羧化酶的序列比较得出结论,亮氨酸/异亮氨酸位点可能是APPs和CHDs两类除草剂作用的关键位点.Southern杂交分析的结果显示,该基因在谷子基因组中只有一个拷贝.     

An acetohydroxy acid synthase mutant reveals a single site involved in multiple herbicide resistance

Acetohydroxy acid synthase (AHAS) is an essential enzyme for many organisms as it catalyzes the first step in the biosynthesis of the branched-chain amino acids valine, isoleucine, and leucine. The enzyme is under allosteric control by these amino acids. It is also inhibited by several classes of herbicides, such as the sulfonylureas, imidazolinones and triazolopyrimidines, that are believed to bind to a relic quinone-binding site. In this study, a mutant allele of AHAS3 responsible for sulfonylurea resistance in a Brassica napus cell line was isolated. Sequence analyses predicted a single amino acid change (557 TrpLeu) within a conserved region of AHAS. Expression in transgenic plants conferred strong resistance to the three classes of herbicides, revealing a single site essential for the binding of all the herbicide classes. The mutation did not appear to affect feedback inhibition by the branched-chain amino acids in plants.     

Acetolactate synthase mutation conferring imidazolinone-specific herbicide resistance in Amaranthus hybridus

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of branched-chain amino acids in plants and is the target of several herbicides. ALS inhibitors have enjoyed popularity as herbicides due to numerous attributes, although their current adequacy in weed control programs is hampered by herbicide resistance. Most cases of ALS-inhibitor resistance have resulted from selection of an altered target site. The study herein reports on an alanine by threonine amino acid substitution at position 122 of ALS as the basis for imidazolinone-specific resistance in an A. hybridus population from Illinois. In vitro inhibition of enzymatic activity (I(50)) required 1000-fold greater concentration of imazethapyr in the resistant population compared with a susceptible control. This mutation represents the second ALS alteration associated with herbicide resistance in a natural A. hybridus population.     

Engineering of cysteine and methionine biosynthesis in potato

Summary. Methionine and cysteine, two amino acids containing reduced sulfur, are not only an important substrate of protein biosynthesis but are also precursors of various other metabolites such as glutathione, phytochelatines, S-adenosylmethionine, ethylene, polyamines, biotin, and are involved as methyl group donor in numerous cellular processes. While methionine is an essential amino acid due to an inability of monogastric animals and human beings to synthesise this metabolite, animals are still able to convert methionine consumed with their diet into cysteine. Thus, a balanced diet containing both amino acids is necessary to provide a nutritionally favourable food or feed source. Because the concentrations of methionine and cysteine are often low in edible plant sources, e.g. potato, considerable efforts in plant breeding and research have been and are still performed to understand the physiological, biochemical, and molecular mechanisms that contribute to their synthesis, transport, and accumulation in plants. During the last decade molecular tools have enabled the isolation of most of the genes involved in cysteine and methionine biosynthesis, and the efficient plant transformation technology has allowed the creation of transgenic plants that are altered in the activity of individual genes. The physiological analysis of these transgenic plants has contributed considerably to our current understanding of how amino acids are synthesised. We focused our analysis on potato (Solanum tuberosum cv. Désirée) as this plant provides a clear separation of source and sink tissues and, for applied purposes, already constitutes a crop plant. From the data presented here and in previous work we conclude that threonine synthase and not cystathionine gamma-synthase as expected from studies of Arabidopsis constitutes the main regulatory control point of methionine synthesis in potato. This article aims to cover the current knowledge in the area of molecular genetics of sulfur-containing amino acid biosynthesis and will provide new data for methionine biosynthesis in solanaceous plants such as potato. Received December 19, 2001 Accepted January 7, 2002     

Amino acid biosynthetic enzymes as targets of herbicide action

Several structurally-unrelated herbicides act by blocking amino acid biosynthesis. Since amino acid metabolism is similar in plants and well-studied, manipulatable microbial systems, the opportunity exists for a particularly productive interaction between microbial and plant molecular biology. Such a symbiosis may lead to new methods for the identification and design of crop protection chemicals.     

Both foliar and residual applications of herbicides that inhibit amino acid biosynthesis induce alternative respiration and aerobic fermentation in pea roots

The objective of this work was to ascertain whether there is a general pattern of carbon allocation and utilisation in plants following herbicide supply, independent of the site of application: sprayed on leaves or supplied to nutrient solution. The herbicides studied were the amino acid biosynthesis‐inhibiting herbicides (ABIH): glyphosate, an inhibitor of aromatic amino acid biosynthesis, and imazamox, an inhibitor of branched‐chain amino acid biosynthesis. All treated plants showed impaired carbon metabolism; carbohydrate accumulation was detected in both leaves and roots of the treated plants. The accumulation in roots was due to lack of use of available sugars as growth was arrested, which elicited soluble carbohydrate accumulation in the leaves due to a decrease in sink strength. Under aerobic conditions, ethanol fermentative metabolism was enhanced in roots of the treated plants. This fermentative response was not related to a change in total respiration rates or cytochrome respiratory capacity, but an increase in alternative oxidase capacity was detected. Pyruvate accumulation was detected after most of the herbicide treatments. These results demonstrate that both ABIH induce the less‐efficient, ATP‐producing pathways, namely fermentation and alternative respiration, by increasing the key metabolite, pyruvate. The plant response was similar not only for the two ABIH but also after foliar or residual application.     

Enhanced levels of free and protein-bound threonine in transgenic alfalfa (Medicago sativa L.) expressing a bacterial feedback-insensitive aspartate kinase gene

Threonine, lysine, methionine, and tryptophan are essential amino acids for humans and monogastric animals. Many of the commonly used diet formulations, particularly for pigs and poultry, contain limiting amounts of these amino acids. One approach for raising the level of essential amino acids is based on altering the regulation of their biosynthetic pathways in transgenic plants. Here we describe the first production of a transgenic forage plant, alfalfa (Medicago sativa L.) with modified regulation of the aspartate-family amino acid biosynthetic pathway. This was achieved by over-expressing the Escherichia coli feedback-insensitive aspartate kinase (AK) in transgenic plants. These plants showed enhanced levels of both free and protein-bound threonine. In many transgenic plants the rise in free threonine was accompanied by a significant reduction both in aspartate and in glutamate. Our data suggest that in alfalfa, AK might not be the only limiting factor for threonine biosynthesis, and that the free threonine pool in this plant limits its incorporation into plant proteins.     

新型白化型除草剂靶标酶对羟苯基丙酮酸双加氧酶及其耐性转基因植物研究进展*

对羟苯基丙酮酸双加氧酶(ρ-hydroxyphenylpyruvate dioxygenase,HPPD;EC 1.13.11.27)催化生物体内对羟苯基丙酮酸与O2作用形成尿黑酸的反应,是植物体中质体醌和生育酚生物合成途径的关键酶。当其活性受到抑制时,植物体中作为类胡萝卜素生物合成途径中最终电子受体和光合链电子传递体的质体醌的生物合成受阻,进而导致类胡萝卜素合成减少,光合链电子传递受阻,致使植物体出现白化症状。目前已经开发了多种以HPPD为靶标的除草剂,该类除草剂及抗除草剂转基因植物研究具有广阔的前景。对这一新型白化型除草剂靶标酶以及耐该类除草剂转基因植物的研究进展作了简要综述。     

Biosynthesis of α-linolenic acid by desaturation of oleic and linoleic acids in several organs of higher and lower plants and in algae

The biosynthesis of α-linolenic acid by successive desaturations of oleic and linoleic acids has been shown to occur in the leaves, roots and seeds of many higher plants. The age and the physiological state of the plant organs are extremely important. This route occurs also in several lower plants including algae. It is concluded that the desaturation pathway is the major route for the biosynthesis of α-linolenic acid in plants.     

谷子叶绿体乙酰辅酶A羧化酶cDNA的克隆和禾本科除草剂靶位点的序列分析

乙酰辅酶A羧化酶是一个生物素羧化酶,它所催化的反应是脂肪酸生物合成中的第一个关键步骤。禾本科植物叶绿体中的乙酰辅酶A羧化酶是两类禾本科除草剂的靶蛋白。从抗除草剂拿捕净和感拿捕净的谷子(Setaria italicaBeauv.)中克隆了两个乙酰辅酶A羧化酶的全长cDNA,分别命名为foxACC-R和foxACC-S,它们推导的蛋白质均编码2 321个氨基酸,然而在第1 780个氨基酸处,foxACC-R编码亮氨酸,而foxACC-S编码异亮氨酸。采用生物信息学方法,我们推断这个cDNA编码的是叶绿体中的乙酰辅酶A羧化酶,并预测了它的功能域和保守区。通过这两个cDNA编码的氨基酸序列与其他乙酰辅酶A羧化酶的序列比较得出结论,亮氨酸/异亮氨酸位点可能是APPs和CHDs两类除草剂作用的关键位点。Southern 杂交分析的结果显示,该基因在谷子基因组中只有一个拷贝。     

正向选择的生物安全标记基因

标记基因的产生方便了植物的转化,随着转基因植物的迅速发展及商品化,人类更关注抗性标记基因的安全性。目前解决的有效途径是发展正向选择系统,使用非抗性的生物安全标记基因,主要包括糖类代谢酶基因（pmi和xylA）、干扰氨基酸代谢酶基因（ak和dapA）、绿色荧光蛋白基因（gfp）、β-葡萄糖苷酸酶基因（gus）、核糖醇操纵子（rtl）和叶绿素生物合成基因（hemL）等。     

Metabolic consequences of overproduction of phosphoenolpyruvate carboxylase in C3 plants

Phosphoenolpyruvate carboxylase (PEPC) has a variety of functions in plants, including a major anaplerotic role in replenishing the tricarboxylic acid cycle with intermediates to meet the demand of carbon skeletons for synthesis of organic acids and amino acids. Various transgenic C3 plants that overproduce PEPC have been produced and analyzed in detail. The results indicate that foreign PEPC is under the control of the regulatory mechanisms intrinsic to the host plant and down-regulated so as not to cause detrimental metabolic effects, although the anaplerotic reaction is slightly enhanced by the foreign PEPC. By use of foreign PEPCs that can avert such regulation, metabolic flow is largely directed toward synthesis of organic acids and amino acids. Observations with transgenic C3 plants also shed light on the interrelation among various metabolic pathways inside the cell.     

Evolution of the biosynthesis of the branched-chain amino acids

The origin of the biosynthetic pathways for the branched-chain amino acids cannot be understood in terms of the backwards development of the present acetolactate pathway because it contains unstable intermediates. We propose that the first biosynthesis of the branched-chain amino acids was by the reductive carboxylation of short branched chain fatty acids giving keto acids which were then transaminated. Similar reaction sequences mediated by nonspecific enzymes would produce serine and threonine from the abundant prebiotic compounds glycolic and lactic acids. The aromatic amino acids may also have first been synthesized in this way, e.g. tryptophan from indole acetic acid. The next step would have been the biosynthesis of leucine from -ketoisovaleric acid. The acetolactate pathway developed subsequently. The first version of the Krebs cycle, which was used for amino acid biosynthesis, would have been assembled by making use of the reductive carboxylation and leucine biosynthesis enzymes, and completed with the development of a single new enzyme, succinate dehydrogenase. This evolutionary scheme suggests that there may be limitations to inferring the origins of metabolism by a simple back extrapolation of current pathways.     

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.     

The relationship between the biosynthetic paths to the amino acids and their coding

The genetic code could not have been fixed until the means for biosynthesis of the amino acids was at hand. The biosynthetic enzymes could not be optimized until the genetic code ceased to be rearranged. Therefore the development of the code and the development of the biosynthesis of the amino acids occurred concurrently. The present day biosynthetic pathways of amino acids, examined from this point of view, help to explain the present set of coded amino acids, in particular the absence of norvaline, norleucine, homoserine, ornithine, and alpha-aminobutyric acid. An order of development of biosyntheses is also proposed. Lysine was first, followed by valine and isoleucine. The more common primordial amino acids did not need biosyntheses so early. The central pathways of metabolism probably developed in response to a need for amino acid biosynthesis.     

The molecular bases for resistance to acetyl co-enzyme A carboxylase (ACCase) inhibiting herbicides in two target-based resistant biotypes of annual ryegrass (Lolium rigidum)

Acetyl-CoA carboxylase (ACCase) (EC.6.4.1.2) is an essential enzyme in fatty acid biosynthesis and, in world agriculture, commercial herbicides target this enzyme in plant species. In nearly all grass species the plastidic ACCase is strongly inhibited by commercial ACCase inhibiting herbicides [aryloxyphenoxypropionate (APP) and cyclohexanedione (CHD) herbicide chemicals]. Many ACCase herbicide resistant biotypes (populations) of L. rigidum have evolved, especially in Australia. In many cases, resistance to ACCase inhibiting herbicides is due to a resistant ACCase enzyme. Two ACCase herbicide resistant L. rigidum biotypes were studied to identify the molecular basis of ACCase inhibiting herbicide resistance. The carboxyl-transferase (CT) domain of the plastidic ACCase gene was amplified by PCR and sequenced. Amino acid substitutions in the CT domain were identified by comparison of sequences from resistant and susceptible plants. The amino acid residues Gln-102 (CAG codon) and Ile-127 (ATA codon) were substituted with a Glu residue (GAG codon) and Leu residue (TTA codon), respectively, in both resistant biotypes. Amino acid positions 102 and 127 within the fragment sequenced from L. rigidum corresponded to amino acid residues 1756 and 1781, respectively, in the A. myosuroides full ACCase sequence. Allele-specific PCR results further confirmed the mutations linked with resistance in these populations. The Ile-to-Leu substitution at position 1781 has been identified in other resistant grass species as endowing resistance to APP and CHD herbicides. The Gln-to-Glu substitution at position 1756 has not previously been reported and its role in herbicide resistance remains to be established.