Indole-3-acetic acid biosynthesis in the biocontrol strain Pseudomonas fluorescens Psd and plant growth regulation by hormone overexpression

Pseudomonas fluorescens is an important biological component of agricultural soils that bestows a number of direct and indirect beneficial attributes to the plants. We analyzed the biocontrol strain P. fluorescens Psd for indole-3-acetic acid (IAA) biosynthesis and studied the effect of its consequent manipulation on its plant-growth-promoting (PGP) potential. While the indole pyruvic acid (IPyA) pathway commonly associated with PGP bacteria was lacking, the indole acetamide (IAM) pathway generally observed in phytopathogens was expressed in strain Psd. Overexpression of IAM pathway genes iaaM-iaaH, from Pseudomonas syringae subsp. savastanoi drastically increased IAA levels and showed a detrimental effect on sorghum root development. On the other hand, heterologous expression of the indole-3-pyruvate decarboxylase/phenylpyruvate decarboxylase gene (ipdC/ppdC) of the IPyA pathway from the PGP bacterium Azospirillum brasilense SM led to enhancement of the IAA level. A more favorable effect of this recombinant strain on sorghum root growth and development suggests that metabolic engineering could be used to generate strains with improved PGP function.     

Characterization of metal-resistant plant-growth promoting Bacillus weihenstephanensis isolated from serpentine soil in Portugal

A metal-resistant bacterial strain SM3 isolated from a serpentine soil in the north-east of Portugal was characterized as Bacillus weihenstephanensis based on the morphological and biochemical characteristics and on the comparative analysis of the partial 16S ribosomal DNA sequence. Bacillus weihenstephanensis SM3 showed a high degree of resistance to nickel (1500 mg l(-1)), copper (500 mg l(-1)) and zinc (700 mg l(-1)) and also to antibiotics (ampicillin, penicillin, kanamycin and streptomycin). Strain SM3 has also exhibited the capability of solubilizing phosphate and producing indole-3-acetic acid (IAA) both in the absence and in the presence of metals (Ni, Cu and Zn). A pot experiment was conducted to elucidate the effects of strain SM3 on plant growth and uptake of Ni, Cu or Zn by Helianthus annuus. Inoculation with strain SM3 increased the shoot and root biomass of H. annuus grown in both non-contaminated and contaminated soil. Furthermore, strain SM3 increased the accumulation of Cu and Zn in the root and shoot systems. A batch experiment was also conducted to assess the metal mobilization potential of strain SM3 in soil. Inoculation with this strain increased the concentrations of water soluble Ni, Cu and Zn in soil. Metal solubilization by this bacterial strain may be an important process to promote the uptake of heavy metals by plants. This study elucidates the multifarious role of strain SM3 in plant growth promotion and its metal mobilizing potential.     

Phenotypic and genomic characterization of the antimicrobial producer Rheinheimera sp. EpRS3 isolated from the medicinal plant Echinacea purpurea: insights into its biotechnological relevance

In recent years, there has been increasing interest in plant microbiota; however, despite medicinal plant relevance, very little is known about their highly complex endophytic communities. In this work, we report on the genomic and phenotypic characterization of the antimicrobial compound producer Rheinheimera sp. EpRS3, a bacterial strain isolated from the rhizospheric soil of the medicinal plant Echinacea purpurea. In particular, EpRS3 is able to inhibit growth of different bacterial pathogens (Bcc, Acinetobacter baumannii, and Klebsiella pneumoniae) which might be related to the presence of gene clusters involved in the biosynthesis of different types of secondary metabolites. The outcomes presented in this work highlight the fact that the strain possesses huge biotechnological potential; indeed, it also shows antimicrobial effects upon well-described multidrug-resistant (MDR) human pathogens, and it affects plant root elongation and morphology, mimicking indole acetic acid (IAA) action.     

Localization of the auxin permease AUX1 suggests two functionally distinct hormone transport pathways operate in the Arabidopsis root apex

Auxins represent an important class of plant hormone that regulate plant development. Plants use specialized carrier proteins to transport the auxin indole-3-acetic acid (IAA) to target tissues. To date, efflux carrier-mediated polar auxin transport has been assumed to represent the sole mode of long distance IAA movement. Localization of the auxin permease AUX1 in the Arabidopsis root apex has revealed a novel phloem-based IAA transport pathway. AUX1, asymmetrically localized to the plasma membrane of root protophloem cells, is proposed to promote the acropetal, post-phloem movement of auxin to the root apex. MS analysis shows that IAA accumulation in aux1 mutant root apices is impaired, consistent with an AUX1 phloem unloading function. AUX1 localization to columella and lateral root cap tissues of the Arabidopsis root apex reveals that the auxin permease regulates a second IAA transport pathway. Expression studies using an auxin-regulated reporter suggest that AUX1 is necessary for root gravitropism by facilitating basipetal auxin transport to distal elongation zone tissues.     

Bio-control and plant growth promotion potential of siderophore producing endophytic Streptomyces from Azadirachta indica A. Juss

Three endophytic actinomycetes strains recovered from surface sterilized root tissues of Azadirachta indica A. Juss. (Meliaceae), plants were selected through tests for their potential as bio-control and plant growth promoting agents. It was also observed that the seed treated with the spore suspension of three selected strains of Streptomyces, significantly promoted plant growth and antagonized the growth of Alternaria alternata, causal agent of early blight disease in tomato plant. It was observed that the three selected strains prolifically produce IAA and siderophores that play vital role in promotion of plant growth and in suppression of Alternaria alternata. Interestingly, Streptomyces strain AzR-051 produced the highest amount of IAA at 13.73 μmol ml(-1) , compared to strains AzR-049 and AzR-010 9.22 μmol ml(-1) and 10.43 μmol ml(-1) respectively. It also produces siderophores higher than the other two strains. Thus these endophytic isolates have the potential as plant growth promoters as well as a bio-control agent, which is a useful trait for crop production in nutrient deficient soils.     

Characterization of cadmium‐resistant rhizobacteria and their promotion effects on Brassica napus growth and cadmium uptake

Excessive cadmium (Cd) accumulation in soil can adversely affect plants, animals, microbes, and humans; therefore, novel and uncharacterized Cd‐resistant plant‐growth‐promoting rhizobacteria (PGPR) are required to address this issue. In the paper, 13 bacteria were screened, their partial 16S rRNA sequences determined, and the isolates, respectively, clustered into Curtobacterium (7), Chryseobacterium (4), Cupriavidus (1), and Sphingomonas (1). Evaluation of PGP traits, including indole‐3‐acetic acid (IAA) production, 1‐aminocyclopropane‐1‐carboxylate (ACC) deaminase activity, siderophore secretion, and cyanhydric acid production, identified Cupriavidus necator GX_5, Sphingomonas sp. GX_15, and Curtobacterium sp. GX_31 as promising candidates for PGPR based on high IAA or ACC deaminase production. Additionally, root‐elongation assays indicated that inoculating GX_5, _15, or _31 increased Brassica napus root length both in the presence and absence of Cd by 19.75–29.96% and 19.15–31.69%, respectively. Pot experiments indicated that inoculating B. napus with GX_5, _15, and _31 significantly increased the dry weight of above‐ground tissues and root biomass by 40.97–85.55% and 18.99–103.13%, respectively. Moreover, these isolates significantly increased Cd uptake in the aerial parts and root tissue of B. napus by 7.38–11.98% and 48.09–79.73%, respectively. These results identified GX_5, _15, or _31 as excellent promoters of metal remediation by using microorganism‐associated phytoremediation.     

COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis

To control organ shape, plant cells expand differentially. The organization of the cellulose microfibrils in the cell wall is a key determinant of differential expansion. Mutations in the COBRA (COB) gene of Arabidopsis, known to affect the orientation of cell expansion in the root, are reported here to reduce the amount of crystalline cellulose in cell walls in the root growth zone. The COB gene, identified by map-based cloning, contains a sequence motif found in proteins that are anchored to the extracellular surface of the plasma membrane through a glycosylphosphatidylinositol (GPI) linkage. In animal cells, this lipid linkage is known to confer polar localization to proteins. The COB protein was detected predominately on the longitudinal sides of root cells in the zone of rapid elongation. Moreover, COB RNA levels are dramatically upregulated in cells entering the zone of rapid elongation. Based on these results, models are proposed for the role of COB as a regulator of oriented cell expansion.     

A halotolerant Enterobacter sp. displaying ACC deaminase activity promotes rice seedling growth under salt stress

Agricultural productivity is proven to be hampered by the synthesis of reactive oxygen species (ROS) and production of stress-induced ethylene under salinity stress. One-aminocyclopropane-1-carboxylic acid (ACC) is the direct precursor of ethylene synthesized by plants. Bacteria possessing ACC deaminase activity can use ACC as a nitrogen source preventing ethylene production. Several salt-tolerant bacterial strains displaying ACC deaminase activity were isolated from rice fields, and their plant growth-promoting (PGP) properties were determined. Among them, strain P23, identified as an Enterobacter sp. based on phenotypic characteristics, matrix-assisted laser desorption ionization-time of flight mass spectrometry data and the 16S rDNA sequence, was selected as the best-performing isolate for several PGP traits, including phosphate solubilization, IAA production, siderophore production, HCN production, etc. Enterobacter sp. P23 was shown to promote rice seedling growth under salt stress, and this effect was correlated with a decrease in antioxidant enzymes and stress-induced ethylene. Isolation of an acdS mutant strain enabled concluding that the reduction in stress-induced ethylene content after inoculation of strain P23 was linked to ACC deaminase activity.     

Isolation of Rhizobacteria from Jatropha curcas and characterization of produced ACC deaminase

Decreased levels of ACC (1-aminocyclopropane-1-carboxylic acid) result in lower levels of endogenous ethylene, which eliminate the potentially inhibitory effects of stress-induced higher ethylene concentrations. It is worth noting the substantial ability of the bacterial species to colonize different environments, including taxonomically distinct plants cultivated in distantly separated geographical regions. For example, Enterobacter cloacae, designated as MSA1 and Enterobacter cancerogenus, designated as MSA2 were recovered from the rhizosphere of Jatropha in the present work. This study first time confirms the ACC deaminase activity in the Enterobacter cancerogenus on the preliminary basis. Several bacterial plant growth-promoting mechanisms were analyzed and detected like phosphate solubilization, siderophore production, IAA production, GA(3) (gibberellic acid) production and ACC deaminase activity in the isolated cultures. Isolates were grown until exponential growth phase to evaluate their ACC deaminase activity and the effect of pH, temperature, salt, metals and substrate concentration after the partial purification of enzyme by ion exchange chromatography. The FOURIER TRANSFORM INFRARED (FT-IR) spectra were recorded for the confirmation of α-ketobutyrate production. By using lineweaver Burk plot K(m) and V(max) value for ACC deaminase of both the organism was calculated in the different fractions. In this work, we discuss the possible implications of these bacterial mechanisms on the plant growth promotion or homeostasis regulation in natural conditions.     

Isolation, characterization and beneficial effects of rice associated plant growth promoting bacteria from Zanzibar soils

This study was undertaken to isolate and characterize plant growth promoting bacteria (PGPB) occurring in four soils of Zanzibar, Tanzania as well as to evaluate their potential use as biofertilizers for rice. A total of 12 PGPB strains were isolated from rice and studied for growth characteristics, carbon/nitrogen source utilization patterns using QTS-24 kits, phosphate solubilization, indole acetic acid (IAA) production, antibiotic resistance patterns and growth at different pH, temperature and salt concentrations. All the isolates were motile and gram negative except Z3-4. Acetylene reduction activity was detected in all isolates ranging from 5.9-76.4 nmole C2H2 reduced/h x mg protein while 9 isolates produced IAA ranged from 20-90.8 mg/l. Most of the isolates showed resistance against different environmental stresses like 10-40 degrees C temperature, 0.2-1 M salt concentration and 4-8.5 pH range. Only one isolate Z2-7 formed clear zones on Pikovskaia's medium showing its ability to solubilize phosphates. Z3-2 was used to develop fluorescent antibodies to check the cross reactivity of the isolates. Inoculation of these bacterial isolates resulted in higher plant biomass, root area, and total N and P contents on Tanzanian rice variety BKN PRAT3036B under controlled conditions. Bacillus sp. Z3-4 and Azospirillum sp. Z3-1 are effective strains and, after further testing under field conditions, can be used for inoculum production of rice in Tanzania. The plant growth promoting effects of these PGPRs suggest that these can be exploited to improve crop productivity of rice in Tanzania.     

Effects of indole-3-acetic acid on croton oil- and arachidonic acid-induced mouse ear edema

The indole-3-acetic acid (IAA) is a plant growth hormone (auxin) being considered as a tryptophan metabolite in animals. The main purpose of this work was to verify IAA's topical anti-inflammatory action using croton oil- or arachidonic acid-induced mouse ear edema, in comparison to known anti-inflammatory agents. IAA antioxidant activity was also verified by measuring the inhibition of brain homogenate lipid peroxidation with the thiobarbituric acid reactive substances (TBARS) test. IAA inhibited the action of both croton oil- and arachidonic acid-induced edema in a dose-dependent manner (4.0 µmoles IAA inhibited 75.8% in croton oil and 82.5% in arachidonic acid induced ear edema). Both IAA (5.3 mM) and indomethacin (8.0 mM) inhibited TBARS formation. Data suggest that IAA exhibits antiinflammatory effect possibly by its antioxidant activity.     

Extracellular fatty acids facilitate flagella-independent translocation by Stenotrophomonas maltophilia

Stenotrophomonas maltophilia is widespread in natural environments such as soil, sewage and plant rhizospheres. Surfactants frequently function in modulating bacterial surface translocation. In this study, rpfB and rpfF orthologues were identified from S. maltophilia strain WR-C, which was isolated from the clogged zone of a septic system. These genes play a role in the biosynthesis of eight extracellular compounds that facilitated flagella-independent translocation by the wild-type or a flagella-defective mutant. This type of surface translocation has not been reported previously for this organism. These eight compounds include cis-delta 2-11-methyl-dodecenoic acid and seven structural derivatives. Two are saturated fatty acids; the others are unsaturated fatty acids with double bonds at position 2. These fatty acids vary in chain length from 12 to 14 carbons and in the position of the branched methyl group. Our results demonstrated that independently cis-delta 2-11-methyl-dodecenoic acid and 11-methyl-dodecanoic acid promoted flagella-independent translocation by S. maltophilia strain WR-C by acting as wetting agents.     

Activity,distribution and function of indole-3-acetic acid biosynthetic pathways in bacteria

The capacity to produce the phytohormone indole-3-acetic acid (IAA) is widespread among bacteria that inhabit diverse environments such as soils, fresh and marine waters, and plant and animal hosts. Three major pathways for bacterial IAA synthesis have been characterized that remove the amino and carboxyl groups from the α-carbon of tryptophan via the intermediates indolepyruvate, indoleacetamide, or indoleacetonitrile; the oxidized end product IAA is typically secreted. The enzymes in these pathways often catabolize a broad range of substrates including aromatic amino acids and in some cases the branched chain amino acids. Moreover, expression of some of the genes encoding key IAA biosynthetic enzymes is induced by all three aromatic amino acids. The broad distribution and substrate specificity of the enzymes suggests a role for these pathways beyond plant-microbe interactions in which bacterial IAA has been best studied.     

Mitigation of drought stress in rice crop with plant growth-promoting abiotic stress-tolerant rice phyllosphere bacteria

In the search of effective drought-alleviating and growth-promoting phyllosphere bacteria, a total of 44 bacterial isolates were isolated from the leaf surface of drought-tolerant rice varieties, Mattaikar, Nootripattu, Anna R(4), and PMK3, and screened for their abiotic stress tolerance by subjecting their growth medium to temperature, salinity, and osmotic stress. Only eight isolates were found to grow and proliferate under different abiotic stress conditions. These isolates were identified using 16S ribosomal DNA gene sequence and submitted to the NCBI database. All the bacterial isolates were identified as Bacillus sp., except PB24, which was identified as Staphylococcus sp., and these isolates were further screened for plant growth-promoting (PGP) traits such as IAA production, GA production, ACC deaminase activity, and exopolysaccharide production under three different osmotic stress conditions adjusted using polyethylene glycol (PEG 6000). Additionally, mineral solubilization was measured under the normal condition. Bacillus endophyticus PB3, Bacillus altitudinis PB46, and Bacillus megaterium PB50 were found to have multifarious PGP traits. Consecutively, the performance of an individual strain to improve the plant growth was investigated under the osmotic stress (25% PEG 6000) and nonstress condition by inoculating them into rice seeds using hydroponics culture. Furthermore, the drought-alleviating potency of bacterial strains was assessed in the rice plants using pot experiment (−1.2 MPa) through bacterial foliar application during the reproductive stage. Finally, as a result of seed inoculation and foliar spray, the application of B. megaterium PB50 significantly improved the plant growth under osmotic stress, protected plants from physical drought through stomatal closure, and improved carotenoid, total soluble sugars, and total protein content. Metabolites of PB50 were profiled under both stress and nonstress conditions using gas chromatography–mass spectroscopy.     

Participating mechanism of a major contributing gene ysnE for auxin biosynthesis in Bacillus amyloliquefaciens SQR9

The phytohormone indole-3-acetic acid (IAA) has been demonstrated to contribute to the plant growth-promoting effect of rhizobacteria, but the IAA biosynthesis pathway in rhizobacteria remains unclear. The ysnE gene, encoding a putative tryptophan acetyltransferase, has been demonstrated to be involved in and strongly contribute to IAA production in Bacillus, but the mechanism is unknown. In this study, to investigate how ysnE participates in IAA biosynthesis in the plant growth-promoting rhizobacterium Bacillus amyloliquefaciens SQR9, differences in the produced IAA biosynthesis intermediates between wild-type SQR9 and ΔysnE were analyzed and compared, and the effects of different intermediate compounds on the production of IAA and the accumulation of other intermediates were also investigated. The results showed that the mutant ΔysnE produced more indole-3-lactic acid (ILA) and tryptamine (TAM) than the SQR9 wild-type strain (nearly 1.6- and 2.1-fold), while the production of tryptophol (TOL) was significantly decreased by 46%. When indole-3-pyruvic acid (IPA) served as the substrate, the concentration of ILA in the ΔysnE fermentation broth was much higher than that of the wild type, while IAA and TOL were significantly lower, and ΔysnE was lower than SQR9 in IAA and TOL with the addition of TAM. The TOL content in the ΔysnE fermentation broth was much lower than that in the wild-type SQR9 with the addition of ILA. We suggest that ysnE may be involved in the IPA and TAM pathways and play roles in indole acetaldehyde (IAAld) synthesis from IPA and TAM and in the conversion of ILA to TOL.     

Inactivation of auxin in tobacco transformed with the indoleacetic acid-lysine synthetase gene of Pseudomonas savastanoi

The iaaL gene of Pseudomonas syringae, subspecies savastanoi, encodes an indoleacetic acid (IAA)-lysine synthetase. To determine the effects of converting IAA to IAA-lysine in whole plants, the iaaL gene was fused to a constitutive plant promoter and introduced into tobacco plants. Biochemical analyses show that endogenous IAA is reduced by up to 19-fold in iaaL plants. Tobacco plants expressing the iaaL gene display reduced apical dominance, reduced rooting, and inhibition of vascular differentiation. The phenotypic effects of iaaL gene expression are reverted by crossing iaaL plants with plants that overproduce IAA. These data indicate that iaaL can act as an anti-auxin gene in vivo and confirm the role of auxin in the control of apical dominance, root growth, and vascular differentiation in whole plants.     

Transcriptome analysis of Armillaria gallica 012 m in response to auxin

Gastrodia elata is an achlorophyllous and fully mycoheterotrophic orchid which obtains carbon and other nutrients from Armillaria species in its life cycle. Many researchers suggested that plant hormones, as signing molecules, play a central role in the plant–fungi interaction. In the process of Armillaria gallica 012 m cultivation, both exogenous indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) distinctly stimulated the growth of mycelia in solid media. The differential expression genes (DEGs) of A. gallica 012 m with IAA versus blank control (BK) and IBA versus BK were investigated. The results showed that more than 80% of DEGs of the IAA group were coincident with the DEGs of the IBA group, and more than half of upregulated DEGs and most of the downregulated DEGs of the IAA group coincided with those DEGs of the IBA group. Above research implied that A. gallica 012 m could perceive IAA and IBA, and possess similar responses and signaling pathways to IAA and IBA. The overlapping differential genes of the IAA group and IBA group were analyzed by GO term, and the results showed that several DEGs identified were related to biological processes including positive regulation of the biological process and biological process. The downregulated NmrA-like and FKBP_C genes might be benefit to the growth of mycelia. Those results can explain that exiguous IAA and IBA improved the growth of A. gallica to some extent. We speculate that IAA and IBA are signaling molecules, and regulate the expression of growth-related genes of A. gallica 012 m by the same signaling pathway.     

Fitness of Japanese encephalitis virus to Neuro-2a cells is determined by interactions of the viral envelope protein with highly sulfated glycosaminoglycans on the cell surface

Genetically different subpopulations were identified and purified from Japanese Encephalitis virus (JEV). Those with small plaques (SPs; <2 mm in diameter), derived from strains of T1P1, CJN, and CC27, were more competent than those with large plaques (LPs; >5 mm in diameter) when passaged in Neuro-2a cells. Differences in amino acids between SPs and LPs from each strain were shown in the viral envelope (E) protein. The amino acid at E-306 was Glu in LP but was substituted by Lys in SP in the T1P1 strain. A similar substitution occurred at E-138 in the CJN strain. However, the amino acid was Asp in LP but was substituted by Asn in SP at E-389 in the CC27 strain. All SPs were shown to have a higher affinity to the cellular membrane when compared to LPs, and this resulted in more-efficient infection of Neuro-2a cells, suggesting that the differential fitness of JEV variants to Neuro-2a cells appeared in the early phase of infection. In addition, glycosaminoglycans (GAGs) on the surface of many mammalian cells have been demonstrated to be critical for infection by JEV, especially SP variants. The present results suggest that T1P1-SP1 viruses infected Neuro-2a cells more efficiently in spite of the sparse distribution of cell surface GAGs. We conclude that highly sulfated forms of GAGs expressed by Neuro-2a cells play an important role in selecting JEV variants with specific mutations in the E glycoprotein.