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.     

Effectiveness of rhizobacteria containing ACC deaminase for growth promotion of peas (Pisum sativum) under drought conditions

A series of experiments were conducted to assess the effectiveness of rhizobacteria containing 1-aminocyclopropane- 1-carboxylate (ACC) deaminase for growth promotion of peas under drought conditions. Ten rhizobacteria isolated from the rhizosphere of different crops (peas, wheat, and maize) were screened for their growth promoting ability in peas under axenic condition. Three rhizobacterial isolates, Pseudomonas fluorescens biotype G (ACC-5), P. fluorescens (ACC-14), and P. putida biotype A (Q-7), were selected for pot trial on the basis of their source, ACC deaminase activity, root colonization, and growth promoting activity under axenic conditions. Inoculated and uninoculated (control) seeds of pea cultivar 2000 were sown in pots (4 seeds/pot) at different soil moisture levels (25, 50, 75, and 100% of field capacity). Results revealed that decreasing the soil moisture levels from 100 to 25% of field capacity significantly decreased the growth of peas. However, inoculation of peas with rhizobacteria containing ACC deaminase significantly decreased the "drought stress imposed effects" on growth of peas, although with variable efficacy at different moisture levels. At the lowest soil moisture level (25% field capacity), rhizobacterial isolate Pseudomonas fluorescens biotype G (ACC-5) was found to be more promising compared with the other isolates, as it caused maximum increases in fresh weight, dry weight, root length, shoot length, number of leaves per plant, and water use efficiency on fresh and dry weight basis (45, 150, 92, 45, 140, 46, and 147%, respectively) compared with respective uninoculated controls. It is highly likely that rhizobacteria containing ACC deaminase might have decreased the drought-stress induced ethylene in inoculated plants, which resulted in better growth of plants even at low moisture levels. Therefore, inoculation with rhizobacteria containing ACC deaminase could be helpful in eliminating the inhibitory effects of drought stress on the growth of peas.     

Isolation and characterization of ACC deaminase-producing fluorescent pseudomonads, to alleviate salinity stress on canola (Brassica napus L.) growth

Salinity stress is of great importance in arid and semi-arid areas of the world due to its impact in reducing crop yield. Under salinity stress, the amount of 1-aminocyclopropane-1-carboxylate (ACC), a precursor for ethylene production in plants, increases. Here, we conducted research under the hypothesis that isolated ACC deaminase-producing Pseudomonas fluorescens and Pseudomonas putida can alleviate the stressful effects of salinity on canola (Brassica napus L.) growth. The experiments were conducted in the Soil and Water Research Institute, Tehran, Iran. Seven experimental stages were conducted to isolate and characterize ACC deaminase-producing Pseudomonas fluorescens strains and to determine factors enhancing their growth and, consequently, their effects on the germination of canola seeds. Under salinity stress, in 14% of the isolates, ACC deaminase activity was observed, indicating that they were able to utilize ACC as the sole N-source. Bacterial strains differed in their ability to synthesize auxin and hydrogen cyanide compounds, as well as in their ACC deaminase activity. Under salinity stress, the rate of germinating seeds inoculated with the strains of ACC deaminase-producing Pseudomonas fluorescens and Pseudomonas putida, and seedling growth was significantly higher. These results indicate the significance of soil biological activities, including the activities of plant growth-promoting bacteria, in the alleviation of soil stresses such as salinity on plant growth.     

Isolation of ACC deaminase producing PGPR from rice rhizosphere and evaluating their plant growth promoting activity under salt stress

Aims

Bacteria possessing ACC deaminase activity reduce the level of stress ethylene conferring resistance and stimulating growth of plants under various biotic and abiotic stresses. The present study aims at isolating efficient ACC deaminase producing PGPR strains from the rhizosphere of rice plants grown in coastal saline soils and quantifying the effect of potent PGPR isolates on rice seed germination and seedling growth under salinity stress and ethylene production from rice seedlings inoculated with ACC deaminase containing PGPR.

Methods

Soils from root region of rice growing in coastal soils of varying salinity were used for isolating ACC deaminase producing bacteria and three bacterial isolates were identified following polyphasic taxonomy. Seed germination, root growth and stress ethylene production in rice seedlings following inoculation with selected PGPR under salt stress were quantified.

Results

Inoculation with selected PGPR isolates had considerable positive impacts on different growth parameters of rice including germination percentage, shoot and root growth and chlorophyll content as compared to uninoculated control. Inoculation with the ACC deaminase producing strains reduced ethylene production under salinity stress.

Conclusions

This study demonstrates the effectiveness of rhizobacteria containing ACC deaminase for enhancing salt tolerance and consequently improving the growth of rice plants under salt-stress conditions.     

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.     

Effect of a Nickel-Tolerant ACC Deaminase-Producing Pseudomonas Strain on Growth of Nontransformed and Transgenic Canola Plants

Four bacterial strains were isolated from soils at nickel-contaminated sites based on their ability to utilize 1-aminocyclopropane-1-carboxylate (ACC) as a sole source of nitrogen. The four isolates were all identified as Pseudomonas putida Biovar B, and subsequent testing revealed that they all exhibited traits previously associated with plant growth promotion (i.e., indoleacetic acid and siderophore production and ACC deaminase activity). These four strains were also tolerant of nickel concentrations of up to 13.2 mM in the culture medium. The strain, HS-2, selected for further characterization, was used in pot experiments to inoculate both nontransformed and transgenic canola plants (expressing a bacterial ACC deaminase gene in its roots). Plants inoculated with the HS-2 strain produced an increase in plant biomass as well as in nickel (Ni) uptake by shoots and roots. The results suggest that this strain is a potential candidate to be used as an inoculant in both phytoremediation protocols and in plant growth promotion.     

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.     

Synergistic interactions between the ACC deaminase-producing bacterium Pseudomonas putida UW4 and the AM fungus Gigaspora rosea positively affect cucumber plant growth

Bacteria producing 1-aminocyclopropane-1-carboxylate (ACC) deaminase modulate plant ethylene levels. Decreased ethylene levels increase plant tolerance to environmental stresses and promote legume nodulation. On the contrary, the role of ethylene in mycorrhizal symbiosis establishment is still controversial. In this work, the ACC deaminase-producing strain Pseudomonas putida UW4 AcdS+ and its mutant AcdS(-), impaired in ACC deaminase synthesis, were inoculated alone or in combination with the AM fungus Gigaspora rosea on cucumber. Mycorrhizal and bacterial colonization as well as plant growth and morphometric parameters were measured. The influence of each microorganism on the photosynthetic efficiency was evaluated on the second and fourth leaf. The strain AcdS+, but not the AcdS(-) mutant, increased AM colonization and arbuscule abundance. The mycorrhizal fungus, but not the bacterial strains, promoted plant growth. However, the AcdS+ strain, inoculated with G. rosea, induced synergistic effects on plant biomass, total root length and total leaf projected area. Finally, the photosynthetic performance index was increased by the strain UW4 AcdS+ inoculated in combination with G. rosea BEG9. These results suggest a key role of this enzyme in the establishment and development of AM symbiosis.     

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.     

焦化厂地肤根内解芘细菌的筛选及促生潜力

从多环芳烃耐受植物根内分离具多环芳烃降解功能的内生细菌并研究其促生特性,为内生菌协同宿主植物修复多环芳烃污染土壤提供基础.以长期受多环芳烃污染的焦化厂区生长的地肤为材料,从其根内分离出以芘和1-氨基环丙烷-1-羧酸(ACC)为唯一碳源和氮源的内生细菌8株.通过芘降解试验,筛选得到3株高效芘降解内生细菌KSE4、KSE7和KSE8,经鉴定分别为芽孢杆菌属、假单胞菌属和鞘氨醇菌属.通过液体培养试验,研究了3株菌在芘胁迫下产ACC脱氨酶的能力和对地肤种子萌发的影响.结果表明: 随着芘浓度(0~15 mg·L^-1)的升高,ACC脱氨酶活性降低,其中KSE7的效果最好,在芘浓度为15 mg·L^-1时,地肤发芽率和芽长分别比对照提高了44.8%和61.1%,在地肤-微生物修复焦化厂污染土壤的修复中具有一定的应用潜力.     

Detection and Characterization of ACC Deaminase in Plant Growth Promoting Rhizobacteria

The enzyme 1-aminocyclopropane-1-carboxylate deaminase converts ACC, the precursor of the plant hormone ethylene to α-ketobutyrate and ammonium. The enzyme has been identified in few soil bacteria, and is proposed to play a key role in plant growth promotion. In this study, the isolates of plant growth promoting rhizobacteria were screened for ACC deaminase activity based on their ability to grow on ACC as a sole nitrogen source. The selected isolates showed the presence of other plant growth promoting characteristics such as IAA production, phosphate solubilization and siderophore production. The role of ACC deaminase in lowering ethylene production under cadmium stress condition was also studied by measuring in vitro ethylene evolution by wheat seedlings treated with ACC deaminase positive isolates. Nucleic acid hybridization confirmed the presence of ACC deaminase gene (acdS) in the bacterial isolates.     

接种泡囊假单胞菌对土壤生物性质及A. corsicum吸收Ni的影响

采用室内模拟试验方法,研究了在水稻土、元江土和墨江土中添加泡囊假单胞菌（Pseulormanas vesicularis）后土壤中微生物种群数量、土壤酶活性和镍超积累植物Alyssum corsicum对土壤镍的富集效果.土壤接种泡囊假单胞菌70d后,水稻土中DTPA提取态镍较对照土中的明显减少、元江土和墨江土中的有所减少;土壤中细菌、真菌和放线菌数量增加,5种土壤酶活性提高.试验结果表明,水稻土、元江土、墨江土添加泡囊假单菌后植物地上部生物量较对照分别增加了29%、309%和43%,进而提高了A.corsicum自土壤中富集镍的效率：水稻土中增加54%,元江土中增加306%,墨江土中增加32%.泡囊假单胞菌这一新用途的发现,可为植物修复微生物制剂和基因工程菌的开发提供本土的微生物的菌种资源.     

Isolation, characterization, and use for plant growth promotion under salt stress, of ACC deaminase-producing halotolerant bacteria derived from coastal soil

In total, 140 halotolerant bacterial strains were isolated from both the soil of barren fields and the rhizosphere of six naturally growing halophytic plants in the vicinity of the Yellow Sea, near the city of Incheon in the Republic of Korea. All of these strains were characterized for multiple plant growth promoting traits, such as the production of indole acetic acid (IAA), nitrogen fixation, phosphorus (P) and zinc (Zn) solubilization, thiosulfate (S2O3) oxidation, the production of ammonia (NH3), and the production of extracellular hydrolytic enzymes such as protease, chitinase, pectinase, cellulase, and lipase under in vitro conditions. From the original 140 strains tested, on the basis of the latter tests for plant growth promotional activity, 36 were selected for further examination. These 36 halotolerant bacterial strains were then tested for 1- aminocyclopropane-1-carboxylic acid (ACC) deaminase activity. Twenty-five of these were found to be positive, and to be exhibiting significantly varying levels of activity. 16S rRNA gene sequencing analyses of the 36 halotolerant strains showed that they belong to 10 different bacterial genera: Bacillus, Brevibacterium, Planococcus, Zhihengliuella, Halomonas, Exiguobacterium, Oceanimonas, Corynebacterium, Arthrobacter, and Micrococcus. Inoculation of the 14 halotolerant bacterial strains to ameliorate salt stress (150 mM NaCl) in canola plants produced an increase in root length of between 5.2% and 47.8%, and dry weight of between 16.2% and 43%, in comparison with the uninoculated positive controls. In particular, three of the bacteria, Brevibacterium epidermidis RS15, Micrococcus yunnanensis RS222, and Bacillus aryabhattai RS341, all showed more than 40% increase in root elongation and dry weight when compared with uninoculated saltstressed canola seedlings. These results indicate that certain halotolerant bacteria, isolated from coastal soils, have a real potential to enhance plant growth under saline stress, through the reduction of ethylene production via ACC deaminase activity.     

Regulation of Ethylene Biosynthesis Under Salt Stress in Red Pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) by 1-Aminocyclopropane-1-Carboxylic Acid (ACC) Deaminase-producing Halotolerant Bacteria

The present study was carried out to understand the mechanism of salt stress amelioration in red pepper plants by inoculation of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase-producing halotolerant bacteria. In general, ethylene production, ACC concentration, ACC synthase (ACS), and ACC oxidase (ACO) enzyme activities increased with increasing levels of salt stress. Treatment with halotolerant bacteria reduced ethylene production by 47–64%, ACC concentration by 47–55% and ACO activity by 18–19% in salt-stressed (150 mmol NaCl) red pepper seedlings compared to uninoculated controls. ACS activity was lower in red pepper seedlings treated with Bacillus aryabhattai RS341 but higher in seedlings treated with Brevibacterium epidermidis RS15 (44%) and Micrococcus yunnanensis RS222 (23%) under salt-stressed conditions as compared to uninoculated controls. A significant increase was recorded in red pepper plant growth under salt stress when treated with ACC deaminase-producing halotolerant bacteria as compared to uninoculated controls. The results of this study collectively suggest that salt stress enhanced ethylene production by increasing enzyme activities of the ethylene biosynthetic pathway. Inoculation with ACC deaminase-producing halotolerant bacteria plays an important role in ethylene metabolism, particularly by reducing the ACC concentration, although a direct effect on reducing ACO activity was also observed. It is suggested that growth promotion in inoculated red pepper plants under inhibitory levels of salt stress is due to ACC deaminase activity present in the halotolerant bacteria.     

Combined use of Alkane-Degrading and Plant Growth-Promoting Bacteria Enhanced Phytoremediation of Diesel Contaminated soil

Inoculation of plants with pollutant-degrading and plant growth-promoting microorganisms is a simple strategy to enhance phytoremediation activity. The objective of this study was to determine the effect of inoculation of different bacterial strains, possessing alkane-degradation and 1-amino-cyclopropane-1-carboxylic acid (ACC) deaminase activity, on plant growth and phytoremediation activity. Carpet grass (Axonopus affinis) was planted in soil spiked with diesel (1% w/w) for 90 days and inoculated with different bacterial strains, Pseudomonas sp. ITRH25, Pantoea sp. BTRH79 and Burkholderia sp. PsJN, individually and in combination. Generally, bacterial application increased total numbers of culturable hydrocarbon-degrading bacteria in the rhizosphere of carpet grass, plant biomass production, hydrocarbon degradation and reduced genotoxicity. Bacterial strains possessing different beneficial traits affect plant growth and phytoremediation activity in different ways. Maximum bacterial population, plant biomass production and hydrocarbon degradation were achieved when carpet grass was inoculated with a consortium of three strains. Enhanced plant biomass production and hydrocarbon degradation were associated with increased numbers of culturable hydrocarbon-degrading bacteria in the rhizosphere of carpet grass. The present study revealed that the combined use of different bacterial strains, exhibiting different beneficial traits, is a highly effective strategy to improve plant growth and phytoremediation activity.     

Inoculation of 1-aminocyclopropane-1-carboxylate deaminase–producing bacteria along with biosurfactant application enhances the phytoremediation efficiency of Medicago sativa in hydrocarbon-contaminated soils

Phytoremediation efficiency of Alfa alfa (Medicago sativa) was evaluated in hydrocarbon-contaminated soil with the combined application of 1-aminocyclopropane-1-carboxylate (ACC) deaminase–producing Bacillus sp. PVMX4 and an isolated biosurfactant from this strain. Results on the plant growth–promoting (PGP) traits of Bacillus sp. PVMX4 revealed that phosphate (P) solubilization, indole-3-acetic acid (IAA) production, and ACC deaminase activity were not affected by low-concentration hydrocarbon amendment in the form of crude oil. Bacillus sp. PVMX4 was able to utilize crude oil as a sole carbon source in mineral salt medium (MSM), and this strain synthesized significant quantities of biosurfactant in growth medium quantified by an emulsification index of 69.2 EI_24% and surface tension reduction of 26.2 mN/m at the end of the experimental period. Biosurfactant, when partially purified and characterized by thin-layer chromatography (TLC) and Fourier transform infrared spectroscopy (FT-IR), revealed it to be a lipopeptide-type biosurfactant. Pilot-scale phytoremediation studies conducted under growth chamber conditions in hydrocarbon-contaminated soil using Medicago sativa along with combined application of ACC deaminase–containing bacteria and biosurfactant recorded 76.4% hydrocarbon degradation.     

Plant Growth-Promoting Bacteria Facilitate the Growth of Barley and Oats in Salt-Impacted Soil: Implications for Phytoremediation of Saline Soils

Plant growth-promoting bacteria (PGPB) strains that contain the enzyme 1-amino- cyclopropane-1-carboxylate (ACC) deaminase can lower stress ethylene levels and improve plant growth. In this study, ACC deaminase-producing bacteria were isolated from a salt-impacted (～50 dS/m) farm field, and their ability to promote plant growth of barley and oats in saline soil was investigated in pouch assays (1% NaCl), greenhouse trials (9.4 dS/m), and field trials (6–24 dS/m). A mix of previously isolated PGPB strains UW3 (Pseudomonas sp.) and UW4 (P. sp.) was also tested for comparison. Rhizobacterial isolate CMH3 (P. corrugata) and UW3+UW4 partially alleviated plant salt stress in growth pouch assays. In greenhouse trials, CMH3 enhanced root biomass of barley and oats by 200% and 50%, respectively. UW3+UW4, CMH3 and isolate CMH2 also enhanced barley and oat shoot growth by 100%–150%. In field tests, shoot biomass of oats tripled when treated with UW3+UW4 and doubled with CHM3 compared with that of untreated plants. PGPB treatment did not affect salt uptake on a per mass basis; higher plant biomass led to greater salt uptake, resulting in decreased soil salinity. This study demonstrates a method for improving plant growth in marginal saline soils. Associated implications for salt remediation are discussed.     

Multi-strain Inoculation with PGPR Producing ACC Deaminase is More Effective Than Single-strain Inoculation to Improve Wheat (<i>Triticum aestivum</i>) Growth and Yield

Rhizosphere bacteria that colonize plant roots and confer beneficial effects are referred as plant growth promoting rhizobacteria (PGPR). Among all PGPR, some rhizobacteria have an ability to produce ACC deaminase enzyme. This enzyme catalyzes stress ACC into a-ketobutyrate and ammonia instead of letting it to be converted to ethylene. Ethylene level rises in plants under stress conditions i.e., drought, salinity, poor soil fertility etc. As poor soil fertility is a big hurdle to achieve the optimum yield of crops, inoculation of ACC deaminase PGPR can overcome this problem to some extent. The aim of the current study was to examine the influence of multi-strain and single-strain inoculation of different ACC deaminase producing PGPR on wheat growth and yield. There were three PGPR strains, Enterobacter cloacae, Serratia ficaria and Burkholderia phytofirmans which were used as consortia and single-strain inoculations. The results showed that inoculation of E. cloacae + S. ficaria + B. phytofirmans significantly increased plant height (63%), spike length (61%), number of spikelets spike^-1 (61%), number of grains spike-1 (131%), 1000 grains weight (33%), grains yield (71%), straw yield (71%) and biological yield (68%) of wheat as compared to control. A significant improvement in N (37 and 200%), P (46 and 166%) and K (39 and 61%) of seeds and shoot respectively, validated the efficacious and more effective role of multi-strain (E. cloacae + S. ficaria + B. phytofirmans) inoculation over control. It is obviously concluded that multi-strain ACC deaminase producing PGPR inoculation is a better approach as compared to singlestrain inoculation for the improvement in growth and yield of wheat.     

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.     

Surface display of ACC deaminase on endophytic <Emphasis Type="Italic">Enterobacteriaceae</Emphasis> strains to increase saline resistance of host rice sprouts by regulating plant ethylene synthesis

Background

Most endophytic bacteria in consortia, which provide robust and broad metabolic capacity, are attractive for applications in plant metabolic engineering. The aim of this study was to investigate the effects of engineered endophytic bacterial strains on rice sprout ethylene level and growth under saline stress. A protocol was developed to synthesize engineered strains by expressing bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene on cells of endophytic Enterobacter sp. E5 and Kosakonia sp. S1 (denoted as E5P and S1P, respectively).

Results

Results showed that ACC deaminase activities of the engineered strains E5P and S1P were significantly higher than those of the wild strains E5 and S1. About 32–41% deaminase was expressed on the surface of the engineered strains. Compared with the controls without inoculation, inoculation with the wild and engineered strains increased the deaminase activities of sprouts. Inoculation with the engineered strains increased 15–21% more deaminase activities of sprouts than with the wild strains, and reduced the ethylene concentrations of sprouts more significantly than with wild strains (P < 0.05). Inoculation with the wild and engineered strains promoted the growth of sprouts, while the promoting effects were more profound with the engineered strains than with the wild strains. The engineered strains improved saline resistance of sprouts under salt concentrations from 10 to 25 g L^?1. The engineered strains promoted longer roots and shoots than the wild strains under the salt stresses, indicating that the ACC deaminases on the endophytic bacterial cells could result in plant-produced ACC degradation and inhibit plant ethylene formation.

Conclusions

The protocols of expressing enzymes on endophytic bacterial cells showed greater potentials than those of plant over-expressed enzymes to increase the efficiency of plant metabolic pathways.