Isolation,characterization and localization of extracellular polymeric substances from the cyanobacterium Arthrospira platensis strain MMG-9

Arthrospira platensis is a cyanobacterium known for its nutritional value and secondary metabolites. Extracellular polymeric substances (EPS) are an important trait of most cyanobacteria, including A. platensis. Here, we extracted and analysed different fractions of EPS from a locally isolated strain of A. platensis. Three different fractions of EPS were distinguished. These were EPS released into the medium (REPS), EPS loosely bound to the organism (LEPS) and EPS tightly bound to the organism (TEPS), which were extracted by different procedures. The LEPS fraction was smaller than the other two fractions. The EPS of A. platensis exhibited high diversity. Total protein and carbohydrate content was determined in each of these fractions. The largest amount of total carbohydrates and total proteins was in the TEPS fraction. Eight sugar moieties were detected and analysed in all EPS fractions using HPAE-PAD. Fructose, mannose and ribose were rare sugar residues in all fractions of EPS. With the exception of fructose, all sugars tested for were detected in TEPS. The amount of sugars detected was significantly higher in TEPS compared with the two other fractions, especially for galactose, xylose and glucose. The EPS were localized by confocal laser scanning microscopy (CLSM) after staining with different fluorescent dyes and it was found that A. platensis possessed a thick and smooth layer of EPS around the spiral trichomes.     

Production of indole-3-acetic acid by Thai native orchid-associated fungi

Thirteen endophytic fungi were isolated from roots of three orchid species, Spathoglottis affinis, Paphiopedelum bellatulum and Phaius tankervilleae. Of these, three fungal isolates produced high levels of indole-3-acetic acid (IAA) in culture medium supplemented with 2 mg/ml of L-tryptophan, and were selected for further analysis. Morphological characteristics and a phylogenetic analysis based on an alignment of internal transcribed spacer regions of nuclear rDNA indicated that the fungal isolates CMU-SLP 007 and CMU-NUT 013 belonged to family Tulasnellaceae, genus Tulasnella (the anamorphic genus Epulorhiza) and the fungal isolate CMU-AU 006 belonged to Colletotrichum gloeosporioides. These three fungal isolates produced maximum levels of IAA when grown in a culture medium supplemented with 4 mg/ml of L-tryptophan (C. gloeosporioides CMU-AU 006, 243.56 μg/ml and Tulasnella sp. CMU-SLP 007, 155.63 μg/ml) and 6 mg/ml of L-tryptophan (Tulasnella sp. CMU-NUT 013, 104.03 μg/ml). Thin layer chromatography revealed that all fungal IAA presented the same R_f value as the standard IAA. The biological activity of fungal IAA showed that it increased the length of stem forming roots and the number of roots of kidney bean (Phaseolus vulgaris), promoted seed germination, the length of roots and root to shoot ratio of corn (Zea mays) and increased the elongation of rice (Oryza sativa) coleoptiles when compared with all controls (water and culture medium treatments). In addition, the results of all biological activities using fungal IAA indicated that the quality of fungal IAA were similar to standard IAA.     

Cryopreservation of the edible alkalophilic cyanobacterium Arthrospira platensis

Efficient cryopreservation conditions for the edible alkalophilic cyanobacterium Arthrospira (Spirulina) platensis were investigated using a model strain A. platensis NIES-39. As a result, it was found that more than 60% of cells were viable upon thawing, when they had been frozen at a cooling rate of approximately ?1 °C min^?1 in the presence of 10% (v/v) dimethyl sulfoxide. Further examination with other Arthrospira strains showed that many of them had strain-dependent optimal conditions for cryopreservation. For example, the best freezing conditions for A. platensis SAG 21.99 were snap-freezing in liquid nitrogen in the presence of 5% (v/v) dimethyl sulfoxide, while they were slow cooling at approximately ?1 °C min^?1 in the presence of 10% (v/v) methanol for A. platensis NIES-46, NIES-2308 and UTEX 1926. The variety of successful cryopreservation conditions presented in this study is useful when attempting to cryopreserve various Arthrospira strains.     

Gibberellin-enhanced indole-3-acetic acid biosynthesis: D-Tryptophan as the precursor of indole-3-acetic acid

Stem segments excised from light-grown Pisum sativum L. (cv. Little Marvel) plants elongated in the presence of indole-3-acetic acid and its precursors, except for L-tryptophan, which required the addition of gibberellin A, for induction of growth. Segment elongation was promoted by D-tryptophan without a requirement for gibberellin, and growth in the presence of both D-tryptophan and L-tryptophan with gibberellin A3, was inhibited by the D-aminotransferase inhibitor D-cycloserine. Tryp-tophan racemase activity was detected in apices and promoted conversion of L-tryptophan to the D isomer; this activity was enhanced by gibberellin A3. When applied to apices of intact untreated plants, radiolabeled D-tryptophan was converted to indole-3-acetic acid and indoleacetylaspartic acid much more readily than L-tryptophan. Treatment of plants with gibberellin A3, 3 days prior to application of labeled tryptophan increased conversion of L-tryptophan to the free auxin and its conjugate by more than 3-fold, and led to labeling of N-malonyl-D-tryptophan. It is proposed that gibberellin increases the biosynthesis of indole-3-acetic acid by regulating the conversion of L-tryptophan to D-tryptophan, which is then converted to the auxin.     

Partial characterization of extracellular polysaccharides produced by cyanobacterium Arthrospira platensis

In this study, the physico-chemical characteristics of extracellular polysaccharides (EPS) produced by Arthrospira platensis were evaluated. Elemental analysis and a bicinchoninic acid (BCA) reaction indicated that the EPS were heteropolysaccharides that contain carbohydrate (13%) and protein (55%) moieties. Analysis of the infrared spectrum and elemental analysis revealed the presence of a sulfate group (0.5%). The UV-visible spectrum showed high UV absorption at 190∼230 nm and a shoulder at 260∼280 nm. In addition, this spectrum indicated that EPS can form aggregates with mycosporine-like amino acids and/or scytonemin. Gas chromatography analysis of the carbohydrate portion of the EPS indicated that it was composed of seven neutral sugars: galactose (14.9%), xylose (14.3%), glucose (13.2%), frucose (13.2%), rhamnose (3.7%), arabinose (1%), and mannose (0.3%) and two uronic acids, galacturonic acid (13.5%) and glucuronic acid (0.9%).     

Production of indole-3-acetic acid by several wild-type strains of Ustilago maydis

Indole-3-acetic acid (IAA) was identified and quantitated in spent media from cultures of ten Ustilago maydis strains. IAA was identified by thin-layer chromatography, high performance liquid chromatography (HPLC) and u.v. spectroscopy, and was quantitated by HPLC. All strains produced IAA in a tryptophan (Trp)-supplemented minimal medium at levels of 0.1 to 4.0 g IAA/ml of spent medium as assessed by HPLC. The highest levels of IAA were found in strains I2 and P2. The latter was also capable of producing IAA without addition of Trp to the medium.     

Utilization of the plant hormone indole-3-acetic acid for growth by Pseudomonas putida strain 1290

We have isolated from plant surfaces several bacteria with the ability to catabolize indole-3-acetic acid (IAA). One of them, isolate 1290, was able to utilize IAA as a sole source of carbon, nitrogen, and energy. The strain was identified by its 16S rRNA sequence as Pseudomonas putida. Activity of the enzyme catechol 1,2-dioxygenase was induced during growth on IAA, suggesting that catechol is an intermediate of the IAA catabolic pathway. This was in agreement with the observation that the oxygen uptake by IAA-grown P. putida 1290 cells was elevated in response to the addition of catechol. The inability of a catR mutant of P. putida 1290 to grow at the expense of IAA also suggests a central role for catechol as an intermediate in IAA metabolism. Besides being able to destroy IAA, strain 1290 was also capable of producing IAA in media supplemented with tryptophan. In root elongation assays, P. putida strain 1290 completely abolished the inhibitory effect of exogenous IAA on the elongation of radish roots. In fact, coinoculation of roots with P. putida 1290 and 1 mM concentration of IAA had a positive effect on root development. In coinoculation experiments on radish roots, strain 1290 was only partially able to alleviate the inhibitory effect of bacteria that in culture overproduce IAA. Our findings imply a biological role for strain 1290 as a sink or recycler of IAA in its association with plants and plant-associated bacteria.     

Enhanced biomass and gamma-linolenic acid production of mutant strain Arthrospira platensis

A mutant of Arthrospira platensis PCC 9108, strain M9108, obtained by mutagenesis with UV treatment, was able to mixotrophically grow in an SOT medium containing 40 g of glucose/l. The biomass and specific growth rate of strain M9108 (4.10 g/l and 0.70/d) were 1.9-fold and 1.4-fold higher, respectively, than those of the wild type (2.21 g/l and 0.58/d) under mixotrophic culture condition. In addition, when compared with the wild type, the content of gamma- linolenic acid (GLA) in the mutant was increased when glucose concentration was increased. Compared with the wild type, the GLA content of the mutant was 2-fold higher in autotrophic culture and about 3-fold higher in mixotrophic culture. Thus, the mutant appears to possess more efficient facility to assimilate and metabolize glucose and to produce more GLA than its wild-type strain.     

The biosynthesis of indole-3-acetic acid byFrankia

Summary High perfomance liquid chromatography (HPLC) of the products of [5-³H] tryptophan metabolism byFrankia sp. Avc I1 indicates that small amounts of [³H] indole-3-acetic acid (IAA) are excreted into the growth medium.Frankia has a limited capacity for the catabolism of [2-¹⁴C]IAA and the product that accumulates is different from that detected inRhizobium japonicum cultures following inoculation with [2-¹⁴C]IAA. The data imply that the rate of turnover of IAA is much more rapid inRhizobium thanFrankia and that the two organisms employ different routes for the catabolism of IAA.     

Optimization of medium for indole-3-acetic acid production using Pantoea agglomerans strain PVM

Aims: To optimize the medium components for the production of indole‐3‐acetic acid (IAA) by isolated bacterium Pantoea agglomerans strain PVM. Methods and Results: Present study deals with the production of an essential plant hormone IAA by a bacterial isolate P. agglomerans strain PVM identified by 16S rRNA gene sequence analysis. The medium containing 8 g l^?1 of meat extract and 1 g l^?1 of l ‐tryptophan (precursor) at optimum pH 7, 30°C and 48‐h incubation gave the maximum production of IAA (2·191 g l^?1). Effect of IAA synthesized on in vitro root induction in Nicotiana tobacum (leaf) explants was compared with that of control. IAA was characterized by high‐performance thin‐layer chromatography, high‐performance liquid chromatography and gas chromatography–mass spectroscopy. Conclusions: Pantoea agglomerans strain PVM was a good candidate for the inexpensive and utmost production of IAA in short period, as it requires simple medium (meat extract and l ‐tryptophan). Significance and Impact of the Study: The present report first time showed the rapid, cost‐effective and maximum production of IAA. No reports are available on the optimization of particular medium components for the production of IAA. This study demonstrates a novel approach for in vitro root induction in N. tobacum (leaf) explants.     

Uptake and metabolism of indole-3-butyric acid and indole-3-acetic acid by Petunia cell suspension culture

The uptake and metabolism of indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) were studied in suspension cell cultures of Petunia hybrida. The initial uptake of ³H-IBA was much higher than that of ³H-IAA, and after 10 min of incubation with labeled IBA and IAA, 4.6 pM vs 0.35 (39% vs 12% of total applied radioactivity) respectively, were found in the cell extracts. The uptake of IBA reached a plateau of 6.0 pM (62%) after 2 h while that of IAA increased continuously up to 1.5 pM (46%) after 24 h. Following the addition of 40 µM of unlabeled auxin more IBA was taken in initially than IAA (39% vs 12%), but the level almost equalized after 24 h of incubation when IBA uptake reached 890 nM (55%) and IAA 840 nM (46%).IBA was metabolized very rapidly by Petunia cell suspension to new compounds. HPLC of the cell extracts demonstrated a new metabolite after only 2 min of incubation, and after 30 min 60% of the radioactivity was in the new metabolite vs 10% in the IBA. The new compound was resolved by autofluorography to two metabolites but after 24 h only one metabolite was present. The IBA metabolites were identified tentatively as IBA aspartic acid (IBAasp) and IBA glucose (IBAglu). In the medium IBA disappeared at a fast rate and after 24h most of the radioactivity was present in the new metabolite, probably IBAasp. IAA was also converted rapidly to two new metabolites and both were still present after 24 h. No attempt was made to identify the metabolites of IAA. IAA metabolism proceeded at a slower rate, and autofluorography showed that while free IBA disappeared after 0.5 h, free IAA was still present after 1 h of incubation. We postulate that Petunia cells conjugate IBA rapidly to IBAglu which in turn is converted to form IBAasp which probably acts as a slow release hormone. Only intact cells were able to metabolize IBA and the reaction was affected by low temperature and anaerobic conditions. The fast rate of IBA uptake, the need for whole cells for the metabolism to proceed, and the fast change of IBA to a new metabolite in the medium, all suggest that both uptake and metabolism of IBA in Petunia cells occur on the cell surface.     

Metabolism of indole-3-acetic acid in rice: identification and characterization of N-beta-D-glucopyranosyl indole-3-acetic acid and its conjugates

A search was made for conjugates of indole-3-acetic acid (IAA) in rice (Oryza sativa) using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) in order to elucidate unknown metabolic pathways for IAA. N-beta-d-Glucopyranosyl indole-3-acetic acid (IAA-N-Glc) was found in an alkaline hydrolysate of rice extract. A quantitative analysis of 3-week-old rice demonstrated that the total amount of IAA-N-Glc was equal to that of IAA. A LC-ESI-MS/MS-based analysis established that the major part of IAA-N-Glc was present as bound forms with aspartate and glutamate. Their levels were in good agreement with the total amount of IAA-N-Glc during the vegetative growth of rice. Further detailed analysis showed that both conjugates highly accumulated in the root. The free form of IAA-N-Glc accounted for 60% of the total in seeds but could not be detected in the vegetative tissue. An incorporation study using deuterium-labeled compounds showed that the amino acid conjugates of IAA-N-Glc were biosynthesized from IAA-amino acids. IAA-N-Glc and/or its conjugates were also found in extracts of Arabidopsis, Lotus japonicus, and maize, suggesting that N-glucosylation of indole can be the common metabolic pathway of IAA in plants.     

Evidence for production of the phytohormone indole-3-acetic acid by cyanobacteria

The ability of cyanobacteria to produce the phytohormone indole-3-acetic acid (IAA) was demonstrated. A colorimetric (Salkowski) screening of 34 free-living and symbiotically competent cyanobacteria, that represent all morphotypes from the unicellular to the highly differentiated, showed that auxin-like compounds were released by about 38% of the free-living as compared to 83% of the symbiotic isolates. The endogenous accumulation and release of IAA were confirmed immunologically (ELISA) using an anti-IAA antibody on 10 of the Salkowski-positive strains, and the chemical authenticity of IAA was further verified by chemical characterization using gas chromatography-mass spectrometry in Nostoc PCC 9229 (isolated from the angiosperm Gunnera) and in Nostoc 268 (free-living). Addition of the putative IAA precursor tryptophan enhanced IAA accumulation in cell extracts and supernatants. As the genome of the symbiotically competent Nostoc PCC 73102 contains homologues of key enzymes of the indole-3-pyruvic acid pathway, a transaminase and indolepyruvate decarboxylase (IpdC), the putative ipdC gene from this cyanobacterium was cloned and used in Southern blot analysis. Out of 11 cyanobacterial strains responding positively in the Salkowski/ELISA test, ipdC homologues were found in 4. A constitutive and possibly tryptophan-dependent production of IAA via the indole-3-pyruvic acid pathway is therefore suggested. The possible role of IAA in cyanobacteria in general and in their interactions with plants is discussed.     

Bioelectrical reactions of Nitellopsis obtusa induced by indole-3-acetic acid

The complex of bioelectrical paramenters (membrane potential, membrane resistance and capacitance) of internodal cells of Nitellopsis obtusa was measured over a wide range of IAA concentration (10⁻¹⁰ to 10⁻⁴ M ) with two intracellular microelectrodes. Primary effects of IAA at a concentration as low as 10⁻¹⁰ M were observed. The optimum range of IAA action was from 10⁻⁹ to 10⁻⁶ M . The type of IAA-induced electroresponse depended on the initial level of membrane potential, which characterized the energetic state of the plasmalemma. In the energized state (ca −200 mV) N. obtusa cells appeared to have 3 typical reactions: hyperpolarization (membrane potential less than K⁺-equilibrium potential), depolarization (membrane potential higher than K⁺-potential) and absence of response at K⁺-electrochemical equilibrium. Membrane capacitance was found constant at 0.74 ± 0.05 μF cm⁻², but membrane resistance increased up to 50% independently of the sign of the electrogenic reaction. Increase of membrance capacitance and decrease of the membrane resistance was a feature of the de-energized state (ca −135 mV) and may be explained by lower viscosity of membrane lipids, which interacted with IAA. The complex of parameter, including cytoplasmic steaming taken as an indicator of energy supply, is discussed as indicating slow IAA penetration combined with a primary action of IAA on the plasmalemma receptor sites.     

Stimulation of indole-3-acetic acid production in Rhizobium by flavonoids

Flavonoids activate nod gene expression in Rhizobium resulting in the synthesis of Nod signals which trigger organogenesis in the host plant. This paper shows that nod-inducers also stimulate the production of the phytohormone IAA (indole-3-acetic acid).     

The Nitrilase ZmNIT2 converts indole-3-acetonitrile to indole-3-acetic acid

We isolated two nitrilase genes, ZmNIT1 and ZmNIT2, from maize (Zea mays) that share 75% sequence identity on the amino acid level. Despite the relatively high homology to Arabidopsis NIT4, ZmNIT2 shows no activity toward beta-cyano-alanine, the substrate of Arabidopsis NIT4, but instead hydrolyzes indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA). ZmNIT2 converts IAN to IAA at least seven to 20 times more efficiently than AtNIT1/2/3. Quantitative real-time polymerase chain reaction revealed the gene expression of both nitrilases in maize kernels where high concentrations of IAA are synthesized tryptophan dependently. Nitrilase protein and endogenous nitrilase activity are present in maize kernels together with the substrate IAN. These results suggest a role for ZmNIT2 in auxin biosynthesis.     

Tryptophan-dependent indole-3-acetic acid biosynthesis by ‘IAA-synthase’ proceeds via indole-3-acetamide

Plants are suggested to produce their major growth promoting phytohormone, indole-3-acetic acid (IAA), via multiple redundantly operating pathways. Although great effort has been made and plenty of possible routes have been proposed based on experimental evidence, a complete pathway for IAA production has yet to be demonstrated. In this study, an in-vitro approach was taken to examine the conversion of l-tryptophan (l-trp) to IAA by gas chromatography-mass spectrometry (GC-MS). Especially the influence of putative reaction intermediates on the enzymatic conversion of l-trp to IAA was analyzed. Among the substances tested only indole-3-acetamide (IAM) showed a pronounced effect on the l-trp conversion. We additionally report that IAM is synthesized from l-trp and that it is further converted to IAA by the utilized cell free Arabidopsis extract. Together, our results underscore the functionality of an IAM-dependent auxin biosynthesis pathway in Arabidopsis thaliana.     

Metabolism of indole-3-acetic acid and indole-3-methanol in wheat leaf segments

Indole-3-methanol is a product of indole-3-acetic acid metabolism in wheat leaves ( Triticum compactum Host., cv. Little Club). It leads either to the production of the corresponding aldehyde and carboxylic acid, to the production of a polar glucoside which releases indole-3-methanol on β-glucosidase treatment, or to an unidentified apolar product on mild alkaline hydrolysis in aqueous methanol. With reference to a published pathway of indole-3-acetic acid degradation, the results provide evidence for a prominent role of indole-3-methanol and also for the occurrence of co-oxidation processes in wheat leaves involving indole-3-acetic acid and phenolic cosubstrates.     

Changes in indole-3-acetic acid,indole-3-acetic acid oxidase,and peroxidase isoenzymes in the seeds of developing peach fruits

Changes in indole-3-acetic acid (IAA) content of peach (Prunus persica L. Batsch cv. Merry) seeds were followed during fruit development. The highest concentration of IAA, 2.7 g/g fresh weight, was found at the beginning of Stage III of fruit development, approximately 50–60 days after anthesis. The IAA-decarboxylating capacity of crude extracts of seeds was also greatest at 55–60 days after anthesis. Four soluble peroxidase isoenzymes were found on anionic electrophoresis. There were no marked changes in two isoenzymes (R _f 0.23 and 0.51), which were present in all three stages of fruit growth. There was a marked increase in a band atR _f 0.59 between Stages II and III, and a decrease in a band atR _f 0.68 from Stages II to III. Neither band (R _f 0.59 and 0.68) was present at Stage I.     

Growth inhibition of the cyanobacterium Spirulina (Arthrospira) platensis by 1.7 MHz ultrasonic irradiation

Ultrasonic waves of high frequency (1.7 MHz) and low intensity (0.6 W cm^–2) were employed to prevent cyanobacterial cells from growing fast and the effects of this growth inhibition were investigated. At least five minutes of ultrasonic irradiation was essential for effective inhibition. The growth rate of irradiated cells was reduced to 38.9% of the control during short-term culture. Longer exposure did not significantly enhance the inhibition. For a particular level of energy input, distributed ultrasonic exposure (more short intermittent exposures) was more effective in inhibiting growth than fewer, but longer exposures. For instance, the final biomass decreased to 30.1% of the control after ultrasonic irradiation for 4 minutes every 3 days, whereas it only decreased to 60% of the control with exposure for 12 minutes every 11 days. It is suggested that distributed ultrasonic irradiation is a practical method to prevent cyanobacterial cells from fast growth. A possible explanation for the inhibition is discussed in relation to cell structure, the absorption spectrum of intact cells, chlorophyll level and oxygen evolution.