Regulation of Auxin Levels in Coleus blumei by Ethylene

An investigation of the effects of ethylene pretreatment on several facets of auxin metabolism in Coleus blumei Benth “Scarlet Rainbow” revealed a number of changes presumably induced by the gas. Transport of indoleacetic acid-1-¹⁴C in excised segments of the uppermost internode was inhibited by about 50%. Decarboxylation of indoleacetic acid-1-¹⁴C by enzyme breis was not affected by the pretreatment. Levels of extractable native auxin in upper leaf and apical bud tissue of the pretreated plants were approximately one-half of those present in untreated plants. The rate of formation of auxin from tryptophan by enzyme breis from pretreated plants was approximately one-half that occurring in incubation mixtures containing the enzyme system from untreated plants. The conjugation of indoleacetic acid-1-¹⁴C in a form characterized chromatographically as indoleacetylaspartic acid was increased 2-fold in the upper stem region of plants pretreated with ethylene.     

Labeled indole-macromolecular conjugates from growing stems supplied with labeled indoleacetic Acid : I. Fractionation

Pea (Pisum sativum var. Alaska) and bean (Phaseolus vulgaris var. Red Kidney) stem sections treated with indoleacetic acid-1-¹⁴C, indoleacetic acid-2-¹⁴C, and indoleacetic acid-5-³H were homogenized, extracted with phenol, and the water-soluble, ethanol-insoluble material subjected to further fractionation. Following an 18-hour incubation period in indoleacetic acid-1-¹⁴C, most of the label was found as nonindole-¹⁴C in high molecular weight polysaccharide, as phenol extraction is specific for both RNA and polysaccharides. With indoleacetic acid-2-¹⁴C and -5-³H, and to a lesser extent with indoleacetic acid-1-¹⁴C, radioactive indoles were obtained by hydrolysis from a heterogeneous fraction between about 500 and 30,000 molecular weight, possibly polysaccharide in nature. Indoleacetic acid accounted for 8% and indole aldehyde accounted for 21% of the total radioactivity in the extract.     

The interrelation between elongation and IAA-1-14C metabolism in wheat coleoptile sections

It has been shown that intensive elongation of wheat coleoptile sections is correlated with indoleacetic acid-1-¹⁴C metabolism. In tissues actively growing the disappearance of indoleacetic acid-1-¹⁴C and formation of conjugates and metabolites (indoleacetamide, indoleacetyl-β-D-glucose, indoleacetyl aspartic acid) is twice as high as in sections where growth has been stopped by physical constraint (plaster) or in tissue which did not elongate by an unknown reason.     

Indoleacetic Acid synthesis in soybean cotyledon callus tissue

Growth of an auxin-requiring soybean cotyledon callus tissue (Glycine max L., Merr. var. Acme) was promoted by tryptophan, tryptamine, indole, indoleacetamide and, to a very slight degree, anthranilic acid. When tryptophan-3-¹⁴C was supplied in the growth medium, labeled indoleacetic acid (IAA) was found in both the tissue and the medium. Medium, from which the cells had been removed, was also found to convert labeled tryptophan to IAA. Soybean callus contained 0.044 μmole/g free tryptophan, but this is apparently not available for conversion to IAA. These results suggest that while exogenously supplied trytophan could elevate a specific internal pool where IAA synthesis occurs some of the growth on a tryptophan medium can be accounted for by external conversion.     

Conversion of indole-3-ethanol to indole-3-acetic Acid in cucumber seedling shoots

Indoleethanol-¹⁴C was applied to intact cucumber seedlings and to hypocotyl segments. The presence of indoleacetic acid-¹⁴C in tissue extracts was demonstrated by thin layer radiochromatography. There was no evidence of conversion of indoleacetic acid to indoleethanol. It is suggested that the growth-promoting activity of indoleethanol is due to its conversion to indoleacetic acid.     

Re-sorption of organic compounds by roots of Zea mays L. and its consequences in the rhizosphere

The exudation of soluble carbon compounds from Zea mays roots was investigated over a 10 day growth period under sterile and non-sterile solution culture conditions. The results showed that plants grown in sterile static solution culture, where C was allowed to accumulate, released 8 times less C than plants grown under culture conditions in which the solutions were replaced daily. The increased C loss from plant cultures in which exudates were removed daily was attributable to, (a) the reduced potential for root re-sorption of previously lost C, and (b), increasing diffusion gradients between the root and the surrounding bathing solution increasing passive leakage of exudates from the roots. In treatments where C was removed daily from the root-bathing solution, 86% of the total C lost was of a soluble low molecular weight nature, whereas, in sterile and non-sterile static cultures, allowing the accumulation of C over 10 days, this was reduced to 67.5 and 48% respectively. The main C fluxes operating in a solution culture system (efflux and influx of C by both roots and microorganisms) were examined using a computer simulation model to describe movement of soluble sugar-C in both sterile and non-sterile conditions. In sterile static cultures where C was allowed to accumulate in solution over a 10 day growth period, 98% of the C exuded was re-absorbed by the plant. Where C was removed daily from the root-bathing solution this was reduced to 86%. The predicted patterns of C accumulation were similar to those found in the experiments. Simulations showed that the pattern of accumulation and final equilibrium concentrations were dependent on the rate of exudation, the spatial characteristics of exudation, solution volume, root growth rate and the presence of a microbial population. Simulations under non-sterile conditions showed that roots can compete with microorganisms for exudates in solution indicating the possible importance of re-sorption in a soil environment. The results clearly indicate that roots are capable of regulating the net amount of C released into a solution culture with the amount of C collected being highly dependent on the experimental conditions employed. The possible implications of soluble C influx on processes operating within the rhizosphere and in experimental systems is discussed.     

Inhibition of ent-Kaurene Oxidation and Growth by alpha-Cyclopropyl-alpha-(p-methoxyphenyl)-5-pyrimidine Methyl Alcohol

Growth of Alaska peas (Pisum sativum) is inhibited more than 60% by alpha-cyclopropyl-alpha-(p-methoxyphenyl)-5-pyrimidine methyl alcohol (ancymidol) treatment. This growth inhibition can be reversed completely by gibberellic acid application. Cell-free enzyme preparations from pea shoot tips and wild cucumber (Marah oreganus) endosperm were used to test the effects of this substituted pyrimidine on the incorporation of mevalonic acid-(14)C into ent-kaurene and ent-kaurenol, respectively. Ancyidol (10(-6)m) completely blocks the conversion of ent-kaurene to ent-kaurenol. This result was confirmed with the wild cucumber endosperm system by testing the direct conversion of labeled ent-kaurene to ent-kaurenol. Ancymidol at higher concentrations (10(-3)m) inhibits the incorporation of mevalonic acid-(14)C into ent-kaurene to a lesser extent. It is concluded that one mode of action of this growth regulator is the inhibition of gibberellin biosynthesis.     

Tryptophan Decarboxylase Activity in Developing Cucumber Seedlings

The rate of decarboxylation of DL-tryptophan-carboxyl-¹⁴C in homogenates of cotyledons, hypocotyls and roots of sterile and non-sterile cucumber seedlings of 4, 8 and 11 days was measured. Tryptophan decarboxylating activity is highest in hypocotyls, lowest in cotyledons. In all organs the activity decreases with age. This enzyme activity does not parallel the IAA level in the organs during ageing.     

Competition strategies for the decolorization of a textile-reactive dye with the white-rot fungi Trametes versicolor under non-sterile conditions

A variety of white-rot fungi can oxidize textile dyes under sterile conditions; however, an important consideration for their use in treating wastewater containing textile dyes is whether similar degrees of treatment can be achieved under non-sterile conditions. Four strategies were investigated for their potential in optimizing the use of the fungus Trametes versicolor in non-sterile culture for treating wastewater containing the diazo textile dye C.I. Reactive Black 5 (RB5). Three strategies with suspended culture were designed to increase the decolorization activity in suspended culture from a given amount of T. versicolor inoculum based on its tolerance of low pH (pH reduction in medium), production of extracellular enzymes (use of suspended enzymes alone), and its ability to produce enzymes independent of growth (nitrogen limitation in medium). The results showed that reduction of the medium pH to 3 did not suppress bacterial growth, while enzyme production by T. versicolor ceased. The use of the extracellular enzymes alone would allow the decoupling of the process of fungal growth from wastewater treatment; however, the enzyme activity of an enzyme suspension decreased rapidly under non-sterile conditions. The strategy of limiting nitrogen in the medium to suppress bacterial growth has potential together with the fourth strategy, the cultivation of fungi on organic solids to produce inocula for a decolorization process under non-sterile conditions. A high degree of decolorization of RB5 under non-sterile conditions was achieved with T. versicolor grown on grains as sole substrate. The rate of decolorization was dependent on the amount of fungal inoculum used.     

Interactions of phenolic acids, metallic ions and chelating agents on auxin-induced growth

By growth experiments in indoleacetic acid-1-¹⁴C (IAA), and determination of the ¹⁴CO₂ evolved, it has been shown directly that polyphenols synergize IAA-induced growth by counteracting IAA decarboxylation. Sinapic and ferulic acids act like polyphenols. Endogenous polyphenols doubtless exert the same influence in intact plants. Monophenols stimulate the decarboxylation of IAA under conditions where they depress growth. When Mn⁺⁺ is present as well, this effect is enhanced. All these growth effects are paralleled by effects on the isolated IAA oxidizing enzyme of Avena.     

Mitigation of growth retardation effect of plant defense activator,acibenzolar-<Emphasis Type="Italic">S</Emphasis>-methyl,in amaranthus plants by plant growth-promoting rhizobacteria

Four rhizobacterial strains and acibenzolar-S-methyl (ASM), a chemical activator, which suppressed foliar blight of amaranthus (Amaranthus tricolor L.) caused by Rhizoctonia solani Kühn were evaluated for their effect on plant growth. The experiments were performed both under sterile and non-sterile soil conditions, in the presence or absence of the pathogen. In all cases, plants treated with ASM showed significant reduction in growth, as determined by shoot length, and shoot and root dry weight when compared to other treatments. The growth retardation effect of ASM was more profound with respect to shoot length. Reduction in shoot length was least when plants were treated with a combination of the chemical activator and Pseudomonas putida 89B61 under non-sterile soil conditions in the absence of the pathogen. Both under sterile and non-sterile soil conditions, in the presence of the pathogen, reduction in shoot length due to application of ASM was diminished significantly when plants were treated with rhizobacterial strain Pseudomonas fluorescens PN026R. Combined use of plant growth-promoting rhizobacteria (PGPR) and ASM was found to be beneficial as the growth retardation effect of the plant defense activator was reduced by the growth-promoting ability of the rhizobacteria.     

Effect of rearing conditions on encephalomyocarditis (EMC) virus-induced diabetes in mice

To compare the incidence in the diabetic syndrome in mice reared under sterile and non-sterile conditions, both conventional and SPF DBA/2 N male mice were inoculated intraperitoneally with two doses (10(4) TCID50/0.1 ml or 10(5) TCID50/0.1 ml) of encephalomyocarditis (EMC) virus M variant. The incidence of diabetic symptoms was higher in mice reared under sterile conditions than under non-sterile conditions. It appeared that a difference in animal rearing conditions would affect the sensitivity to EMC virus in mice.     

The effects of spent mushroom compost and municipal solid waste compost on Phytophthora drechsleri in vivo and in vitro

Leached spent mushroom compost (SMC), municipal solid waste compost (MSWC) and their extracts, were tested to suppress Phytophthora drechsleri in cucumber plants. The composts were mixed with sand-loam soil in sterile and non-sterile types and were used to assess suppressiveness against P. drechselri in greenhouse experiments. The extracts of composts, in both sterile and non-sterile types, were applied to evaluate their effect in suppression of pathogen in vitro. The results of the experiments showed that all applications rate of non-sterile SMC were significantly effective in the control of the pathogen. However the sterile SMC amendments did not have a positive effect on the pathogen suppression in vitro or in vivo, as it was expected. In greenhouse experiments, both composts were effective in controlling pathogen at the rate of 15%, but the treatments amended with higher rate of MSWC did not show a positive effect. The treatments amended with MSWC (15%) and SMC (25%) showed the most suppressive effect in controlling the pathogen. The extract of leached-SMC could inhibit P. drechselri in petri dish.     

Indoleacetic Acid biosynthesis in Avena coleoptile tips and excised bean shoots

Avena coleoptiles did not elongate when incubated with tryptophan under sterile conditions. Indole, anthranilic acid, and tryptamine promoted elongation. Under the same conditions, the tissue converted tryptophan-¹⁴C to IAA-¹⁴C. More IAA-¹⁴C was produced from indole-¹⁴C than from tryptophan-¹⁴C; however, the free tryptophan content of the tissue was also greatly increased by the indole treatment. Tryptophan-¹⁴C was readily taken up by the tissue but was mainly incorporated into protein and did not increase the free tryptophan level. When bean shoots were labeled with tryptophan-¹⁴C or indole-¹⁴C, the label incorporation into IAA-¹⁴C was very nearly the same. In this tissue the free tryptophan level in the tryptophan-¹⁴C and indole-¹⁴C treatments was also about equal. These results suggest that failure of exogenously supplied tryptophan to promote the elongation of Avena coleoptiles is a result of its predominant incorporation into protein and consequent unavailability for conversion to IAA.     

Biosynthesis and Metabolism of Indol-3yl-acetic acid: II. IN VIVO EXPERIMENTS WITH 14C-LABELLED PRECURSORS OF IAA IN TOMATO AND BARLEY SHOOTS

Our previous investigation of the naturally occurring indolecompounds in barley and tomato shoots suggested that the biosynthesisof indol-3yl-acetic acid (IAA) from tryptophan might proceedvia either the indol-3yl-pyruvic acid or tryptamine pathwaysin both species. The results further indicated that the indol-3yl-lacticacid pathway for IAA formation might also be operative in tomato.In the present study, tryptophan-3-¹⁴C and tryptamine-2-¹⁴Cwere fed to excised shoots of both barley and tomato, and indol-3yl-lacticacid-3-¹⁴C was also fed to shoots of tomato. All three compoundswere found to give rise to radioactive IAA with little dilutionin specific activity. Feeding tryptophan-3-¹⁴C also resultedin the labelling of indol-3yl-pyruvic acid, indol-3yl-acetaldehyde,and tryptamine, which were isolated and chemically identifiedfrom both species, and radioactive indol-3yl-lactic acid andtryptophol were also produced in tomato. Indol-3yl-acetaldehydewas found to be labelled in both species after administrationof tryptamine-2-¹⁴C, while the principal metabolite of indol-3yl-lacticacid-3-¹⁴C was radioactive tryptophan. These findings, alongwith the results from a quantitative study of the radioactivemetabolites, indicate that both the indol-3yl-pyruvic acid andtryptamine pathways can operate in both species, while the formationof IAA from indol-3yl-lactic acid in tomato probably occursindirectly, via tryptophan. These conclusions were supportedby the demonstration of the enzymes, L-tryptophan transaminase,L-trypto-phan decarboxylase, and indol-3yl-acetaldehyde dehydrogenasein cell-free extracts of both tissues, and of indol-3yl-pyruvicacid decarboxylase in the tomato extract. No indol-3yl-lacticacid decarboxylase activity was observed in the extracts fromeither tissue.     

Induction of indoleacetic Acid synthetases in tobacco pith explants

Formation of indoleacetic acid synthetases in tobacco pith explants was determined by following the growth of tissue cultures under conditions of indole-3-acetic acid (IAA) deprivation and by measuring the enzymatic conversion of tryptophan to IAA in the cultures. The pith explants obtained from the parent plant (Nicotiana glauca) and from basal regions of the tumor-prone hybrid (N. glauca × N. langsdorffii) both show a requirement for exogenous IAA for growth initiation in culture. The parent pith requires the constant presence of added IAA for continued growth, but hybrid pith, after initial treatment with IAA, will grow without further additions. IAA synthetases are detected in the cell homogenates of hybrid pith explants cultured with either continuous or initial IAA addition. These observations indicate that IAA may induce its own production. In contrast, IAA synthetases are not found in the parent pith under comparable culture conditions. Besides IAA, nonhormonal compounds such as indole and tryptophan are also capable of stimulating growth of hybrid pith, possibly through the induction of IAA synthetases needed for IAA formation. Indole and tryptophan are, however, inactive in growth promotion of the parent pith. These results suggest that the genomic expression of IAA synthetase formation is more stringently controlled in N. glauca than in the tumorprone hybrid.     

Pathways of IAA production from tryptophan by plants and by their epiphytic bacteria: Metabolism of indolepyruvic acid and indolelactic acid

Metabolites of indolepyruvic acid and indolelactic acid were investigated using 2 systems: a bacterial (pea stem homogenates containing the epiphytic bacteria) and a plant system (pea stem sections under sterile conditions). The products of spontaneous indolepyruvic acid decomposition in aqueous solution and during chromatography were investigated, too. Biological indolepyruvic acid conversion yields, besides those substance amounts which occur spontaneously, indoleacetic acid, indoleethanol (tryptophol) and (only in the sterile plant system) indoleacetaldehyde. An inhibitor extract from pea stems decreases the indoleacetic acid and increases the indoleethanol and indoleacetaldehyde gain. Indolelactic acid is not metabolized in the sterile plant sections. Indolelactic acid oxidation by the bacteria-containing homogenate yields indolepyruvic acid and is inhibited by the inhibitor extract.     

Interactions between Plants and Epiphytic Bacteria Regarding Their Auxin Metabolism

Proofs of different kind are presented of the existence of highly active bacteria producing IAA from tryptophan on plant surfaces and in plant homogenates. Both homogenates and washing solutions of nonsterile pea plant parts are active in producing IAA from tryptophan. Activity is much enhanced by the addition of glucose or by preincubating the preparations; it is abolished by sterile filtration, by some bactericidic and bacteriostatic substances, by chloramphenicol, streptomycin, and albucid (penicillin being only partly effective). Preparations of sterile plants do not produce IAA from tryptophan within the detection limit of the Salkowski test. The bacteria are even present on seed surfaces, in the air, and in aceton or ammonium sulfate precipitations of homogenates. Main products of the bacterial tryptophan conversion, as demonstrated by paper chromatography, are indolepyruvic acid, indoleacetic acid, indolecarboxaldehyde, and indolecarboxylic acid. In presence of glucose indolepyruvic acid is by far dominating. Many hitherto known results about tryptophan conversion to IAA by higher plants are likely to be falsified by epiphytic bacteria.     

The role of indoleacetaldehyde in IAA production from tryptophan by plants and by their epiphytic bacteria

Tryptophan, tryptamine, or indolepyruvic acid were applied to 2 systems: a bacterial (pea stem sections containing the epiphytic bacteria) and a plant system (pea stem sections under sterile conditions). In the plant system, the production of indoleacetic acid and indoleethanol (tryptophol) from each applied indole derivative is clearly reduced by the aldehyde reagents bisulfite and dimedon, respectively. Indoleacetaldehyde is chromatographically detected after alkaline liberation from its bisulfite addition product. In the bacterial system, the production of indoleacetic acid and indoleethanol is likewise reduced by bisulfite and dimedon. However, after tryptophan or tryptamine application, we could not detect indoleacetaldehyde in the described way. In one case only, namely tryptamine application to the bacterial system, indoleethanol production (contrary to indoleacetic acid production) is scarcely reduced by the aldehyde reagents. This indicates a bacterial pathway tryptamine → indoleethanol which bypasses indoleacetaldehyde.     

Soil colonization by Trametes versicolor grown on lignocellulosic materials: Substrate selection and naproxen degradation

The ability of Trametes versicolor to degrade soil pollutants has been widely studied. However, the use of such fungus in real soil applications has lead to dissimilar results mainly due to soil colonization limitations. Therefore, it is important to investigate techniques to improve the survival of this white rot fungus in soils. In the present study, several processed and unprocessed low-cost lignocellulosic substrates were employed as inoculum carriers for fungal growth prior to application in soil for bioaugmentation. The fungal growth was determined by means of laccase activity and ergosterol determinations; additionally, the degrading capacity was measured by the naproxen degradation test (ND24). Although T. versicolor was able to colonize all materials, the colonization and enzymatic production was higher on processed agricultural wastes with relative low C/N ratios than in raw lignocellulosic substrates. Soil colonization was successful under both sterile and non-sterile conditions when amended with processed agricultural wastes, yielding even higher laccase production in non-sterile conditions. Moreover, T. versicolor was able to degrade significant amounts of spiked naproxen after 24 h in sterile and non-sterile soil cultures, showing the best results when using a material based on wheat straw as carrier.