Understanding the molecular basis of plant growth promotional effect of Pseudomonas fluorescens on rice through protein profiling

Background  

Plant Growth Promoting Rhizobacteria (PGPR), Pseudomonas fluorescens strain KH-1 was found to exhibit plant growth promotional activity in rice under both in-vitro and in-vivo conditions. But the mechanism underlying such promotional activity of P. fluorescens is not yet understood clearly. In this study, efforts were made to elucidate the molecular responses of rice plants to P. fluorescens treatment through protein profiling. Two-dimensional polyacrylamide gel electrophoresis strategy was adopted to identify the PGPR responsive proteins and the differentially expressed proteins were analyzed by mass spectrometry.     

Understanding the Jasmine phenotype of rice through metabolite profiling and sensory evaluation

Introduction

Aromatic rices are culturally and economically important for many countries in Asia. Investigation of the volatile compounds emitted by rice during cooking is the key to understanding the flavour of elite aromatic rice varieties.

Objectives

The objectives of this study were to compare Jasmine-type aromatic rices from the Greater Mekong Subregion and Australia in terms of their metabolomics and sensory profiles and to draw out associations between the volatile organic compounds and human sensory perception of rice aroma.

Methods

A set of aromatic rice varieties from South East Asia and Australia, along with non-aromatic controls, was grown in tropical and temperate areas of Australia. Untargeted metabolite profiling of volatile compounds, from the heated rice flour, by static headspace extraction and separation by two dimensional gas chromatography time-of-flight mass spectrometry was performed. Volatile compounds were also assayed in the standard references used in the sensory evaluation and compared to the compounds detected in the headspace of rice.

Results

While 2-acetyl-1-pyrroline (2-AP) was a discriminating compound, we identified several of its structural homologues, and a number of other metabolites that were consistently detected in fragrant Jasmine rice. 2-AP producing rice varieties have different sensory properties and these variations were defined by the discriminating compounds identified in each rice type.

Conclusions

The results of this study are valuable in understanding the aspects of aromatic rice that are important to consumers, and in the identification of compounds that breeding programs can use to select for pleasant aromas, enabling breeding programs to target markets with greater accuracy.

     

Adaptation of Pseudomonas fluorescens to the plant rhizosphere

Saprophytic Pseudomonas are common root-colonizing bacteria that can improve plant health. Efficient exploitation of these bacteria in agriculture requires knowledge of traits that enhance ecological performance in the rhizosphere. Here, I describe the development and application of a promoter-trapping technology (IVET) that enables the isolation of Pseudomonas fluorescens genes that show elevated levels of expression in the rhizosphere. Using IVET, 20 P. fluorescens genes were identified that are induced during rhizosphere colonization, and their patterns of expression were analysed in laboratory media and in the rhizosphere. Fourteen genes showed significant homology to sequences in GenBank that are involved in nutrient acquisition, stress response, or secretion; six showed no homology. Seven of the rhizosphere-induced ( rhi ) genes have homology to known non- Pseudomonas genes. One of the rhi genes ( hrcC ) is a component of a type III secretion pathway, not previously known in non-parasitic bacteria. Together, these genes provide a view of the rhizosphere environment as perceived by a rhizosphere colonist, and suggest that the nature of the association between P. fluorescens and the plant root may be more complex and intimate than previously thought.     

Mechanism of zinc resistance in a plant growth promoting Pseudomonas fluorescens strain

Bacterial systems have evolved a number of mechanisms, both active and passive, to manage toxic concentrations of heavy metals in their environment. The present study is aimed at describing the zinc resistance mechanism in a rhizospheric isolate, Pseudomonas fluorescens strain Psd. The strain was able to sustain an external Zn²⁺ concentration of up to 5 mM in the medium. The strategy for metal management by the strain was found to be extracellular biosorption with a possible role of exopolysaccharides in metal accumulation. The attainment of equilibrium in biosorption reaction was found to be dependent on initial Zn²⁺ concentration, with the reaction reaching equilibrium faster (50 min) at high initial Zn²⁺ concentration. Biosorption kinetics of the process was adjusted to pseudo-first order rate equation. With the help of Langmuir and Freundlich adsorption isotherms, it was established that Zn²⁺ biosorption by the bacterium is a thermodynamically favourable process.     

Influence of pressure on growth of Pseudomonas fluorescens

Summary The effect of increased pressure on the growth of the bacterium Pseudomonas fluorescens has been investigated in an airlift-fermenter (10 l) up to 8 bars. It could be established, that increased hydrostatic pressure has a strong effect on the metabolism of the bacterium.     

Biological control of rice root-knot nematode,Meloidogyne graminicola through mixture of Pseudomonas fluorescens strains

The plant growth promoting rhizobacterium, Pseudomonas fluorescens strains PF1, TDK1, and PY15 were evaluated individually and in combinations for their efficacy against root-knot nematode, Meloidogyne graminicola, in rice plants under in vitro, glass house and field conditions. Culture filtrates of these strains either individually or as mixture inhibited egg hatching and caused mortality of juveniles of M. graminicola in vitro. The efficacy was more pronounced when filtrates of the strain were used as mixtures than as individual strains. Mixtures of P. fluorescens strains signficantly reduced M. graminicola infestation when applied as bacterial suspensions through seed treatment. The higher activity of peroxidase and chitinase enzymes was observed in plants treated with P. fluorescens mixtures than the plants treated with individual strains, two strain mixtures and untreated control. In field trials on rice, talc formulations of the P. fluorescens strains individually as well as mixtures were evaluated as seed treatment, soil treatment and combination of both. A mixture of the three strains was the most effective when applied either as seed + soil treatment or as seed treatment alone. The introduced P. fluorescens strains survived endophytically on rice roots. The application of the P. fluorescens mixture PF1 + TDK1 + PY15 in seed + soil treatment resulted in higher grain yield which provided a 27.3% increase over the control followed by P. fluorescens mixture PF1 + TDK1 + PY15 in seed treatment alone, which increased the grain yield of rice by 24.7% compared to the control.     

Understanding protein trafficking in plant cells through proteomics

The functions of approximately one-third of the proteins encoded by the Arabidopsis thaliana genome are completely unknown. Moreover, many annotations of the remainder of the genome supply tentative functions, at best. Knowing the ultimate localization of these proteins, as well as the pathways used for getting there, may provide clues as to their functions. The putative localization of most proteins currently relies on in silico-based bioinformatics approaches, which, unfortunately, often result in erroneous predictions. Emerging proteomics techniques coupled with other systems biology approaches now provide researchers with a plethora of methods for elucidating the final location of these proteins on a large scale, as well as the ability to dissect protein-sorting pathways in plants.     

Understanding protein trafficking in plant cells through proteomics

The functions of approximately one-third of the proteins encoded by the Arabidopsis thaliana genome are completely unknown. Moreover, many annotations of the remainder of the genome supply tentative functions, at best. Knowing the ultimate localization of these proteins, as well as the pathways used for getting there, may provide clues as to their functions. The putative localization of most proteins currently relies on in silico-based bioinformatics approaches, which, unfortunately, often result in erroneous predictions. Emerging proteomics techniques coupled with other systems biology approaches now provide researchers with a plethora of methods for elucidating the final location of these proteins on a large scale, as well as the ability to dissect protein-sorting pathways in plants.     

The effect of essential oils of basil on the growth of Aeromonas hydrophila and Pseudomonas fluorescens

Basil essential oils, including basil sweet linalool (BSL) and basil methyl chavicol (BMC), were screened for antimicrobial activity against a range of Gram-positive and Gram-negative bacteria, yeasts and moulds using an agar well diffusion method. Both essential oils showed antimicrobial activity against most of the micro-organisms examined except Clostridium sporogenes , Flavimonas oryzihabitans , and three species of Pseudomonas . The minimum inhibitory concentration (MIC) of BMC against Aeromonas hydrophila and Pseudomonas fluorescens in TSYE broth (as determined using an indirect impedance method) was 0·125 and 2% (v/v), respectively; the former was not greatly affected by the increase of challenge inoculum from 10³ to 10⁶ cfu ml⁻¹. Results with resting cells demonstrated that BMC was bactericidal to both Aer. hydrophila and Ps. fluorescens . The growth of Aer. hydrophila in filter-sterilized lettuce extract was completely inhibited by 0·1% (v/v) BMC whereas that of Ps. fluorescens was not significantly affected by 1% (v/v) BMC. In addition, the effectiveness of washing fresh lettuce with 0·1 or 1% (v/v) BMC on survival of natural microbial flora was comparable with that effected by 125 ppm chlorine.     

Pyrroloquinoline quinone is a plant growth promotion factor produced by Pseudomonas fluorescens B16

Pseudomonas fluorescens B16 is a plant growth-promoting rhizobacterium. To determine the factors involved in plant growth promotion by this organism, we mutagenized wild-type strain B16 using OmegaKm elements and isolated one mutant, K818, which is defective in plant growth promotion, in a rockwool culture system. A cosmid clone, pOK40, which complements the mutant K818, was isolated from a genomic library of the parent strain. Tn3-gusA mutagenesis of pOK40 revealed that the genes responsible for plant growth promotion reside in a 13.3-kb BamHI fragment. Analysis of the DNA sequence of the fragment identified 11 putative open reading frames, consisting of seven known and four previously unidentified pyrroloquinoline quinone (PQQ) biosynthetic genes. All of the pqq genes showed expression only in nutrient-limiting conditions in a PqqH-dependent manner. Electrospray ionization-mass spectrometry analysis of culture filtrates confirmed that wild-type B16 produces PQQ, whereas mutants defective in plant growth promotion do not. Application of wild-type B16 on tomato (Solanum lycopersicum) plants cultivated in a hydroponic culture system significantly increased the height, flower number, fruit number, and total fruit weight, whereas none of the strains that did not produce PQQ promoted tomato growth. Furthermore, 5 to 1,000 nm of synthetic PQQ conferred a significant increase in the fresh weight of cucumber (Cucumis sativus) seedlings, confirming that PQQ is a plant growth promotion factor. Treatment of cucumber leaf discs with PQQ and wild-type B16 resulted in the scavenging of reactive oxygen species and hydrogen peroxide, suggesting that PQQ acts as an antioxidant in plants.     

Evaluation of multiple plant growth promoting traits of an isolate of Pseudomonas fluorescens strain Psd

P. fluorescens strain Psd was isolated from the rhizosphere of Vigna mungo and evaluated for its multiple plant growth promoting and biocontrol properties against F. oxyspornum. Interestingly, this strain not only produces a range of antimicrobial compounds but also solubilizes complexed phosphates and synthesizes phytohormone (IAA). These properties can be assessed to elucidate the agronomic significance and rhizospheric competence of this soil isolate. Biocontrol action has been demonstrated in vitro against some other rhizospheric bacteria, and a phytopathogenic fungus along with wild type E. coli K-12. Genetic evidence for the antimicrobial status of strain Psd has been derived in terms of elucidating a unique combination of phenazine and pyrrolnitrin biosynthesis genes, not reported for any other P. fluorescens strain. The conserved part of antibiotics biosynthesis operon has been PCR amplified, cloned, sequenced and phylogenetic relationship based on similar genes from a few known Pseudomonads has been derived. The properties possessed by strain Psd may enable the bacterium to establish itself successfully in the rhizosphere.     

Effect of carbon dioxide on growth of Pseudomonas fluorescens.

In minimal medium at 30 degrees C, growth of Pseudomonas fluorescens was stimulated when the pressure (p) of CO2 in solution was 100 mm of Hg, but at higher concentrations the growth rate declined linearly with increasing pCO2. All concentrations of CO2 were inhibitory for growth in complex medium, and at 30 degrees C the maximum degree of inhibition was attained when pCO2 was 250 mm of Hg. The degree of inhibition at a constant pCO2 in solution increased with decreasing temperature. The degree of inhibition was directly proportional to temperature for growth in complex medium, but not in minimal medium. The inhibition of cell respiration by CO2 was the same whether cells had been grown in air or in the presence of CO2, indicating that adaptive enzyme synthesis does not occur in response to CO2.     

Effect of carbon dioxide on growth of Pseudomonas fluorescens.

In minimal medium at 30 degrees C, growth of Pseudomonas fluorescens was stimulated when the pressure (p) of CO2 in solution was 100 mm of Hg, but at higher concentrations the growth rate declined linearly with increasing pCO2. All concentrations of CO2 were inhibitory for growth in complex medium, and at 30 degrees C the maximum degree of inhibition was attained when pCO2 was 250 mm of Hg. The degree of inhibition at a constant pCO2 in solution increased with decreasing temperature. The degree of inhibition was directly proportional to temperature for growth in complex medium, but not in minimal medium. The inhibition of cell respiration by CO2 was the same whether cells had been grown in air or in the presence of CO2, indicating that adaptive enzyme synthesis does not occur in response to CO2.     

Understanding the molecular basis of Apert syndrome

Apert syndrome, first described in 1906, is one of the most severe of the craniosynostosis syndromes and is further characterized by midface hypoplasia, syndactyly, and other visceral abnormalities. Affected individuals generally require lifelong management by a multidisciplinary team of health care specialists. Apert syndrome results almost exclusively from one or the other of two point mutations in fibroblast growth factor receptor 2. Tremendous scientific advances have been made recently in understanding the molecular basis for Apert syndrome through clinical genetic, biochemical, and structural approaches. In this review, the authors provide the clinician with a basic overview of these findings and their therapeutic implications.     

Complete genome sequence of the plant commensal Pseudomonas fluorescens Pf-5

Pseudomonas fluorescens Pf-5 is a plant commensal bacterium that inhabits the rhizosphere and produces secondary metabolites that suppress soilborne plant pathogens. The complete sequence of the 7.1-Mb Pf-5 genome was determined. We analyzed repeat sequences to identify genomic islands that, together with other approaches, suggested P. fluorescens Pf-5's recent lateral acquisitions include six secondary metabolite gene clusters, seven phage regions and a mobile genomic island. We identified various features that contribute to its commensal lifestyle on plants, including broad catabolic and transport capabilities for utilizing plant-derived compounds, the apparent ability to use a diversity of iron siderophores, detoxification systems to protect from oxidative stress, and the lack of a type III secretion system and toxins found in related pathogens. In addition to six known secondary metabolites produced by P. fluorescens Pf-5, three novel secondary metabolite biosynthesis gene clusters were also identified that may contribute to the biocontrol properties of P. fluorescens Pf-5.     

Enhancement of plant stem growth by flocculation of the antibiotic-producing bacterium, Pseudomonas fluorescens S272, on the roots

The antibiotic-producing bacterium, Pseudomonas fluorescens, is assumed to be important in protecting plants from soilborne diseases. S. fluorescens S272, a hyper-producing strain of pyoluteorin (PT) and 2,4-diacetylphloroglucinol (DG), had previously been isolated from soil. The present paper reported that the growth of water-cultivated Kaiware radish was promoted to 120-140% of its normal level by the coaddition of an S272 culture broth (0.01-1% v/v) and a polysaccharide flocculant (1-100 ppm) from Klebsiella pneumoniae H12. Tight adhesion of S272 cells to the root tissue was microscopically observed. The growth promotion is assumed to have been caused by antibiotic effects for the following two reasons: 1) PT (4 mg/l) and DG (24 mg/l) addition to a radish culture enhanced stem growth to 130% of the normal level; 2) a culture solution containing the S272 culture broth (0.01-1% v/v) markedly inhibited the decomposition of hypersensitive chrysanthemum leaves. A soil-cultivation experiment with Gomphrena globosa under natural conditions also exhibited enhanced stem length (160%) by coaddition of the S272 culture broth and H12 polysaccharide. These results suggest that polysaccharide-enhanced adhesion of P. fluorescens S272 cells might be useful for promoting plant growth through the increased antibiotic effect.     

Development of a cold resistant mutant of plant growth promoting Pseudomonas fluorescens and its functional characterization

A cold resistant mutant of plant growth promoting Pseudomonas fluorescens GRS(1), was isolated following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. The mutant was able to grow and promote root and shoot elongation of wheat both at 25 and 10 degrees C, a temperature at which the wild type was unable to proliferate and function. In an effort to determine the mechanistic basis for this behavior, it was observed that following growth at 10 degrees C, the mutant synthesized some new proteins. SDS-polyacrylamide gel electrophoresis of the protein synthesized by the mutant revealed the presence of one major protein with a molecular mass of 13.5 kDa. However, at this point the function of this protein in not known.     

The effect of immobilization on rhamnolipid production by Pseudomonas fluorescens

Pseudomonas fluorescens in free suspension and immobilized to a commercially available biosupport (Biofix) and a biosorbent (Drizit), were used as bioremediation agents in an aqueous system with petrol (Slovene diesel) as the carbon source. Analysis of cellular growth and estimation of rhamnolipid production was carried out on the free suspension of the free and immobilized systems over 5 d. An increase in growth and rhamnolipid production was seen in the immobilized systems in comparison to the free system. EDTA was shown to be an inhibitor of rhamnolipid production. Its addition to the aqueous suspensions of all systems resulted in a fall in production of the surface-active agent in all cases, with no corresponding decrease in growth. This indicates the bacteria can rely on contact between the cell and the oil droplet for hydrocarbon transport into the cell. The data from this study indicated that immobilization resulted in a combination of increased contact between cell and hydrocarbon droplets and enhanced levels of rhamnolipid production.