Tolerance of transgenic canola expressing 1-aminocyclopropane-1-carboxylic acid deaminase to growth inhibition by nickel.

Plant growth-promoting bacteria are useful to phytoremediation strategies in that they confer advantages to plants in contaminated soil. When plant growth-promoting bacteria contain the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, the bacterial cell acts as a sink for ACC, the immediate biosynthetic precursor of the plant growth regulator ethylene thereby lowering plant ethylene levels and decreasing the negative effects of various environmental stresses. In an effort to gain the advantages provided by bacterial ACC deaminase in the phytoremediation of metals from the environment two transgenic canola lines with the gene for this enzyme were generated and tested. In these transgenic canola plants, expression of the ACC deaminase gene is driven by either tandem constitutive cauliflower mosaic virus (CaMV) 35S promoters or the root specific rolD promoter from Agrobacterium rhizogenes. Following the growth of transgenic and non-transformed canola in nickel contaminated soil, it was observed that the rolD plants demonstrate significantly increased tolerance to nickel compared to the non-transformed control plants.     

Regulation of Expression of the prb-1b / ACC Deaminase Gene by UV-8 in Transgenic Tomatoes

Transgenic tomato plants with 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase gene from Enterobacter cloacae UW4 under the control of a pathogenesis-related promoter (prb-1b) from tobacco were challenged by abiotic stresses to determine the expression patterns ofthe transgene. No ACC deaminase RNA or protein was detected by RT-PCR and in western blots prepared from leaf proteins of transgenic plants after wounding or treatment with α-amino butyric acid, xylanase, ethephon, salicylic acid, jasmonic acid, ethylene, or ethylene plus jasmonic acid. However, expression of the ACC deaminase transgene was observed in leaves and roots oftransformed tomato lines exposed to UV light. The UV response required a minimum of 48 h of exposure and was specific to UV-8 light.     

ACC deaminase from plant growth-promoting bacteria affects crown gall development

In addition to the well-known roles of indoleacetic acid and cytokinin in crown gall formation, the plant hormone ethylene also plays an important role in this process. Many plant growth-promoting bacteria (PGPB) encode the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which can degrade ACC, the immediate precursor of ethylene in plants, to alpha-ketobutyrate and ammonia and thereby lower plant ethylene levels. To study the effect of ACC deaminase on crown gall development, an ACC deaminase gene from the PGPB Pseudomonas putida UW4 was introduced into Agrobacterium tumefaciens C58, so that the effect of ACC deaminase activity on tumour formation in tomato and castor bean plants could be assessed. Plants were also coinoculated with A. tumefaciens C58 and P. putida UW4 or P. putida UW4-acdS- (an ACC deaminase minus mutant strain). In both types of experiments, it was observed that the presence of ACC deaminase generally inhibited tumour development on both tomato and castor bean plants.     

Effect of transferring 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase genes into Pseudomonas fluorescens strain CHA0 and its gacA derivative CHA96 on their growth-promoting and disease-suppressive capacities

Pseudomonas fluorescens strain CHA0, a root colonizing bacterium, has a broad spectrum of biocontrol activity against plant diseases. However, strain CHA0 is unable to utilize 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of plant ethylene, as a sole source of nitrogen. This suggests that CHA0 does not contain the enzyme ACC deaminase, which cleaves ACC to ammonia and alpha-ketobutyrate, and was previously shown to promote root elongation of plant seedlings treated with bacteria containing this enzyme. An ACC deaminase gene, together with its regulatory region, was transferred into P. fluorescens strains CHA0 and CHA96, a global regulatory gacA mutant of CHA0. ACC deaminase activity was expressed in both CHA0 and CHA96. Transformed strains with ACC deaminase activity increased root length of canola plants under gnotobiotic conditions, whereas strains without this activity had no effect. Introduction of ACC deaminase genes into strain CHA0 improved its ability to protect cucumber against Pythium damping-off, and potato tubers against Erwinia soft rot in small hermetically sealed containers. In contrast, ACC deaminase activity had no significant effect on the ability of CHA0 to protect tomato against Fusarium crown and root rot, and potato tubers against soft rot in large hermetically sealed containers. These results suggest that (i) ACC deaminase activity may have lowered the level of plant ethylene thereby increasing root length; (ii) the role of stress-generated plant ethylene in susceptibility or resistance depends on the host-pathogen system, and on the experimental conditions used; and (iii) the constructed strains could be developed as biosensors for the role of ethylene in plant diseases.     

Perspectives of bacterial ACC deaminase in phytoremediation

Phytoremediation of contaminated soil and water environments is regulated and coordinated by the plant root system, yet root growth is often inhibited by pollutant-induced stress. Prolific root growth could maximize rates of hyperaccumulation of inorganic contaminants or rhizodegradation of organic pollutants, and thus accelerate phytoremediation. Accelerated ethylene production in response to stress induced by contaminants is known to inhibit root growth and is considered as a major limitation in improving phytoremediation efficiency. Recent work shows that bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase regulates ethylene levels in plants by metabolizing its precursor ACC into alpha-ketobutyric acid and ammonia. Plants inoculated with ACC deaminase bacteria or transgenic plants that express bacterial ACC deaminase genes can regulate their ethylene levels and consequently contribute to a more extensive root system. Such proliferation of roots in contaminated soil can lead to enhanced uptake of heavy metals or rhizodegradation of xenobiotics.     

Reduced symptoms of Verticillium wilt in transgenic tomato expressing a bacterial ACC deaminase

Ethylene evolved during compatible or susceptible disease interactions may hasten and/or worsen disease symptom development; if so, the prevention of disease-response ethylene should reduce disease symptoms. We have examined the effects of reduced ethylene synthesis on Verticillium wilt (causal organism, Verticillium dahliae) of tomato by transforming tomato with ACC deaminase, which cleaves ACC, the immediate biosynthetic precursor of ethylene in plants. Three promoters were used to express ACC deaminase in the plant: (i) CaMV 35S (constitutive expression); (ii) rolD (limits expression specifically to the site of Verticillium infection, i.e. the roots); and (iii) prb-1b (limits expression to certain environmental cues, e.g. disease infection). Significant reductions in the symptoms of Verticillium wilt were obtained for rolD- and prb-1b-, but not for 35S-transformants. The pathogen was detected in stem sections of plants with reduced symptoms, suggesting that reduced ethylene synthesis results in increased disease tolerance. The effective control of formerly recalcitrant diseases such as Verticillium wilt may thus be obtained by preventing disease-related ethylene production via the tissue-specific expression of ACC deaminase.     

通过表达ACC脱氨酶基因控制番茄果实的成熟

乙烯在跃变型果实的成熟过程中起着触发呼吸跃变和促进果实成熟的作用。细菌来源的1-氨基环丙烷-1-羧酸(ACC)脱氨酶能降解乙烯的直接前体ACC,从而抑制植物体内乙烯的合成。我们用PCR方法从假单孢杆菌中克隆到ACC脱氨酶基因并通过农杆菌介导的方法将其转入番茄(Lycopersicun esculentum)中。再生植株经Southern blot检测证明,ACC脱氨酶基因已整合到番茄基因组中并稳定表达。转基因番茄果实成熟期的推迟时间与体内乙烯的抑制程度有相关性。转基因番茄植株乙烯的合成降低80%左右,果实在离体条件下可保鲜75d左右。研究ACC脱氢酶基因在植物体内的作用可阐明高等植物体内乙烯的作用机理并为培育耐贮藏果蔬品种打下基础。     

Pseudomonas brassicacearum strain Am3 containing 1-aminocyclopropane-1-carboxylate deaminase can show both pathogenic and growth-promoting properties in its interaction with tomato

The role of bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity in the interaction between tomato (Lycopersicon esculentum=Solanum lycopersicum) and Pseudomonas brassicacearum was studied in different strains. The phytopathogenic strain 520-1 possesses ACC deaminase activity, an important trait of plant growth-promoting rhizobacteria (PGPR) that stimulates root growth. The ACC-utilizing PGPR strain Am3 increased in vitro root elongation and root biomass of soil-grown tomato cv. Ailsa Craig at low bacterial concentrations (10(6) cells ml-1 in vitro and 10(6) cells g-1 soil) but had negative effects on in vitro root elongation at higher bacterial concentrations. A mutant strain of Am3 (designated T8-1) that was engineered to be ACC deaminase deficient failed to promote tomato root growth in vitro and in soil. Although strains T8-1 and 520-1 inhibited root growth in vitro at higher bacterial concentrations (>10(6) cells ml-1), they did not cause disease symptoms in vitro after seed inoculation, or in soil supplemented with bacteria. All the P. brassicacearum strains studied caused pith necrosis when stems or fruits were inoculated with a bacterial suspension, as did the causal organism of this disease (P. corrugata 176), but the non-pathogenic strain Pseudomonas sp. Dp2 did not. Strains Am3 and T8-1 were marked with antibiotic resistance and fluorescence to show that bacteria introduced to the nutrient solution or on seeds in vitro, or in soil were capable of colonizing the root surface, but were not detected inside root tissues. Both strains showed similar colonization ability either on root surfaces or in wounded stems. The results suggest that bacterial ACC deaminase of P. brassicacearum Am3 can promote growth in tomato by masking the phytopathogenic properties of this bacterium.     

Perspective of plant growth promoting rhizobacteria (PGPR) containing ACC deaminase in stress agriculture

Ethylene is a gaseous plant growth hormone produced endogenously by almost all plants. It is also produced in soil through a variety of biotic and abiotic mechanisms, and plays a key role in inducing multifarious physiological changes in plants at molecular level. Apart from being a plant growth regulator, ethylene has also been established as a stress hormone. Under stress conditions like those generated by salinity, drought, waterlogging, heavy metals and pathogenicity, the endogenous production of ethylene is accelerated substantially which adversely affects the root growth and consequently the growth of the plant as a whole. Certain plant growth promoting rhizobacteria (PGPR) contain a vital enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, which regulates ethylene production by metabolizing ACC (an immediate precursor of ethylene biosynthesis in higher plants) into α-ketobutyrate and ammonia. Inoculation with PGPR containing ACC deaminase activity could be helpful in sustaining plant growth and development under stress conditions by reducing stress-induced ethylene production. Lately, efforts have been made to introduce ACC deaminase genes into plants to regulate ethylene level in the plants for optimum growth, particularly under stressed conditions. In this review, the primary focus is on giving account of all aspects of PGPR containing ACC deaminase regarding alleviation of impact of both biotic and abiotic stresses onto plants and of recent trends in terms of introduction of ACC deaminase genes into plant and microbial species.     

Real time expression of ACC oxidase and PR-protein genes mediated by Methylobacterium spp. in tomato plants challenged with Xanthomonas campestris pv. vesicatoria

Biotic stress like pathogenic infection increases ethylene biosynthesis in plants and ethylene inhibitors are known to alleviate the severity of plant disease incidence. This study aimed to reduce the bacterial spot disease incidence in tomato plants caused by Xanthomonas campestris pv. vesicatoria (XCV) by modulating stress ethylene with 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity of Methylobacterium strains. Under greenhouse condition, Methylobacterium strains inoculated and pathogen challenged tomato plants had low ethylene emission compared to pathogen infected ones. ACC accumulation and ACC oxidase (ACO) activity with ACO related gene expression increased in XCV infected tomato plants over Methylobacterium strains inoculated plants. Among the Methylobacterium spp., CBMB12 resulted lowest ACO related gene expression (1.46 Normalized Fold Expression), whereas CBMB20 had high gene expression (3.42 Normalized Fold Expression) in pathogen challenged tomato. But a significant increase in ACO gene expression (7.09 Normalized Fold Expression) was observed in the bacterial pathogen infected plants. In contrast, Methylobacterium strains enhanced β-1,3-glucanase and phenylalanine ammonia-lyase (PAL) enzyme activities in pathogen challenged tomato plants. The respective increase in β-1,3-glucanase related gene expressions due to CBMB12, CBMB15, and CBMB20 strains were 66.3, 25.5 and 10.4% higher over pathogen infected plants. Similarly, PAL gene expression was high with 0.67 and 0.30 Normalized Fold Expression, in pathogen challenged tomato plants inoculated with CBMB12 and CBMB15 strains. The results suggest that ethylene is a crucial factor in bacterial spot disease incidence and that methylobacteria with ACC deaminase activity can reduce the disease severity with ultimate pathogenesis-related protein increase in tomato.     

植物修复重金属污染及内生细菌效应

土壤和水体的重金属污染已严重危害人类生存环境与健康。由于受重金属污染的环境分布广泛,迫切需要开发经济的清除环境重金属的技术。植物修复是通过绿色植物降解或移除环境污染物,有望成为重金属污染环境的原位修复技术。植物内生菌是指定殖于健康植物的各种组织和器官内部的细菌,被感染的宿主植物不表现出外在病症,耐重金属的内生菌在多种超富集植物中存在。在植物修复过程中,野生型内生菌或基因工程内生菌的抗性系统能降低重金属植物毒性,促进其迁移金属。耐重金属内生菌还可以通过固氮、溶解矿物元素及产生类植物激素、铁载体和ACC脱氨酶等产物促进植物的生长。主要综述目前植物-内生菌相互作用及其潜在的促进植物修复重金属污染的研究进展。     

Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria

One of the major mechanisms utilized by plant growth-promoting rhizobacteria (PGPR) to facilitate plant growth and development is the lowering of ethylene levels by deamination of 1-aminocyclopropane-1-carboxylic acid (ACC) the immediate precursor of ethylene in plants. The enzyme catalysing this reaction, ACC deaminase, hydrolyses ACC to α -ketobutyrate and ammonia. Several bacterial strains that can utilize ACC as a sole source of nitrogen have been isolated from rhizosphere soil samples. All of these strains are considered to be PGPR based on the ability to promote canola seedling root elongation under gnotobiotic conditions. The treatment of plant seeds or roots with these bacteria reduces the amount of ACC in plants, thereby lowering the concentration of ethylene. Here, a rapid procedure for the isolation of ACC deaminase-containing bacteria, a root elongation assay for evaluating the effects of selected bacteria on root growth, and a method of assessing bacterial ACC deaminase activity are described in detail. This should allow researchers to readily isolate new PGPR strains adapted to specific environments.     

Transformation of Azospirillum brasilense Cd with an ACC deaminase gene from enterobacter cloacae UW4 fused to the Tet r gene promoter improves its fitness and plant growth promoting ability

It has been reported that PGPB, containing ACC deaminase, can cleave the plant ethylene precursor ACC and thereby lower ethylene concentration in a developing or stressed plant, protecting it against the deleterious effects of stress ethylene and facilitating the formation of longer roots. In a previous work we have demonstrated expression of the ACC deaminase gene (acdS) from Enterobacter cloacae UW4 under the control of the lac promoter in Azospirillum brasilense Cd. With the inference that a construct including the ACC deaminase gene under the control of a constitutive promoter weaker than the lac promoter might impose less metabolic load on Azospirillum and improve its fitness, it was decided to clone acdS under the control of a tetracycline resistance gene promoter. The ACC deaminase structural gene was fused to the Tet ^r gene promoter by overlap extension using PCR, cloned in pRK415, and transferred into A. brasilense Cd. The resulting transformants showed lower ACC deaminase activity than those with the lac promoter controlled acdS gene. However, acdS under the control of the Tet ^r gene promoter imposed lesser metabolic load on Azospirillum brasilense Cd. The result was significantly increased IAA synthesis and greater bacterial growth rate, as well as increased ability to survive on the surface of tomato leaves and to promote the growth of tomato seedlings.     

Azospirillum lipoferum strain AZm5 containing 1-aminocyclopropane-1-carboxylic acid deaminase improves early growth of tomato seedlings under nitrogen deficiency

In this study we evaluated the ability of two wild strains of Azospirillum, A. lipoferum AZm5 and A. brasilense VS9, to produce ACC deaminase. We tested the effects of a deficiency and medium doses of nitrogenous fertilizers on the growth and physiology of tomato plants (Lycopersicon esculentum Mill cv. ACE VF55) inoculated with both Azospirillum strains independently. Tomato plants were evaluated by root elongation assay and grown in pot soil culture with different nitrogen levels (0 kg N ha^–1 and 170 kg N ha^–1). The root:shoot ratio (R:S) and some ecophysiological traits were determined after 42 days of plant growth. Results showed very different physiological characteristics in both strains. We found three relevant aspects related to the AZm5 strain: it produces high amounts of cytokinins, it contains the gene acdS, which encodes ACC deaminase, and it promotes plant growth. We conclude that AZm5 maybe useful to increase N uptake in N-deficient soil by production of cytokinins and the promotion of ACC deaminase activity, which favored leaf expansion and higher leaf N investment. Therefore, for tomato culture, a simultaneous biofertilization with AZm5 and a relatively low fertilization with N (170 kg N ha^–1) to promote AZm5 activity could be advantageous.     

Expression of sarcotoxin IA gene via a root-specific tob promoter enhanced host resistance against parasitic weeds in tomato plants

We have developed a simple genetic engineering strategy for conferring resistance against parasitic weeds on host plants. Transgenic tomato plants expressing the sarcotoxin IA gene were grown either in polyethylene bags (PE) or in pots inoculated with Orobanche aegyptiaca seeds. The results indicate that transgenic plants exhibited strong inhibition of parasite growth and significantly increased yield as compared with non-transgenic ones. In both PE and pot systems most of the parasite tubercles attached to the transgenic root plants turned necrotic and developed abnormally. Integration and expression of the gene were confirmed by Southern blot, RT-PCR and Western blot analysis. Our results indicate that the insect gene produced in the plant cells was selectively toxic to the parasite and non-toxic to the host plant.     

The interconversion of ACC deaminase and <Emphasis Type="SmallCaps">d</Emphasis>-cysteine desulfhydrase by directed mutagenesis

Progress in DNA sequencing of plant genomes has revealed that, in addition to microorganisms, a number of plants contain genes which share similarity to microbial 1-aminocyclopropane-1-carboxylate (ACC) deaminases. These enzymes cleave ACC, the immediate precursor of ethylene in plants, into ammonia and alpha-ketobutyrate. We therefore sought to isolate putative ACC deaminase cDNAs from tomato plants with the objective of establishing whether the product of this gene is a functional ACC deaminase. In the work reported here, it was demonstrated that the enzyme encoded by the putative ACC deaminase cDNA does not have the ability to break the cyclopropane ring of ACC, but rather it utilizes D: -cysteine as a substrate, and in fact encodes a D: -cysteine desulfhydrase. Kinetic characterization of the tomato enzyme indicates that it is similar to other, previously characterized, D: -cysteine desulfhydrases. Using site-directed mutagenesis, it was shown that altering only two amino acid residues within the predicted active site served to change the enzyme from D: -cysteine desulfhydrase to ACC deaminase. Conversely, by altering two amino acid residues at the same positions within the active site of ACC deaminase from Pseudomonas putida UW4 the enzyme was converted into D: -cysteine desulfhydrase. Therefore, it is possible that a change in these two residues may have occurred in an ancestral protein to result in two different enzymatic activities.